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Peroxidase-Mimicking Activity of Biogenic Gold Nanoparticles Produced from Prunus nepalensis Fruit Extract: Characterizations and Application for the Detection of Mycobacterium bovis
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Peroxidase-Mimicking Activity of Biogenic Gold Nanoparticles Produced from Prunus nepalensis Fruit Extract: Characterizations and Application for the Detection of Mycobacterium bovis
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  • Bhaskar Das
    Bhaskar Das
    School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
    Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela 769008, India
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  • Javier Lou-Franco
    Javier Lou-Franco
    School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
    More by Javier Lou-Franco
  • Brendan Gilbride
    Brendan Gilbride
    School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
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  • Matthew G. Ellis
    Matthew G. Ellis
    School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
    Nanophotonics Centre, University of Cambridge, Cambridge CB3 0HE, U.K.
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  • Linda D. Stewart
    Linda D. Stewart
    School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
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  • Irene R. Grant
    Irene R. Grant
    School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
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  • Paramasivan Balasubramanian
    Paramasivan Balasubramanian
    Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela 769008, India
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    Orcidhttps://orcid.org/0000-0002-3821-5029
  • Cuong Cao*
    Cuong Cao
    School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
    Material and Advanced Technologies for Healthcare, Queen’s University of Belfast, Belfast BT7 1NN, U.K.
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0001-8621-8403
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ACS Applied Bio Materials

Cite this: ACS Appl. Bio Mater. 2022, 5, 6, 2712–2725
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https://doi.org/10.1021/acsabm.2c00180
Published May 11, 2022

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Abstract

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In the present study, a facile, eco-friendly, and controlled synthesis of gold nanoparticles (Au NPs) using Prunus nepalensis fruit extract is reported. The biogenically synthesized Au NPs possess ultra-active intrinsic peroxidase-like activity for the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. Chemical analysis of the fruit extract demonstrated the presence of various bioactive molecules such as amino acids (l-alanine and aspartic acids), organic acids (benzoic acid and citric acid), sugars (arabinose and glucose), phenolic acid, and bioflavonoids (niacin and myo-inositol), which likely attributed to the formation of stable biogenic Au NPs with excellent peroxidase-mimicking activity. In comparison with the natural horseradish peroxidase (HRP) enzyme, the biogenic Au NPs displayed a 9.64 times higher activity with regard to the reaction velocity at 6% (v/v) H2O2, presenting a higher affinity toward the TMB substrate. The Michaelis–Menten constant (KM) values for the biogenic Au NPs and HRP were found to be 6.9 × 10–2 and 7.9 × 10–2 mM, respectively, at the same concentration of 100 pM. To investigate its applicability for biosensing, a monoclonal antibody specific for Mycobacterium bovis (QUBMA-Bov) was directly conjugated to the surface of the biogenic Au NPs. The obtained results indicate that the biogenic Au NPs-QUBMA-Bov conjugates are capable of detecting M. bovis based on a colorimetric immunosensing method within a lower range of 100 to 102 cfu mL–1 with limits of detection of ∼53 and ∼71 cfu mL–1 in an artificial buffer solution and in a soft cheese spiked sample, respectively. This strategy demonstrates decent specificity in comparison with those of other bacterial and mycobacterial species. Considering these findings together, this study indicates the potential for the development of a cost-effective biosensing platform with high sensitivity and specificity for the detection of M. bovis using antibody-conjugated Au nanozymes.

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1. Introduction

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In recent years, green synthesis of nanomaterials exploiting natural resources is one of the most promising alternatives to the existing physical and chemical methods as this approach does not require high energy consumption, sophisticated instrumentation, and chemical reagents. For example, gold nanoparticles (Au NPs) have been biogenically produced using microorganisms, (1−3) agricultural waste (e.g., mango peels), (4) or plant and fruit extracts, (5−10) demonstrating that the synthesis process is not only rapid, readily scalable, and cost-effective but also eco-friendly and not harmful to biological systems. In addition, the biosynthesized Au NPs have also been reported to possess excellent catalytic activities with improved stability and biocompatibility. (11) The synthesis mechanism using plant extracts involves the reduction of metal ions due to the presence of natural products comprising functional groups such as phenolic acids, proteins, polyphenol, bioactive alkaloids, terpenoids, and sugars, which play a further role in stabilizing the nanoparticles with improved catalytic efficiency. (12) Dauthal and Mukhopadhyay demonstrated the biogenic synthesis of stable Au NPs using Prunus domestica extracts with excellent catalytic activity for the reduction of 4-nitrophenol. (13) Furthermore, biogenic Au NPs with excellent peroxidase-mimicking activity have also been reported for various biosensing applications. Kumar et al. demonstrated the size-dependent green synthesis of Au NPs with peroxidase-like activity and subsequent utilization in the development of a colorimetric biosensor for the detection of glutathione from human blood serum. (14) Similarly, Li and co-workers synthesized stable Au NPs using kiwi extract with higher stability and intrinsic peroxidase-mimicking activity for the colorimetric detection of cysteine. (15) Additionally, new breakthroughs in biogenic synthesis of nanomaterials have allowed for applications beyond catalysis and biosensing, for example, applications in drug delivery and in bioimaging have been reported. (16)
In this present study, a single-step, facile, and green synthesis of Au NPs at room temperature (RT) using fruit extract of Prunus nepalensis (or locally known as “Sohiong”) is presented. P. nepalensis has barely been explored among the species within the Rosaceae family, native to different parts of Northeast India. P. nepalensis fruit extract possesses a wide range of pharmacological properties as it contains a high concentration of phytochemicals and bioactive compounds and has been used as an astringent, a hepatoprotective agent, or an antioxidant, while the leaves of P. nepalensis are also used as a diuretic agent for edema. (17,18) In this study, we describe the first report of the reducing ability of P. nepalensis fruit extract for a cost-effective and eco-friendly one-step biogenic synthesis of Au NPs. The chemical analysis revealed the excellent antioxidant potential of the fruit extract. Further spectroscopic analysis demonstrated the presence of various bioactive molecules such as amino acids (l-alanine, aspartic acids, oxoproline, aminobutanoic acid, and asparagine), organic acids (benzoic acid, malic acid, and citric acid), sugars (arabinose, glucose, and fructose), phenolic acid (protocatechuic acid), saturated fatty acid (palmitic acid), and bioflavonoids (niacin and myo-inositol) in the fruit extract, which potentially acted to enhance the stability and nanozyme activity of the Au NPs. Moreover, the biogenic Au NPs exhibit excellent catalytic efficiency, which is comparable to that of the horseradish peroxidase (HRP) enzyme, a natural enzyme, but with improved environmental stability. (19) Application of enzyme-mimicking nanoparticles for colorimetric biosensor development has gained popularity as it overcomes the limitations of natural enzymes (i.e., less stability in harsh environmental conditions and cost effectiveness) in the case of biosensor development for point-of-care (POC) applications. (20,21) However, enhanced catalytic efficiency, cost-effective fabrication, biocompatibility, and high stability remain of paramount concern for effective biosensor development. Over the last few years, Mycobacterium bovis is the main causative organism responsible for bovine tuberculosis (bTB) outbreaks, a chronic granulomatous, respiratory disease that affects a cow’s lung tissues and lymph nodes. (22) The route of transmission to humans occurs predominantly through close contact with the infected animals or consumption of M. bovis-contaminated animal products. (23) To reduce the economic costs of bTB and eliminate the public health risks associated with the consumption of contaminated dairy products, rapid identification of pathogenic mycobacterial species including M. bovis is essential for unambiguous diagnosis and an effective disease control strategy. (24) Thus, there is a need for rapid, cost-effective, user-friendly, and sensitive biosensors for M. bovis. Furthermore, in this study, based on the excellent intrinsic peroxidase-mimicking phenomenon, as a proof of concept, the biogenic Au NPs were utilized for the development of a colorimetric approach for the qualitative detection of M. bovis. This biosensing strategy could be broadly applied for the development of rapid, cost-effective, and on-site detection of pathogens, biomarkers, or toxins indicating human or animal disease or contamination in food and water.

2. Experimental Section

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2.1. Materials and Reagents

2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH), the Tris base (C4H11NO3), hydrochloric acid (HCl), l-ascorbic acid (C6H8O6), phosphate-buffered saline (PBS), trichloroacetic acid (C2HCl3O2), ferric chloride (FeCl3), Folin and Ciocalteu’s phenol reagent, sodium carbonate (Na2CO3), gallic acid (C7H6O5), sodium nitrite (NaNO2), aluminum chloride (AlCl3), sodium hydroxide (NaOH), catechin (C15H14O6), hydrogen tetrachloroaurate (III) (HAuCl4·3H2O), hydrogen peroxide (H2O2, 30%), the HRP enzyme, 1,4-dithiothreitol (DTT), sodium acetate (NaOAc), and ethylenediaminetetraacetic acid (EDTA) were all purchased from Sigma-Aldrich (UK). 3,3′,5,5′-Tetramethylbenzidine (TMB) was purchased from Thermo Fisher Scientific (UK). PD-10 desalting columns were purchased from GE Healthcare Life Sciences. Solid cheese (cheddar) was purchased from a local supermarket (Tesco, Belfast, UK).

2.2. Synthesis of Biogenic Au NPs Using P. nepalensis Fruit Extract

The P. nepalensis fruit was collected from Meghalaya (25.4670° N, 91.3662° E) in the Northeast region of India. After thoroughly washing and sun-drying, 5 g of dried fruit pulp was cut into small pieces and mixed with 100 mL of distilled water at 60 °C for 10 min under stirring conditions. A solution of P. nepalensis fruit extract was prepared by centrifuging at an 8505g force for 15 min (Sorvall Legend RT Refrigerated Benchtop Centrifuge, Germany), the resultant solution was filtered using Whatman Grade No. 40 filter paper, and the filtered fruit extract was stored at 4 °C until required (the fruit extract was processed and prepared fresh for every experiment performed during the study). For green synthesis of Au NPs, 80 mL of 1 mM aqueous solution of HAuCl4·3H2O was added to 20 mL of a 10-fold dilution (in aqueous) of the primary crude extract of P. nepalensis. The reaction mixture of the Au salt solution with fruit extract (natural pH of 4.3) was kept at RT overnight under shaking conditions. Optimization of the synthesis process is presented in the Supporting Information.

2.3. Optical and Morphological Characterizations of Biogenic Au NPs

Optical analysis of the synthesized Au NPs was performed using a Cary 60 ultraviolet–visible (UV–vis) spectrophotometer (Agilent Technologies, USA). The particle morphology was determined using transmission electron microscopy (TEM-TECNAI TF 30 G2 Super-Twin by FEI, USA) and field emission scanning electron microscopy (FESEM, Nova nanoSEM 450, Czech Republic). The crystal structure of the materials was investigated using the X-ray diffraction (XRD) technique (D/Max 2005, Rigaku, Japan). The surface charge or zeta potential (Zp) of biogenic Au NPs was measured using dynamic light scattering (DLS) techniques (Zetasizer Nano ZS90, Malvern, UK).

2.4. Chemical Analysis of P. nepalensis Extract

To allow us to postulate the mechanism behind the biogenic synthesis of Au NPs using P. nepalensis fruit extract with excellent catalytic efficiency, detailed chemical analysis of the fruit extract was performed. For determining the free radical scavenging activity and reducing ability of P. nepalensis extract, several characterization assays were carried out, including (i) a DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) assay, (ii) a reducing power assay, and (iii) total phenolic and (iv) flavonoid content estimation. Detailed description of the free radical scavenging assays is presented in the Supporting Information. To determine the presence of organic bioactive compounds and their different functional groups, P. nepalensis fruit extract was characterized using two different analytical spectroscopic techniques. A Fourier transform infrared (FTIR) spectrophotometer (Shimadzu 8201PC, Japan) was used to investigate the presence of the different types of functional groups responsible for the biosynthesis of Au NPs based on the different mode of vibrations. Gas chromatography–mass spectrometry (GC–MS) analysis of the fruit extract was carried out using a GC–MS system (GC: Agilent Technologies 7890N, Palo Alto, California, USA, and MS: quadrupole analyzer, Agilent Technologies 5975C MSD, Palo Alto, California, USA), consisting of a Zebron 7HG-G030-11, ZB-5MSplus capillary column (30 m length, 0.25 mm internal diameter, and 0.25 μm film thickness), with a column temperature range of 0–325 °C (20 °C min–1) and an injector temperature of 300 °C.

2.5. Characterization of Peroxidase-Mimicking Activity of Biogenic Au NPs

Peroxidase-like activity of biogenic Au NPs was determined using TMB as a chromogenic substrate. Experiments were carried out using a 100 pM concentration of synthesized biogenic Au NPs in a final reaction volume of 1 mL, with 1 mM TMB and 6% (v/v) H2O2. The reaction mixture was incubated at RT for 10 min, and full-spectrum analysis was carried out using a UV–vis spectrophotometer. Time-dependent UV–vis full spectral analysis of the catalyzed reaction using the same conditions was assessed in a 1 mL final reaction volume for 90 min. To understand the reaction mechanism of TMB oxidation using biogenic Au NPs, 1 mM TMB was catalyzed using the same conditions as those mentioned earlier. The reaction kinetics were monitored at three different wavelengths of 370, 450, and 650 nm for a 20 min time course. Furthermore, to understand the role of P. nepalensis fruit extract in the oxidation of chromogenic substrate TMB in the presence of H2O2, a simple experiment was carried out (see the Supporting Information for more details).

2.5.1. Reaction Kinetics of Peroxidase-Mimicking Activity of Biogenic Au NPs

The reaction kinetics study for the catalytic oxidation of TMB, demonstrating the peroxidase-like activity of biogenic Au NPs, was carried out by recording the absorption values at 370 nm using a microplate reader (Tecan Safire 2, Switzerland). Biogenic Au NPs and HRP were incubated with TMB (0.1–1.0 mM) at various H2O2 concentrations [0.0075–10% (v/v)] at RT for 10 min. The kinetic parameters were calculated based on the Michaelis–Menten equation, eq 1
(1)
where V is the initial velocity, Vmax is the maximal reaction velocity, [S] is the substrate concentration, and KM is the Michaelis constant. For further information on the calculation of parameters, see the Supporting Information. To compare the effect of reaction buffer pH on the catalytic oxidation of TMB, 0.2 M NaOAc buffer solutions with the pH ranging from 2 to 12 were used in the reaction mixtures containing biogenic Au NPs and HRP, respectively, at RT. Similarly, to determine the influence of incubation temperature on catalytic activity, reaction solutions were incubated in a water bath from 20 to 60 °C, while the pH of the reaction mixture was kept constant at 4. The absorption values were measured at 370 nm after 10 min incubation.

2.6. Preparation of the Indirect Enzyme-Linked Immunosorbent Assay for the Detection of M. bovis

2.6.1. Preparation of Au NPs-QUBMA-Bov Conjugates

For the preparation of biogenic Au NPs-QUBMA-Bov conjugates, thiolation of the M. bovis-specific monoclonal IgG antibody (QUBMA-Bov) was performed as previously reported using the DTT reduction method. (25) The M. bovis-specific monoclonal antibody (mAb), QUBMA-Bov, was sourced from a previous study. (26) In a typical experiment, QUBMA-Bov (0.5 mg) was dissolved in PBS (10 mM, pH 7)/2.5 mM EDTA (500 μL) with a concentration of 1 mg mL–1. Ten microliters of 0.5 M DTT was added to PBS (10 mM, pH 7)/2.5 mM EDTA, and the solution was mixed with 500 μL of QUBMA-Bov solution. The reaction mixture was then incubated at RT for 30 min on a rotator. (25) After the incubation, the mixture was purified through the PD-10 desalting column to remove the excess unutilized DTT molecules. One milliliter of freshly synthesized biogenic Au NPs (4 nM) was centrifuged at 11,200g for 30 min, and the pellet was resuspended in PBS (10 mM, pH 7)/2.5 mM EDTA buffer solution. Thiol (SH) group-containing reduced QUBMA-Bov (1 mL) obtained from the DTT reduction method was added to the biogenic Au NP solution (1 mL, 4 nM), and the mixture was incubated on a rotator (RT, 16 h). After the incubation, the mixture was centrifuged at a 7168g force for 30 min and resuspended again in 1 mL of distilled water, and this process was repeated twice to remove all the unbound thiolated antibodies. The purified biogenic Au NPs-QUBMA-Bov conjugates were further characterized using a UV–vis spectrophotometer.

2.6.2. Catalytic Efficiency of Au NPs-QUBMA-Bov Conjugates

To determine the peroxidase-mimicking activity of Au NPs-QUBMA-Bov conjugates, the catalytic experiments were carried out using the same reaction conditions as those mentioned in the previous experiments (Section 2.5), and the absorbance of the reaction solution was measured at 370 nm at 1 min intervals for 20 min using a microplate reader (Tecan Safire 2, Switzerland).

2.6.3. Preparation of M. bovis Cell Solution

M. bovis AF2122/97 was cultured in Middlebrook 7H9 broth consisting of 10% (v/v) oleic acid-albumin dextrose-catalase (both from Difco) (Middlebrook 7H9/OADC broth) until the stationary phase was achieved and then harvested by centrifugation followed by washing in PBS (pH 7.4). To inactivate the cultures, mycobacterial cell suspensions were subjected to a 10 kGy dose of gamma radiation (Gammabeam 650 cobalt irradiator, AFBI, Belfast). After the radiation treatment, samples of each suspension were cultured on Middlebrook 7H10 agar plates and read after 56 days to ensure complete inactivation of all pathogenic species. Irradiated cells were diluted in PBS (pH 7.4) to 106 cfu/mL suspensions and stored at −80 °C. (27)

2.6.4. Indirect Enzyme-Linked Immunosorbent Assay

For the indirect enzyme-linked immunosorbent assay (iELISA), the general procedure is summarized as follows: 96-well microtiter plates (NUNC Maxisorp) were coated with M. bovis cells at the final concentration of 104 cfu mL–1 in 100 μL of sodium carbonate/bicarbonate buffer (pH 9.6) for 16 h at 4 °C. After the incubation, M. bovis-coated wells were washed three times with washing buffer [PBS, pH 7.4, and Tween-20 0.05% (v/v) (PBST)] to remove unbound cells, and the 96-well plate wells were blocked by adding 300 μL/well of blocking buffer [1% (w/v) BSA/PBST] and incubating for 1 h at RT with gentle shaking. After the incubation, wells were washed three times with PBST and kept ready for the assay. For performing the iELISA experiment, M. bovis and Au NPs-QUBMA-Bov conjugates were preincubated, as detailed briefly, and standard suspensions containing M. bovis cells at concentrations 0, 10, 102, 103, 104, and 105 cfu mL–1 were prepared in washing buffer (PBST), and 100 μL of Au NPs-QUBMA-Bov conjugates (diluted 1:5 in PBST) was added to each M. bovis cell suspension and incubated for 30 min at RT on a rotator mixer. After 30 min of incubation, 100 μL of a solution containing different concentrations of M. bovis and Au NPs-QUBMA-Bov conjugates was added to the respective wells of the microtiter plate and incubated for 30 min at 37 °C at shaking conditions. After the incubation, the wells were washed three times with washing buffer (PBST). Then, 100 μL of the TMB (1 mM) enzyme substrate along with 60 μL of H2O2 [30% (v/v)] was added to each well and incubated for 15 min at RT on a plate shaker. Color development was stopped by adding 20 μL of the stop solution (2.5 M sulfuric acid). The optical density (OD) was measured at 450 nm using a microplate reader (Tecan Safire 2, Switzerland) within 5 min after stopping the reaction. The absorbance spectra corresponding to each concentration of M. bovis were recorded. The OD versus concentration of M. bovis calibration curve was obtained by calculating the average reading of three experiments performed under the same conditions. The limit of detection (LOD) of the biosensing strategy was calculated based on the method mentioned by Long and Winefordner, 1983, using the calibration curve data. (28) The standard deviation value (STDV) for the negative control (NC) was multiplied by 3 (three replicates), and then, it was subtracted from the absorbance value for the NC sample to calculate the Y for the lowest detectable concentration. Finally, the X (bacteria concentration) in correspondence with the Y value was calculated to identify the final LOD concentration. To determine the specificity of the Au NPs-QUBMA-Bov conjugates, the iELISA was performed using four other different bacterial cultures (105 cfu/mL) including irradiated Mycobacterium smegmatis mc2155 and Mycobacterium tuberculosis H37Rv cultures, as well as Escherichia colik12 ER2738 and E. coli BL21 (DE3). Two hundred microliters of washing buffer (PBST) without M. bovis was incubated with 100 μL of Au NPs-QUBMA-Bov and used as the NC (blank).

2.6.5. Real Sample Analysis

A homogenized solution of solid cheese (cheddar) was spiked with different concentrations of M. bovis (100 to 105 cfu mL–1). To prepare the solid cheese matrix, 1g of solid cheese was homogenized by using a mortar and pestle in 10 mL of PBST. The cheese solution was then centrifuged at a 664g force for 5 min to exclude bigger food particles. (29) The supernatant was spiked with different concentrations of M. bovis cells, and the iELISA was performed as described above.

3. Results and Discussion

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3.1. Synthesis and Characterization of Biogenic Au NPs

3.1.1. UV–Vis Spectrophotometric Analysis

Biosynthesis of Au NPs was followed by the color change in the reaction solution with the appearance of a ruby red color and a change in the surface plasmon resonance (SPR) UV–vis spectrophotometer analysis. The color change occurred after overnight incubation of the Au salt precursor assisted by P. nepalensis fruit extract, indicating the formation of Au NPs (Figure 1A inset), attributed to the cumulative oscillation of free surface electrons of metal nanoparticles induced by an interacting electromagnetic field. The absorption spectrum of Au NPs was recorded within the range of 350–700 nm. Figure 1A shows the UV–vis spectrum of unwashed Au NPs recorded with an SPR value at 520 nm, indicating the formation of Au NPs. Further, there was no change in the SPR value after washing steps were carried out, which indicates the stability of the synthesized biogenic Au NPs. The optimized conditions for the Au NP synthesis were found to be the natural pH of the fruit extract (pH 4.3), RT (25–28 °C), a 1 mM precursor salt concentration, and a 10 times (1:10) diluted aqueous fruit extract solution (Figure S1). To investigate the probable synthesis mechanism of Au NPs using P. nepalensis fruit extract, chemical characterization of the fruit extract was performed using the total phenolic content measurement, DPPH scavenging activity assay, and reducing power assay, and spectroscopic analysis of the fruit extract was performed using FTIR spectroscopy and GC–MS analysis.

Figure 1

Figure 1. Characterization of biogenic Au NPs. (A) UV–vis spectrum of biologically synthesized Au NPs using P. nepalensis fruit extract. The inset shows the color formation of the reaction mixture due to the reduction of the gold salt to form Au NPs: (i) gold salt solution, (ii) P. nepalensis fruit extract, (iii) crude Au NPs, and (iv) washed Au NPs. (B) XRD analysis of Au NPs synthesized by P. nepalensis fruit extract.

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3.1.2. XRD Analysis

The phase and crystal structure of the biologically synthesized Au NPs obtained from P. nepalensis extract were investigated using the XRD technique, leading to a better understanding of the atomic positions in its atomic structure. Figure 1B shows the corresponding XRD pattern of the Au NPs in the 2θ range of 20–80°. The XRD spectrum resulted in three intense peaks at 2θ = 38.1, 44.4, and 64.7, which can be indexed as (111), (200), and (220) reflections, respectively, of the fcc (face-centered cubic) structure of metallic gold (JCPDS no. 04-0784). (30) The absence of any other peak position suggested the formation of monophasic gold nanostructures with pure crystallinity.

3.1.3. TEM Analysis

Enzyme-mimicking properties of metal nanoparticles strongly depend on their structure and morphology. Figure 2 shows the TEM images of biogenic Au NPs obtained via synthesis using P. nepalensis fruit extract, which further confirms the spherical structure of the Au NPs (Figure 2A–C). The size distribution of biogenic Au NPs varies from 2 to 15 nm, with an average particle size of 6 ± 3 nm (Figure 2D). The selected area electron diffraction (SAED) pattern of individual Au NPs shows the lattice pattern structure for the Au NP sample. Most of the plates were truncated and close to the single-crystalline structure corresponding to the fcc structure (Figure 2A inset), which corresponds to the XRD results. TEM energy-dispersive X-ray spectrometry (EDX) was used to understand the chemical composition of the nanoparticles. As shown in Figure 2E, Au has demonstrated a clear peak, clear evidence of the presence of gold (Au) traces in the NP sample with a specific concentration. Furthermore, the FESEM result (Supporting Information, Figure S2) suggested the formation of uniform spherical Au NPs. This observation was in accordance with the data obtained from UV–vis spectrum of Au NPs. The surface charge of the synthesized Au NPs was investigated using zeta potential analysis (Supporting Information, Figure S3), and the surface charge was found to be −20.6 mV, which confirms the presence of a negative charge on the surface of the Au NPs. The negatively charged Au NPs displayed more affinity toward the positively charged TMB substrate for peroxidase-mimicking activity, while also providing the repulsive forces that help prevent Au NPs from aggregating.

Figure 2

Figure 2. TEM images. (A) Bright field images (inset: SAED pattern). (B) Dark field image. (C) High-angle annular dark field image. (D) Particle size analysis performed on approximately 100 particles using ImageJ software and (E) EDX analysis of biogenic Au NPs synthesized from P. nepalensis fruit extract.

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3.2. Characterization of Peroxidase-Mimicking Activity of Biogenic Au NPs

Figure 3 illustrates the evidence of the catalytic activity of biogenic Au NPs, which catalyze the oxidation of TMB in the presence of H2O2. The Figure 3A inset demonstrates the formation of a blue color in the presence of 1 mM TMB and 6% H2O2 due to the peroxidase-mimicking activity of biogenic Au NPs. Moreover, the images clearly show that the formation of blue color due to the TMB oxidation requires Au NPs to catalyze the reaction as for the NC samples without Au NPs, (i) only TMB or (ii) TMB with 6% H2O2, the solution cannot be converted into the blue-colored oxidized TMB form [Figure 3A, insets (i) and (ii)]. Figure 3A showing the full UV–vis spectrum is clear evidence of the TMB oxidation reaction catalyzed by biogenic Au NPs, demonstrated by the formation of charge transfer complexes at different absorption peaks: 370, 450, and 650 nm for biogenic Au NPs in the presence of TMB and H2O2 (green line). (31,32) Furthermore, biogenic Au NPs showed their SPR peak at 520 nm (Figure 3A, blue line). However, there is no peak formation found for TMB or TMB with 6% H2O2, which indicates that no color formation occurred due to the absence of the TMB oxidation reaction without biogenic Au NPs (Figure 3A black and red line). Figure 3B demonstrates the time-dependent UV–vis full spectral scanning kinetic analysis of the TMB oxidation reaction in the presence of biogenic Au NPs along with 1 mM TMB and 6% H2O2. Within 10 min of the reaction time, the formation of the charge transfer complex shows its highest intensity for the absorption peaks at 370 and 650 nm (Figure 3B). To understand the full reaction mechanism of the TMB oxidation process catalyzed by biogenic Au NPs, a kinetic analysis of the TMB oxidation was performed with 1 mM TMB, 6% (v/v) H2O2, and 100 pM biogenic Au NPs at set conditions. The reaction kinetics were monitored at three different wavelengths of 370, 450, and 650 nm using a UV–vis spectrophotometer until 20 min (detailed results and discussion in the Supporting Information, Figure S4). The results show an increase in absorbance at 370 nm (Figure S4B) in a time-dependent manner in comparison with that at 450 and 650 nm (Figure S4C,D), while keeping all the reaction parameters similar for every three reactions. Based on this, we proposed to utilize the peak at 370 nm for characterizing the peroxidase-mimicking activity of biogenic Au NPs in kinetic analysis. This result provides significant insights into the peroxidase-mimicking activity of the biogenic Au NPs obtained from P. nepalensis extract.

Figure 3

Figure 3. (A) UV–vis spectroscopic analysis of the catalyzed reaction of TMB/H2O2 in the absence and presence of Au NPs. Figure 3A inset: (i) TMB, (ii) TMB + H2O2, (iii) Au NPs, and (iv) TMB + H2O2 +Au NPs. (B) UV–vis full spectral analysis of the catalyzed reaction of 1 mM TMB/6% H2O2 in the presence of 100 pM Au NPs.

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3.2.1. Kinetic Analysis of Peroxidase-Mimicking Activity of Biogenic Au NPs in Comparison with that of HRP

Kinetic analysis of the TMB oxidation using biogenic Au NPs was performed by mixing various concentrations of TMB/H2O2 with a fixed concentration of Au NPs and monitoring the oxidation process using absorption analysis at 370 nm. Figure 4A clearly shows the increment in the reaction velocity in accordance with the increased H2O2 concentration with respect to the TMB concentration in the presence of biogenic Au NPs, which confirms that peroxidase-mimicking activity strongly depends on the H2O2 concentration (detailed study in Supporting Information, Figure S5). However, in the case of a natural enzyme (i.e., HRP), the reaction velocity decreased significantly with the increasing concentration of H2O2 from 0.0075 to 10% (Figure 4B). To determine the maximum conversion rate of the substrate to its product by HRP and biogenic Au NPs at the point of the reaction where all the active sites are fully saturated, the maximal velocity (Vmax) was calculated. At lower concentrations of H2O2, such as 0.0075%, the maximum rate of the reaction is found to be 1.68 × 10–7 M·min–1 for biogenic Au NPs, which significantly increased with the higher concentrations of H2O2 (10%) up to 5.34 × 10–6 M·min–1. Further details of the kinetic parameters are summarized in the Supporting Information. From a practical perspective, clinical samples may contain a certain amount of natural peroxidase enzymes, which could potentially interfere with the enzyme-mimicking activity of the Au NPs. Thus, for a better understanding of the kinetic interferences, kinetic analysis of HRP was also performed. Figure 4B clearly shows the catalytic oxidation of TMB using HRP with a maximum reaction velocity of 5.57 × 10–6 M·min–1 at 0.0075% H2O2. However, the activity of HRP decreases significantly (5.83 times) when the H2O2 concentration exceeds 0.0075% (i.e., Vmax = 9.86 × 10–7 M·min–1 at 1% H2O2, which decreases accordingly with the increased concentration of H2O2) (details of the kinetic parameters are summarized in Supporting Information, Table S1). The maximum reaction velocity of TMB oxidation using HRP is much smaller with the increasing concentration of H2O2 in comparison with that of the biogenic Au NPs. For example, at a moderate level of H2O2 concentration (i.e., 6% H2O2), the maximum reaction velocity of biogenic Au NPs is 9.64 times higher than that of HRP (Table S1). The reduced reaction rate is most likely due to the inactivation of HRP at a higher concentration of H2O2. (33) Furthermore, this finding can be used to adjust the catalytic response. Therefore, to avoid potential interference from contaminating peroxidase enzymes in clinical samples and to enhance the nanozyme activity of biogenic Au NPs, 6% H2O2 was chosen for all experiments henceforth. The effect of pH and temperature for the intrinsic peroxidase-like activity of biogenic Au NPs is shown in Figure 4C,D. The results demonstrate that the optimal pH of the reaction solution for biogenic Au NPs is in the range of 3.5–4, which is in agreement with previous reports indicating that in acidic conditions, the intrinsic peroxidase-like activity of biogenic Au NPs increased significantly to catalyze the TMB oxidation (Figure 4C). (34,35) Moreover, at pH values above the acidic condition (above a pH of 4.0), the catalytic activity decreased, possibly due to the increased redox potential of the substrate, consequently making them less susceptible for oxidation. (36) Similarly, the effect of temperature on peroxidase-mimicking activity of biogenic Au NPs is shown in Figure 4D. The peroxidase-mimicking activity reaches the maximum at a range of 35–40 °C, which is similar to that of the natural enzyme HRP. (37)

Figure 4

Figure 4. Typical kinetic analysis of TMB oxidation. (A) Kinetic analysis of TMB oxidation by Au NPs in the presence of varying TMB (0–1 mM) and H2O2 concentrations (1–10%). The concentration of the Au nanozyme was fixed at 100 pM. (B) Kinetic analysis of TMB oxidation by the HRP enzyme in the presence of varying TMB (0–1 mM) and H2O2 concentrations (1–10%). Samples were analyzed in triplicate (n = 3), and the standard deviation was deduced from these data. (C) Kinetic analysis of the HRP enzyme along with Au NPs at different pH values (2–12). (D) Kinetic analysis of the HRP enzyme along with Au NPs at different temperatures (20–60 °C). All the absorbance values were measured at 370 nm.

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To obtain more information on the required substrate concentration for the catalytic reaction, the Michaelis–Menten constant (KM) was calculated. The results shown in the Supporting Information indicates a lower KM value for the biogenic Au NPs in comparison with that for HRP with the increase in the concentration of H2O2 from 0.0075 to 6%, indicating that less substrate is required to initiate the catalysis reaction at a significant rate with a higher affinity (details of the kinetic parameters are summarized in Supporting Information, Table S1). The KM values for both biogenic Au NPs and HRP have changed significantly according to the changes in the H2O2 concentrations. For instance, from 0.0075 to 6% H2O2 concentrations, the changes in the KM values for Au NPs and HRP are 1.17 × 10–2 to 6.9 × 10–2 and 1.1 × 10–1 to 7.9 × 10–2 mM, respectively. The results indicate the higher requirement of the substrate to reach the maximum velocity for HRP-assisted TMB oxidation. For a better understanding of the differences in the reaction rate, the catalytic rate (kcat) was calculated for both Au NPs and HRP. The results reveal the reduced catalytic rate (kcat) of HRP with the increasing concentration of H2O2, that is, the catalytic rate beyond 0.0075% decreased by 5.83 times, whereas in the case of biogenic Au NPs, it increased up to 6.75 times (details of the kinetic parameters are summarized in Supporting Information, Table S1). Figure 5 shows the reduction of the catalytic efficiency (kcat/KM) of the HRP enzyme with the increasing concentration of H2O2, demonstrating that at 0.0075% H2O2 (the lowest concentration), the HRP enzyme can convert a higher concentration of the substrate TMB into the product, which further reduced at 6% (v/v) H2O2. However, for biogenic Au NPs, the catalytic efficiency (kcat/KM) was improved up to 6 times at 6% H2O2, demonstrating the highest conversion rate of the substrate into its product.

Figure 5

Figure 5. Comparison of the catalytic efficiency of HRP and Au NPs. The reaction was carried out in the presence of 0.1 mM TMB and various H2O2 concentrations for 10 min at RT. Concentrations of the natural enzyme and nanozyme were fixed at 100 pM, and the absorbance was measured at 370 nm.

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3.3. Chemical Analysis of P. nepalensis Fruit Extract

3.3.1. Free Radical Scavenging Activity and Antioxidant Potential of P. nepalensis Fruit Extract

A vast repertoire of secondary metabolites with antioxidant properties found in plant products (e.g., fruit) exhibiting redox capacity have been exploited for biogenic synthesis of nanoparticles. (38) Estimation of the reducing capacity, antioxidant potential, and radical scavenging ability of plant extracts can be achieved by different electron and hydrogen atom transfer-based assays such as DPPH, Folin–Ciocalteu, and ferric reducing antioxidant power assays. (39,40) The working principle of these redox reactions is based on the transfer of electrons from the antioxidants present in the plant extract to oxidants such as DPPH or to the metal ions present in the Folin–Ciocalteu reagent. Additionally, this free radical scavenging activity or reducing capacity can be measured by recording the alteration in the absorbance values at specific wavelengths. (41) Chemical analysis of the P. nepalensis fruit extract was performed for a better understanding of the probable role of the bioactive compounds in the biogenic synthesis of Au NPs with higher stability and excellent catalytic efficiency. The basic mechanism behind the green synthesis of metal nanoparticles also relies on the redox reaction where plant or fruit extract with higher total reducing substances are able to produce higher concentration of metal nanoparticles. (41) Therefore, for P. nepalensis fruit extract, the total reducing power assay was performed to determine the electron-donating capability of the biological complex compounds present in the extract, which would act as reducing agents, leading to the formation of Au NPs from its ionic state (i.e., Au3+). Table 1 represents the reducing power ability of P. nepalensis fruit extract in comparison with that of ascorbic acid as standard. The absorbance value for P. nepalensis fruit extract is around 0.447 ± 0.05 (a.u), which is higher than that of ascorbic acid [i.e., 0.300 ± 0.03 (a.u)]. The result indicates the high reduction potential of the fruit extract, which might have resulted in the reduction of Au metal ions to form Au NPs. Similarly, the ability to transfer electrons from antioxidant substances to the unstable free radical molecules due to the antioxidant activity of fruit extracts also plays a significant role in the reduction of metal ions to form metal nanoparticles with excellent catalytic activity. The antioxidant potential of P. nepalensis fruit extract was investigated using the DPPH method (517 nm), as shown in Table 1. DPPH acted as a solid free radical, which is reduced by accepting electron or hydrogen from the antioxidant present in the extract. Based on the color changes of the DPPH solution from purple to yellow, the total percent inhibition of the reduced DPPH radical can be measured spectrophotometrically. (42) The result (Table 1) shows that P. nepalensis fruit extract exhibited substantial inhibition of DPPH with 76.091 ± 0.25% RSA (radical scavenging activity) when compared with that of ascorbic acid as a standard (72.22 ± 0.22% inhibition). It is important to note that the antioxidant potential of fruit extract is directly proportional to the quantitative existence of phenolic compounds. Therefore, the higher antioxidant potential of P. nepalensis might be due to the presence of phenolic compounds and other secondary metabolites present in the extract, such as anthocyanin and flavonoids. (43) Total phenolic content in the fruit extract was found to be 43 ± 0.15 μg GAE mL–1 (Table 1), which was measured spectrophotometrically at 765 nm by taking gallic acid as the standard. The total flavonoid content of P. nepalensis fruit extract was found to be 208.4 ± 0.35 μg CE (catechin equivalent) per mL (Table 1). The antioxidant activity of biological extracts depends on the presence of free OH groups, especially 3-OH groups in flavonoid compounds including flavones, flavonols, and condensed tannins, which are secondary metabolites of plants. (44) Along with higher polyphenols and flavonoid, P. nepalensis is also reported to contain a high amount of anthocyanin. (45,46) Swer et al. demonstrated the presence of a high amount of anthocyanins (984.40 ± 3.84 mg C3G per 100 g DM), with cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside, and malvidin as the major anthocyanins. (47)
Table 1. Free Radical Scavenging Activity and Antioxidant Potential of P. nepalensis Fruit Extract
 free radical scavenging activity and antioxidant potential of P. nepalensis fruit extract
sampleDPPH scavenging ability (%)reducing power (absorbance)total phenolic content (μg GAE mL–1)total flavonoid content (μg CE mL–1)
P. nepalensis aqueous fruit extract76.091 ± 0.250.447 ± 0.0543 ± 0.15208.4 ± 0.35
ascorbic acid72.22 ± 0.220.300 ± 0.03  
The results of the in vitro antioxidant potential and reducing power assay of the P. nepalensis fruit extract suggested that the higher total reducing ability of the fruit extract could be attributed to the presence of phytochemicals with higher antioxidant potentials such as polyphenols and flavonoids. Phenolic compounds present in the P. nepalensis such as catechin, caffeic acid, dicaffeoylquinic acid, cinnamic acid, and neo-chlorogenic acid might have assisted the reduction of Au metal ions to form biogenic Au NPs. (46) For example, Ahmad and Sharma reported the involvement of chlorogenic acid from plant extract in the biogenic synthesis of Ag NPs. (38) On the other hand, adsorption of different flavonoids and other bioactive compounds over the nanoparticle surface might have acted as the capping and stabilizing agents, which resulted in the formation of stable biogenic Au NPs within a size range of 6 ± 3 nm. Interestingly, the interaction between different phytochemicals present in the P. nepalensis extract with the Au metal ions during the synthesis process might have improved the catalytic activity of the Au NPs due to the presence of bioactive functional moieties with high antioxidant properties over the surface of nanoparticles. (13,48) However, the role that fruit extracts may play in the intrinsic peroxidase-like activity of Au nanoparticles is still unclear. Therefore, as a preliminary attempt to ascertain whether fruit extract can oxidize TMB, a simple reaction was performed, and the results suggested the incapability of fruit extract alone to oxidize TMB (Figure S6, detailed result and discussion in the Supporting Information). Nevertheless, the electrostatic attractions between positively or neutrally charged Au NPs and negatively charged secondary metabolites present in the extract resulted in the formation of a negative surface coating on Au NPs, which further possibly improved the affinity toward positively charged TMB during enzyme-mimicking activity. (43,49) The presence of various functional groups on the surface of biogenic Au NPs responsible for the improved stability and catalytic activity was investigated by using spectroscopic analysis.

3.3.2. Spectroscopic Analysis of P. nepalensis Fruit Extract

The FTIR spectrum of Au NPs synthesized by using P. nepalensis was studied to gain a greater understanding about the possible synthesis mechanism and the presence of functional moieties over the nanoparticle surface (Figure 6). The complex nature of the peaks was expected due to the presence of many biological molecules in the system. There is a slight change in the intensity of the peaks in the two different reaction systems. The presence of a broad peak in the range of 3100–3700 cm–1 may correspond to the stretching vibration of NH and OH groups, depicting the traces of phenolic groups. (50) A strong peak at 2926.3 cm–1 in both the spectra represented the C–H stretching of the alkyl group, whereas a slightly intense peak at 2855 cm–1 in Au NP spectra indicated the C–H stretching of aldehyde groups. (49) In addition, there is a strong peak around 2365 cm–1 that corresponds to the presence of carboxylic and phenolic groups. A slightly intense peak around 1600–2000 cm–1 corresponds to the C═O stretching, corresponding to primary amides and carboxylic groups, and the presence of N–H stretching of amines. Stretched peaks at 1027 and 1029 cm–1 represent the presence of the C–N stretching vibration of aliphatic amines in both of the spectra. The peaks around 600 and 800 cm–1 are attributed to R-CH groups. (49) The peak around 668 cm–1 in both the spectra confirms the presence of N–H wags from the primary and secondary amines. FTIR analysis of P. nepalensis fruit extract and biogenic Au NPs suggested the presence of functional groups associated with different organic bioactive compounds such as polyphenols, enzymes, and proteins in the extract as well as on the surface of biologically synthesized Au NPs. Hence, a simple reaction mechanism can be inferred for synthesizing and stabilizing the Au NPs where bioactive compounds (e.g., phenolic compounds) in the extract possibly acted as a reducing agent. Furthermore, different proteins containing free amino groups, for example, cysteine residues attached on the surface of Au NPs via a thiol linkage, may act as a stabilizing agent. On the other hand, the higher antioxidant potential of phenolic compounds on the surface of Au NPs might have resulted in the enhanced catalytic efficacy of nanoparticles, leading to their excellent intrinsic peroxidase-mimicking activity. (7,9,12,41,49,50)

Figure 6

Figure 6. FTIR spectra of biogenic Au NPs synthesized by using P. nepalensis extract.

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GC–MS analysis of the P. nepalensis fruit extract was carried out to investigate the bioactive organic compounds present in it, as shown in Figure S7. In total, more than 40 compounds were detected and identified (a table containing all the detected and identified compounds is given in the Supporting Information, Table S2) by comparing the obtained mass spectra with the standard mass spectra from the NIST and Wiley electronic libraries supplied with the instrument. Table 2 shows the list of biological compounds present in the fruit extract with higher match factors from GC–MS analysis. Chemical analysis of the fruit extract suggested the presence of various bioactive compounds such as amino acids (l-alanine, aspartic acids, oxoproline, aminobutanoic acid, and asparagine), organic acids (benzoic acid, malic acid, and citric acid), sugars (arabinose, glucose, and fructose), phenolic acid (protocatechuic acid), saturated fatty acid (palmitic acid), and bioflavonoids (niacin and myo-inositol). From these results, it can be postulated that after the reduction of Au metal ions assisted by the polyphenols and other bioactive compounds present in the fruit extract, biogenic Au NPs were further stabilized by these amino acids, sugars, and organic acids due to the capping effect. (50,51)
Table 2. List of Biological Compounds Present in the P. nepalensis Extract from GC–MS Results
retention time (min)compound nameformula
5.4558l-alanine, 2TMS derivativeC9H23NO2Si2
6.6430benzoic Acid, TMS derivativeC10H14O2Si
6.8742silanol, trimethyl-, phosphate (3:1)C9H27O4PSi3
6.9946niacin, TBDMS derivativeC12H19NO2Si
8.3407malic acid, 3TMS derivativeC13H30O5Si3
8.5486l-aspartic acid, 3TMS derivativeC13H31NO4Si3
8.5777l-5-oxoproline, 2TMS derivativeC11H23NO3Si2
8.62384-aminobutanoic acid, 3TMS derivativeC13H33NO2Si3
9.4246d-arabinose, tetrakis(trimethylsilyl) ether, ethyloxime (isomer 2)C19H47NO5Si4
9.4560asparagine, 3TMS derivativeC13H32N2O3Si3
10.2792protocatechuic acid, 3TMS derivativeC16H30O4Si3
10.2950citric acid, 4TMS derivativeC18H40O7Si4
10.3842d-fructose, 5TMS derivativeC21H52O6Si5
11.3433palmitic acid, TMS derivativeC19H40O2Si
11.7465myo-inositol, 6TMS derivativeC24H60O6Si6

3.4. iELISA for the Detection of M. bovis Using Biogenic Au NPs

3.4.1. Preparation of Au NPs-QUBMA-Bov Conjugates and Analysis of Peroxidase-Mimicking Activity

Au NPs-QUBMA-Bov conjugates were prepared by attaching the mAb reduced by DTT onto the surface of biogenic Au NPs. DTT reduces the disulfide bonds, leading to the formation of different fragments containing free cystine residues with thiol (−SH) groups, which readily form covalent bonding with biogenic Au NPs. (52,53)Figure 7A shows the UV–vis spectrum of biogenic Au NPs and Au NPs-QUBMA-Bov conjugates. The result demonstrates a red shift (25 nm) in the localized SPR of Au NPs-QUBMA-Bov at 545 nm with a broadening of the peak, which indicates the formation of Au NPs-QUBMA-Bov conjugates. Additionally, the absorption spectra of Au NPs-QUBMA-Bov conjugates have a sharp characteristic absorption peak near the region of 280 nm, which is indicative of the presence of amino acid residues in the mAb (immuno globular proteins). (54) Thus, we confirmed the successful conjugation of the antibody on the Au NP surface. To investigate the efficiency of the enzyme-mimicking activity of synthesized Au NPs-QUBMA-Bov conjugates, the catalytic experiment with 0.1 mM TMB in the presence of 6% (v/v) H2O2 was performed. Figure 7B shows the catalytic efficiency of Au NPs-QUBMA-Bov conjugates in comparison with that of the biogenic Au NPs alone. The result indicates the reduction of the enzyme-mimicking activity of Au NPs-QUBMA-Bov (100 pM) by 32% in comparison with that of biogenic Au NPs after a 20 min reaction time. The reduction in the catalytic efficiency may have occurred due to the interference caused by the antibody coating over the surface of Au NPs during its interaction with TMB. However, the peroxidase-mimicking activity of Au NPs-QUBMA-Bov conjugates can be enhanced by increasing the concentration of Au NPs-QUBMA-Bov for the same substrate concentration (TMB).

Figure 7

Figure 7. (A) UV–vis spectrum of biogenic Au NPs-QUBMA-Bov conjugates in comparison with that of Au NPs only (B) kinetic analysis of the catalyzed reaction of 1 mM TMB/6% (v/v) H2O2 in the presence of 100 pM Au NPs and Au NPs-QUBMA-Bov conjugates.

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3.4.2. iELISA for M. bovis Detection

Based on the outcomes of this study, the intrinsic peroxidase-mimicking activity of biogenic Au NPs could be used to create a simple colorimetric biosensor, which can be used possibly in POC settings. Therefore, for the detection of M. bovis, an immunoassay was developed using highly peroxidase-mimicking biogenic Au NPs conjugated with a M. bovis-specific mAb instead of HRP as a signaling tag. Figure 8A demonstrates the calibration curve for the sensitive detection of M. bovis using biogenic Au NPs-QUBMA-Bov conjugates. The result shows the efficiency of biogenic Au NPs-QUBMA-Bov conjugates for a satisfactory level of detection of M. bovis within a concentration range of 100 to 105 cfu mL–1─, whereas a linear range of detection was found within the range 100 to 102 cfu mL–1. Figure 8A clearly shows the decrease in the absorbance value with the increasing concentration of M. bovis cells, indicating the presence of free Au NPs-QUBMA-Bov conjugates available after the incubation period to bind with the plate coated with M. bovis cells. The higher absorbance response at 100 cfu mL–1 indicates the presence of the maximum concentration of free Au NPs-QUBMA-Bov conjugates available to bind with the M. bovis cells (coated in the plate). On the other hand, with the increase in the concentration of M. bovis cells up to 102 cfu mL–1, the absorbance got decreased significantly, indicating the availability of a low concentration of free Au NPs-QUBMA-Bov conjugates for binding as most of the biogenic Au NPs-QUBMA-Bov conjugates have attached with M. bovis cells during the incubation period. Interestingly, with the further increase in the M. bovis cell concentration (i.e., 103 to 105 cfu mL–1), there were no significant changes in the absorbance value. This is probably attributed to the less availability of the active sites on the Au NPs-QUBMA-Bov surface for their substrate TMB to generate a colorimetric signal as most of the active sites could have been preoccupied with the higher concentration of cells during the incubation period (Figure 8A). We further confirmed the linear response of the biosensing strategy by fitting the absorbance value to the concentration of the target analyte up to 102 cfu mL–1. (55,56) The linear regression equation was determined to be y = 0.1364x + 1.4212R2 = 0.9086. This study reveals the potential applicability of biogenic Au NPs-QUBMA-Bov conjugates for sensitive detection of M. bovis cells within a linear range of 100 to 102 cfu mL–1 with an adequate efficiency in the context of previously reported molecular detection techniques. Further, based on the calibration curve data, the LOD was calculated and found to be ∼53 cfu mL–1. Stewart and co-workers reported immunomagnetic separation (IMS) along with PCR for the detection of M. bovis (from infected animal tissues) with an LOD of 57.7 cfu mL–1 within a linear range of 100 to 105 cfu mL–1 (26) and M. bovis (from infected animals) detection using an IMS-LFD (lateral flow device) with an LOD higher than 104–5 cfu mL–1. (27) In another report, Young et al. developed an RT-PCR assay for M. bovis detection with an LOD of 104 cfu mL–1 from the soil sample. (57) However, there are limited reports of commercially available biosensors for the detection of M. bovis in contaminated food samples, which can be used in POC diagnosis methods. (58) A comparative analysis of different biosensing strategies for the detection of M. bovis and a few other pathogenic bacteria has been provided in the Supporting Information, Table S3. Our findings could possibly be used for the development of LFD-based POC sensing devices for the rapid and sensitive detection of M. bovis. To determine the interference of the experimental matrixes on the detection of M. bovis cells using biogenic Au NPs-QUBMA-Bov conjugates, the iELISA was performed in a food matrix (solid cheese) for real sample analysis. Figure 8B shows the absorbance response of the calibration curve for sensitive detection of M. bovis cells in the food matrix. The results correspond with the previous findings of a calibration curve for M. bovis detection in a laboratory buffer solution. The absorbance response decreases with the increase in the concentration of M. bovis cells from 100 to 105 cfu mL–1, indicating the moderately sensitive detection capability within the linear range of 100 to 102 cfu mL–1 of the analyte. Further, based on the calibration curve data, the LOD was calculated and found to be ∼71 cfu mL–1. We further confirmed the linear response of the biosensing strategy by fitting the absorbance value to the concentration of the target analyte up to 102 cfu mL–1. (55,56) The linear regression equation was determined to be y = 0.6702x + 1.0924, R2 = 0.9508. However, the absorbance value decreases in comparison with the previous results, probably due to the interference of the food molecules present in the reaction solution, which hinders the oxidation of TMB (31) catalyzed by biogenic Au NPs-QUBMA-Bov conjugates.

Figure 8

Figure 8. iELISA sensitivity assay using AuNPs-QUBMA-Bov conjugates to detect M. bovis (A) in a laboratory buffer solution (PBS, pH 7.4, Tween-20 0.05% v/v, PBST) and (B) in a solid cheese matrix solution. (C) iELISA specificity assay using AuNPs-QUBMA-Bov conjugates to detect M. bovis in a laboratory buffer (PBST) solution.

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Apart from the sensitivity, specificity is the most essential key influencing factor ensuring the feasibility of the Au NPs-QUBMA-Bov conjugates-based immunosensing assay. To determine the specificity of the biogenic Au NPs-QUBMA-Bov conjugates, the iELISA was performed against four different bacterial strains: (i) two mycobacterial species, M. smegmatis mc2155 and M. tuberculosis H37Rv and (ii) two common E. colik12 ER2738 and E. coli BL21 (DE3) by comparing the absorbance response with each culture isolate. Figure 8C demonstrates that the absorbance responses from E. colik12, E. coliBL21 DE3, and M. smegmatis are similar to the negative signal (only laboratory buffer/PBST without any bacteria), with the higher absorbance attributed to more availability of free unbound biogenic Au NPs-QUBMA-Bov conjugates after the incubation period. Similarly, the absorbance response for M. bovis has the lowest absorbance value in comparison to others, indicating that Au NPs-QUBMA-Bov gets attached to the M. bovis cells during the incubation period as the mAb conjugated with biogenic Au NPs is specific to M. bovis. The absorbance response for M. tuberculosis is slightly higher than that of M. bovis but less than for the other tested bacteria. These results indicate the specific nature of the mAb toward the Mycobacterium species in comparison to the other bacteria tested (27,54) (Figure 8C).

4. Conclusions

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In conclusion, an eco-friendly, cost effective, convenient, and single-step green synthesis of biogenic Au NPs was achieved by using P. nepalensis fruit extract. To the best of our knowledge, this is the first report for the biogenic synthesis of Au NPs utilizing P. nepalensis fruit extract. Chemical analysis of the fruit extract revealed the presence of various bioactive organic compounds such as polyphenols, amino acids, flavonoids, antioxidants, organic acids, and sugars that probably assisted the reduction of Au3+ ions followed by the stabilization of the synthesized Au NPs. On the other hand, adsorption of these bioactive compounds over the biogenic Au NP surface not only resulted in them acting as a capping agent but also played an important role in improving the catalytic efficiency of Au NPs. The biogenic Au NPs with a uniform size (6 ± 3 nm) and morphology possess ultra-active intrinsic peroxidase-like activity and can catalyze the oxidation of TMB in the presence of H2O2. The peroxidase-like activity of biogenic Au NPs followed Michaelis–Menten kinetics, and the reaction velocity was dependent on the environmental pH, temperature, substrate, and H2O2 concentration. In comparison with the natural enzyme (HRP), biogenic Au NPs showcased a 9.64 times higher maximum reaction velocity at 6% H2O2 with a higher affinity toward substrate TMB. The Michaelis–Menten constant (KM) values for biogenic Au NPs and HRP were found to be 6.9 × 10–2 and 7.9 × 10–2 mM, respectively, at the same nanozyme/enzyme concentration of 100 pM and at 6% H2O2. Under the experimental conditions, the peroxidase-mimicking activity of biogenic Au NPs was suppressed by 32% after mAb conjugation.
As a proof-of-concept for suitable biosensing applications with biogenic Au NPs, an iELISA was developed to detect M. bovis cells using biogenic Au NPs-QUBMA-Bov conjugates, where Au NPs-QUBMA-Bov acted as a peroxidase mimic and a signaling tag. Further experiments were carried out using the iELISA method, and from the absorbance response, it was found that the biogenic Au NPs-QUBMA-Bov conjugates are capable of detection of M. bovis within a lower range of 100 to 102 cfu mL–1 with decent specificity. These findings suggest that biogenic Au NPs can be used to fabricate an efficient and cost-effective colorimetric immunosensing device that can enable a rapid and reliable qualitative detection of analytes with the naked eye through the use of a peroxidase-mimicking property. Furthermore, this developed biosensing strategy could potentially be integrated into the design of LFD with modifications for in situ testing and could be useful in POC diagnostics.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.2c00180.

  • Optimization study of the biogenic synthesis process of Au NPs; chemical analysis of P. nepalensis extract; free radical scavenging activity and antioxidant potential of P. nepalensis fruit extract; reducing power assay; DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) scavenging ability; determination of the total phenolic content; determination of the total flavonoid content; characterization of peroxidase-mimicking activity of biogenic Au NPs; understanding the reaction mechanism of TMB oxidation catalyzed by peroxidase-mimicking biogenic Au NPs; determining the role of P. nepalensis fruit extract in the catalysis of TMB oxidation; additional results and discussion: optimization study, FESEM analysis, and characterization of the peroxidase-mimicking activity of biogenic Au NPs; UV–vis spectra of optimization study of Au NP synthesis using P. nepalensis extract; FESEM image of Au NPs; zeta potential analysis of the biogenic Au NPs; reaction mechanism of the TMB oxidation process; UV–vis absorption spectra of the reaction kinetics of TMB oxidation; role of P. nepalensis fruit extract in the TMB oxidation process; GC–MS analysis of the P. nepalensis fruit extract; UV–vis absorption spectra (370 nm) of the reaction kinetics of TMB oxidation comparative analysis; Michaelis–Menten kinetic parameters calculation; GC–MS analysis of P. nepalensis extract; and comparison between different biosensing assays for the detection of bacteria (PDF)

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Author Information

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  • Corresponding Author
    • Cuong Cao - School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.;  Material and Advanced Technologies for Healthcare, Queen’s University of Belfast, Belfast BT7 1NN, U.K.;  Orcidhttps://orcid.org/0000-0001-8621-8403;  Email: [email protected]
  • Authors
    • Bhaskar Das - School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.;  Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela 769008, India
    • Javier Lou-Franco - School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
    • Brendan Gilbride - School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
    • Matthew G. Ellis - School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.;  Nanophotonics Centre, University of Cambridge, Cambridge CB3 0HE, U.K.
    • Linda D. Stewart - School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
    • Irene R. Grant - School of Biological Sciences, Queen’s University of Belfast, Belfast BT9 5DL, U.K.
    • Paramasivan Balasubramanian - Department of Biotechnology and Medical Engineering, National Institute of Technology Rourkela, Rourkela 769008, India;  Orcidhttps://orcid.org/0000-0002-3821-5029
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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Authors B.D., P.B., and C.C. acknowledge the support from the Commonwealth Split-site Scholarship (2018–2019). Authors B.G., C.C., and L.D.S. acknowledge funding from the PhD studentship support fund from the Department for the Economy for Northern Ireland (DfE). Authors B.D. and P.B. also acknowledge the support from National Institute of Technology Rourkela, Odisha, India and The Ministry of Education, Government of India. The authors J.L. and C.C. acknowledge funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement no. 720325.

References

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This article references 58 other publications.

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    Viruses present a confined environment and unique protein surface topol. (i.e. polarity, residue charge, and surface relief) for nanoparticle synthesis and are amenable to mol. biol. manipulations. Consequently, we have examd. the cowpea chlorotic mottle viruses of unmodified SubE (yeast), (HRE)-SubE engineered with interior HRE peptide epitopes (AHHAHHAAD), and wild-type as viral templates for the potentiated redn. and symmetry directed synthesis of gold nanoparticles. In the first approach, the viral capsid actively potentiated the redn. of AuCl4- by electron transfer from surface tyrosine residues resulting in a gold nanoparticle decorated viral surface. Viral redn. appeared to be selective for gold as a collection of metal precursor substrates of Ag+, Pt4+, Pd4+, and an insol. AuI complex were not reduced to zero-valent nanoclusters by virus. Alternatively, the viral capsid provided a template for the symmetry directed synthesis of Au0 nanoparticles from a non-reducible gold precursor.
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    We report a simple and cost effective way for synthesis of metallic nanoparticles (Au and Ag) using natural precursor clove. Au and Ag nanoparticles have been synthesized by reducing the aq. soln. of AuCl4 and AgNO3 with clove ext. One interesting aspect here is that redn. time is quite small (few minutes instead of hours as compared to other natural precursors). We synthesized gold and silver nanoparticles of different shape and size by varying the ratio of AuCl4 and AgNO3 with respect to clove ext., where the dominant component is eugenol. The evolution of Au and Ag nanoparticles from the redn. of different ratios of AuCl4 and AgNO3 with optimized concn. of the clove ext. has been evaluated through monitoring of surface plasmon behavior as a function of time. The redn. of AuCl4 and AgNO3 by eugenol is because of the inductive effect of methoxy and allyl groups which are present at ortho and para positions of proton releasing -OH group as two electrons are released from one mol. of eugenol. This is followed by the formation of resonating structure of the anionic form of eugenol. The presence of methoxy and allyl groups has been confirmed by FTIR. To the best of our knowledge, use of clove as reducing agent, the consequent very short time (minutes instead of hours and without any scavenger) and the elucidation of mechanism of redn. based on FTIR anal. has not been attempted earlier.
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    Dubey, S. P.; Lahtinen, M.; Sillanpää, M. Green Synthesis and Characterizations of Silver and Gold Nanoparticles Using Leaf Extract of Rosa Rugosa. Colloids Surf., A 2010, 364, 34– 41,  DOI: 10.1016/j.colsurfa.2010.04.023
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    Tai, Y.; Chang, L.-W.; Tran, N. T. T.; Tsai, Y.-C.; Fang, J.-Y. One-Step Synthesis of Highly Biocompatible Multi-Shaped Gold Nanostructures with Fruit Extract. IET Nanobiotechnol. 2011, 5, 52– 59,  DOI: 10.1049/iet-nbt.2010.0028
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    In this study, the authors demonstrate the synthesis of various gold nanostructures through a one-step, green and complete bio-modulation approach. Nanoparticles were successfully synthesized by the addn. of gold aq. soln. to fruit exts., including orange, papaya, peach or lemon. The particles were of various shapes and sizes with high abundance, such as sphere, marigold, triangle and hexagon. The biocompatibility of the presented gold nanostructures was examd.; hemolysis tests revealed a non-toxicity result in blood cell uptake of such gold nanostructures. This study opens the exciting possibility of synthesizing various multi-shaped nanoparticles through a simple and green approach, as well as paving the way for future bio-applications.
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    Das, J.; Velusamy, P. Catalytic Reduction of Methylene Blue Using Biogenic Gold Nanoparticles from Sesbania Grandiflora L. J. Taiwan Inst. Chem. Eng. 2014, 45, 2280– 2285,  DOI: 10.1016/j.jtice.2014.04.005
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    In the present investigation, we report the synthesis of gold nanoparticles (AuNPs) using leaf ext. of Sesbania grandiflora. The processes of nucleation and growth of AuNPs were followed by monitoring the absorption spectra during the reaction. UV-vis spectrum of the aq. medium contg. AuNPs showed a peak at around 534 nm. FE-SEM and TEM micrograph anal. of the AuNPs indicated that they were predominantly spherical, well-dispersed within 7-34 nm. The synthesized AuNPs are obsd. to have an excellent catalytic activity on the redn. of methylene blue by S. grandiflora ext. which is confirmed by the decrease in absorbance max. values of methylene blue (MB) with respect to time using UV-vis spectrophotometer and is attributed as an effective catalyst for degrading chem. dyes.
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    Gold nanoparticles (Au-NPs) were synthesized at room temp. using Prunus domestica (plum) fruit ext. as reducing agent. The UV-visible absorption spectrum showed a characteristic optical absorption peak of Au-NPs at 543 nm. The X-ray diffraction pattern suggested the formation and crystallinity of Au-NPs. Spherical Au-NPs synthesized with an av. particle size of 20 ± 6 nm were confirmed by transmission electron microscopy. Fourier transform IR spectroscopy anal. supported the role of water-sol. polyols and amino acids of plum fruit ext. for bioredn. and stabilization of Au-NPs. The catalytic activity of Au-NPs was investigated for 4-nitrophenol (4-NP) redn. using UV-visible absorption spectroscopy. Biosynthesized Au-NPs showed a dose-dependent catalytic activity. Catalytic redn. followed pseudo-first-order kinetics with respect to 4-NP.
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    An eco-friendly and economic efficient approach has been developed for the photoinduced synthesis of more stable AuNPs using an aq. ext. of Croton bonplandianum (AEC) as a reducing and capping agent. The reaction mixt. of AEC and HAuCl4.xH2O, when exposed to sunlight turned purple which primarily confirmed the biosynthesis of AuNPs. The biosynthesis was monitored using UV-vis spectroscopy which exhibited a sharp SPR band at 530 nm after 16 min of sunlight exposure. The parameters affecting the synthesis of AuNPs such as sunlight exposure, AEC inoculum dose, and HAuCl4.xH2O concn. were also optimized. The HR-TEM study revealed that, as the metal ion concns. increased, the av. size and anisotropic nature of the AuNPs increased. The x-ray diffraction pattern of AuNPs synthesized confirmed the formation of face-centered cubic crystal lattice of metallic gold. The involvement of polyphenolics in the synthesis of AuNPs was confirmed by comparing the FTIR anal. of pure tannic acid, AEC, and pre- and postannealed AuNPs. The XPS anal. corroborated the presence of two individual peaks attributed to the Au 4f7/2 and Au 4f5/2 binding energies which corresponded to the presence of metallic gold. The AuNPs thus obtained showed peroxidase-like mimicking activity which catalyzed the oxidn. of TMB to oxTMB with the development of blue color and absorption spectra at 652 nm. However, the presence of GSH caused further redn. of oxTMB. This detection expt. showed an excellent linear relationship between 1 and 40 μM with a limit of detection of 0.013 μM. In addn. to this, the significant recovery of GSH from human blood serum advocated that the developed system was simple and sensitive for the real sample anal.
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    Li, R. S.; Liu, H.; Chen, B. B.; Zhang, H. Z.; Huang, C. Z.; Wang, J. Stable Gold Nanoparticles as a Novel Peroxidase Mimic for Colorimetric Detection of Cysteine. Anal. Methods 2016, 8, 2494– 2501,  DOI: 10.1039/c6ay00367b
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    Stable gold nanoparticles as a novel peroxidase mimic for colorimetric detection of cysteine
    Li, Rong Sheng; Liu, Hai; Chen, Bin Bin; Zhang, Hong Zhi; Huang, Cheng Zhi; Wang, Jian
    Analytical Methods (2016), 8 (11), 2494-2501CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)
    In this work, a novel method for colorimetric detection of cysteine was proposed based on the inhibition of peroxidase-like behavior of AuNPs. Kiwi juice prepd. gold nanoparticles presented strong catalytic activity, which could catalyze H2O2-TMB to generate a blue product, resulting in strong absorption at 650 nm. In HAc-NaAc (pH 4.0) buffer, cysteine induced the aggregation of gold nanoparticles through covalent Au-S bonds and electrostatic as well as hydrogen bonding, leading to the redn. of the catalytic activity of gold nanoparticles with a weakened blue color product and a lower absorbance. Under optimal conditions, the concns. of cysteine were proportional to the degree of reduced absorbance. This approach offers a new, simple, sensitive and selective assay for cysteine.
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    Il Kim, M.; Park, C. Y.; Seo, J. M.; Kang, K. S.; Park, K. S.; Kang, J.; Hong, K. S.; Choi, Y.; Lee, S. Y.; Park, J. P.; Park, H. G.; Park, T. J. J. In Situ Biosynthesis of a Metal Nanoparticle Encapsulated in Alginate Gel for Imageable Drug-Delivery System. ACS Appl. Mater. Interfaces 2021, 13, 36697– 36708,  DOI: 10.1021/acsami.1c02286
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    Chaudhuri, Dipankar; Ghate, Nikhil Baban; Das, Sourav Panja Abhishek; Manda, Nripendranath
    PLoS One (2015), 10 (12), e0144280/1-e0144280/20CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    The antioxidant and restoration potentials of hepatic injury by Prunus nepalensis Ser. (Steud), a wild fruit plant from the Northeastern region of India, were investigated. The fruit ext. (PNME) exhibited excellent antioxidant and reducing properties and also scavenged the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (IC50 = 30.92 ± 0.40 μg/mL). PNME demonstrated promising scavenging potency, as assessed by the scavenging of different reactive oxygen and nitrogen species. Moreover, the ext. revealed an exceptional iron chelation capacity with an IC50 of 25.64 ± 0.60 μg/mL. The ext. induced significant improvement of hepatic injury and liver fibrosis against iron overload induced hepatotoxicity in mice in a dose-dependent manner, and this effect was supported by different histopathol. studies. The phytochem. constitutions and their identification by HPLC confirmed the presence of purpurin, tannic acid, Me gallate, reserpine, gallic acid, ascorbic acid, catechin and rutin. The identified compds. were investigated for their individual radical scavenging and iron chelation activity; some compds. exhibited excellent radical scavenging and iron chelation properties, but most were toxic towards normal cells (WI-38). On the other hand, crude PNME was found to be completely nontoxic to normal cells, suggesting its feasibility as a safe oral drug. The above study suggests that different phytochems. in PNME contributed to its free radical scavenging and iron chelation activity; however, further studies are required to det. the pathway in which PNME acts to treat iron-overload diseases.
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  19. 19
    Lou-Franco, J.; Das, B.; Elliott, C.; Cao, C. Gold Nanozymes: From Concept to Biomedical Applications. Nano-Micro Lett. 2021, 13, 10,  DOI: 10.1007/s40820-020-00532-z
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    Wang, X.; Hu, Y.; Wei, H. Nanozymes in Bionanotechnology: From Sensing to Therapeutics and Beyond. Inorg. Chem. Front. 2016, 3, 41– 60,  DOI: 10.1039/c5qi00240k
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    Nanozymes in bionanotechnology: from sensing to therapeutics and beyond
    Wang, Xiaoyu; Hu, Yihui; Wei, Hui
    Inorganic Chemistry Frontiers (2016), 3 (1), 41-60CODEN: ICFNAW; ISSN:2052-1553. (Royal Society of Chemistry)
    In the past few decades, researchers have developed lots of artificial enzymes with various materials to mimic the structures and functions of natural enzymes. Recently, nanozymes, nanomaterials with enzyme-like characteristics, are emerging as novel artificial enzymes, and attracting researchers' enormous interest. Remarkable advances have been made in the area of nanozymes due to their unique properties compared with natural enzymes and classic artificial enzymes. Until now, lots of nanomaterials have been studied to mimic various natural enzymes for wide applications. To highlight the recent progress of nanozymes (esp. in bionanotechnol.), here we discuss the diverse applications of nanozymes, which range from sensing, imaging, and therapeutics, to logic gates, pollutant removal, water treatment, etc. Finally, we address the current challenges facing nanozyme research as well as possible directions to fulfill their great potential in future.
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  21. 21
    Das, B.; Franco, J. L.; Logan, N.; Balasubramanian, P.; Kim, M. I.; Cao, C. Nanozymes in Point-of-Care Diagnosis: An Emerging Futuristic Approach for Biosensing. Nano-Micro Letters 2021, 13, 193,  DOI: 10.1007/s40820-021-00717-0
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    Nanozymes in point-of-care diagnosis: an emerging futuristic approach for biosensing
    Das, Bhaskar; Franco, Javier Lou; Logan, Natasha; Balasubramanian, Paramasivan; Kim, Moon Il; Cao, Cuong
    Nano-Micro Letters (2021), 13 (), 193CODEN: NLAEBV; ISSN:2150-5551. (Nano-Micro Letters)
    Nanomaterial-based artifcial enzymes (or nanozymes) have attracted great attention in the past few years owing to their capability not only to mimic functionality but also to overcome the inherent drawbacks of the natural enzymes. Numerous advantages of nanozymes such as diverse enzyme-mimicking activities, low cost, high stability, robustness, unique surface chem., and ease of surface tunability and biocompatibility have allowed their integration in a wide range of biosensing applications. Several metal, metal oxide, metal-org. framework-based nanozymes have been exploited for the development of biosensing systems, which present the potential for point-of-care anal. To highlight recent progress in the feld, in this review, more than 260 research articles are discussed systematically with suitable recent examples, elucidating the role of nanozymes to reinforce, miniaturize, and improve the performance of point-of-care diagnostics addressing the ASSURED (afordable, sensitive, specifc, user-friendly, rapid and robust, equipment-free and deliverable to the end user) criteria formulated by World Health Organization. The review reveals that many biosensing strategies such as electrochem., colorimetric, fuorescent, and immunol. sensors required to achieve the ASSURED stds. can be implemented by using enzyme-mimicking activities of nanomaterials as signal producing components. However, basic system functionality is still lacking. Since the enzyme-mimicking properties of the nanomaterials are dictated by their size, shape, compn., surface charge, surface chem. as well as external parameters such as pH or temp., these factors play a crucial role in the design and function of nanozyme-based point-of-care diagnostics. Therefore, it requires a deliberate exertion to integrate various parameters for truly ASSURED solns. to be realized. This review also discusses possible limitations and research gaps to provide readers a brief scenario of the emerging role of nanozymes in state-of-the-art POC diagnosis system development for futuristic biosensing applications.
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  22. 22
    Taylor, G. M.; Worth, D. R.; Palmer, S.; Jahans, K.; Hewinson, R. G. Rapid Detection of Mycobacterium Bovis DNA in Cattle Lymph Nodes with Visible Lesions Using PCR. BMC Vet. Res. 2007, 3, 12,  DOI: 10.1186/1746-6148-3-12
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    22
    Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR
    Taylor G Michael; Worth Danny R; Palmer Si; Jahans Keith; Hewinson R Glyn
    BMC veterinary research (2007), 3 (), 12 ISSN:.
    BACKGROUND: We have evaluated a sensitive screening assay for Mycobacterium tuberculosis (MTB) complex organisms and a specific assay for detecting Mycobacterium bovis DNA in lymph nodes taken from cattle with evidence of bovine tuberculosis. Underlying these series of experiments was the need for a versatile DNA extraction protocol which could handle tissue samples and with the potential for automation.The target for the screening assay was the multi-copy insertion element IS1081, present in 6 copies in the MTB complex. For confirmation of M. bovis we used primers flanking a specific deletion in the genome of M. bovis known as region of difference 4 (RD4). The sensitivity and specificity of these PCRs has been tested on genomic DNA from MTB complex reference strains, mycobacteria other than tuberculosis (MOTT), spiked samples and on clinical material. RESULTS: The minimum detection limits of the IS1081 method was < I genome copy and for the RD4 PCR was 5 genome copies. Both methods can be readily adapted for quantitative PCR with the use of SYBR Green intercalating dye on the RotorGene 3000 platform (Corbett Research). Initial testing of field samples of bovine lymph nodes with visible lesions (VL, n = 109) highlighted two shortfalls of the molecular approach. Firstly, comparison of IS1081 PCR with the "gold standard" of culture showed a sensitivity of approximately 70%. The sensitivity of the RD4 PCR method was 50%. Secondly, the success rate of spoligotyping applied directly to clinical material was 51% compared with cultures. A series of further experiments indicated that the discrepancy between sensitivity of detection found with purified mycobacterial DNA and direct testing of field samples was due to limited mycobacterial DNA recovery from tissue homogenates rather than PCR inhibition. The resilient mycobacterial cell wall, the presence of tissue debris and the paucibacillary nature of some cattle VL tissue may all contribute to this observation. Any of these factors may restrict application of other more discriminant typing methods.A simple means of increasing the efficiency of mycobacterial DNA recovery was assessed using a further pool of 95 cattle VL. Following modification of the extraction protocol, detection rate with the IS1081 and RD4 methods increased to 91% and 59% respectively. CONCLUSION: The IS1081 PCR is a realistic screening method for rapid identification of positive cases but the sensitivity of single copy methods, like RD4 and also of spoligotyping will need to be improved to make these applicable for direct testing of tissue extracts.
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  23. 23
    Bolaños, C. A. D.; Paula, C. L. d.; Guerra, S. T.; Franco, M. M. J.; Ribeiro, M. G. Diagnosis of Mycobacteria in Bovine Milk: An Overview. Rev. Inst. Med. Trop. Sao Paulo 2017, 59, e40  DOI: 10.1590/S1678-9946201759040
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    23
    Diagnosis of mycobacteria in bovine milk: an overview
    Bolanos, Carmen Alicia Daza; Lechinski de Paula, Carolina; Guerra, Simony Trevizan; Franco, Marilia Masello Junqueira; Ribeiro, Marcio Garcia
    Revista do Instituto de Medicina Tropical de Sao Paulo (2017), 59 (), e40/1-e40/13CODEN: RMTSAE; ISSN:1678-9946. (Instituto de Medicina Tropical de Sao Paulo)
    Tuberculosis remains as the world's biggest threat. In 2014, human tuberculosis ranked as a major infectious disease by the first time, overcoming HIV death rates. Bovine tuberculosis is a chronic disease of global distribution that affects animals and can be transmitted to humans by the consumption of raw milk, representing a serious public health concern. Despite the efforts of different countries to control and eradicate bovine tuberculosis, the high neg. economic impact on meat and milk prodn. chains remains, given the decreased prodn. efficiency (approx. 25%), the high no. of condemned carcasses, and increased animal culling rates. This scenario has motivated the establishment of official programs based on regulations and diagnostic procedures. Although Mycobacterium tuberculosis and Mycobacterium bovis are the major pathogenic species to humans and bovines, resp., nontuberculous mycobacteria within the Mycobacterium genus have become increasingly important in recent decades due to human infections, including the ones that occur in immunocompetent people. Diagnosis of mycobacteria can be performed by microbiol. culture from tissue samples (lymph nodes, lungs) and secretions (sputum, milk). In general, these pathogens demand special nutrient requirements for isolation/growth, and the use of selective and rich culture media. Indeed, within these genera, mycobacteria are classified as either fast- or slow-growth microorganisms. Regarding the latter ones, incubation times can vary from 45 to 90 days. Although microbiol. culture is still considered the gold std. method for diagnosis, mol. approaches have been increasingly used. We describe here an overview of the diagnosis of Mycobacterium species in bovine milk.
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  24. 24
    Dean, A. S.; Forcella, S.; Olea-Popelka, F.; Idrissi, A. E.; Glaziou, P.; Benyahia, A.; Mumford, E.; Erlacher-Vindel, E.; Gifford, G.; Lubroth, J.; Raviglione, M.; Fujiwara, P. A Roadmap for Zoonotic Tuberculosis: A One Health Approach to Ending Tuberculosis. Lancet Infect. Dis. 2018, 18, 137– 138,  DOI: 10.1016/S1473-3099(18)30013-6
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    A roadmap for zoonotic tuberculosis: a One Health approach to ending tuberculosis
    Dean Anna S; Forcella Simona; Olea-Popelka Francisco; Idrissi Ahmed El; Lubroth Juan; Glaziou Philippe; Raviglione Mario; Benyahia Amina; Mumford Elizabeth; Erlacher-Vindel Elisabeth; Gifford Glen; Fujiwara Paula
    The Lancet. Infectious diseases (2018), 18 (2), 137-138 ISSN:.
    There is no expanded citation for this reference.
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  25. 25
    Ji, T.; Muenker, M. C.; Papineni, R. V. L.; Harder, J. W.; Vizard, D. L.; Mclaughlin, W. E. Increased Sensitivity in Antigen Detection with Fluorescent Latex Nanosphere-IgG Antibody Conjugates. Bioconjugate Chem. 2010, 21, 427– 435,  DOI: 10.1021/bc900295v
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    Increased Sensitivity in Antigen Detection with Fluorescent Latex Nanosphere-IgG Antibody Conjugates
    Ji, Tao; Muenker, M. Catherine; Papineni, Rao V. L.; Harder, John W.; Vizard, Douglas L.; McLaughlin, William E.
    Bioconjugate Chemistry (2010), 21 (3), 427-435CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
    IgG antibodies were conjugated to Kodak X-Sight nanospheres to develop fluorescent-labeled antibodies using two different synthetic routes: one involving the DTT redn. method, and the other involving Traut's Reagent modification method. These two methods result in different conjugation efficiencies and different performances in antigen detection. Western blotting shows that the nanosphere-IgG antibody conjugates synthesized using the DTT redn. method are more immunospecific than the conjugates synthesized using Traut's Reagent modification method. In addn., the conjugates synthesized using DTT redn. also show higher antigen detection sensitivity than other com. available fluorescent-IgG antibody conjugates, including Alexa Fluor, Qdot, and CyDye conjugates.
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    Stewart, L. D.; McNair, J.; McCallan, L.; Thompson, S.; Kulakov, L. A.; Grant, I. R. Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium Bovis. J. Clin. Microbiol. 2012, 50, 1598– 1605,  DOI: 10.1128/JCM.05747-11
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    26
    Production and evaluation of antibodies and phage display-derived peptide ligands for immunomagnetic separation of Mycobacterium bovis
    Stewart, Linda D.; McNair, James; McCallan, Lyanne; Thompson, Suzan; Kulakov, Leonid A.; Grant, Irene R.
    Journal of Clinical Microbiology (2012), 50 (5), 1598-1605CODEN: JCMIDW; ISSN:0095-1137. (American Society for Microbiology)
    This study describes the development and optimization of an immunomagnetic sepn. (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extd. surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concn. range, 10 to 105 CFU/mL) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic labs. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrixes, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/mL of lymph node tissue homogenate for IMS-PCR and IMS-culture, resp.). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.
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    Stewart, L. D.; Tort, N.; Meakin, P.; Argudo, J. M.; Nzuma, R.; Reid, N.; Delahay, R. J.; Ashford, R.; Montgomery, W. I.; Grant, I. R. Development of a Novel Immunochromatographic Lateral Flow Assay Specific for Mycobacterium Bovis Cells and Its Application in Combination with Immunomagnetic Separation to Test Badger Faeces. BMC Vet. Res. 2017, 13, 131,  DOI: 10.1186/s12917-017-1048-x
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    27
    Development of a novel immunochromatographic lateral flow assay specific for Mycobacterium bovis cells and its application in combination with immunomagnetic separation to test badger faeces
    Stewart, Linda D.; Tort, Nuria; Meakin, Paul; Argudo, Jose M.; Nzuma, Ruramayi; Reid, Neil; Delahay, Richard J.; Ashford, Roland; Montgomery, W. Ian; Grant, Irene R.
    BMC Veterinary Research (2017), 13 (), 131/1-131/12CODEN: BVRMA9; ISSN:1746-6148. (BioMed Central Ltd.)
    The European badger is an important wildlife reservoir of Mycobacterium bovis implicated in the spread of bovine tuberculosis in the United Kingdom and Ireland. Infected badgers are known to shed M. bovis in their urine and faeces, which may contaminate the environment. To aid bovine tuberculosis control efforts novel diagnostic tests for detecting infected and shedding badgers are needed. We proposed development of a novel, rapid immunochromatog. lateral flow device (LFD) as a non-invasive test to detect M. bovis cells in badger faeces. Its application in combination with immunomagnetic sepn. (IMS) to detect Mycobacterium bovis cells in badger faeces is reported here. A novel prototype LFD for M. bovis cells was successfully developed, with unique specificity for M. bovis and a limit of detection 50% (LOD50%) of 1.7× 104M. bovis cells/mL. When IMS was employed to selectively capture and conc. M. bovis cells from badger faeces prior to LFD testing, the LOD50% of the IMS-LFD assay was 2.8 × 105M. bovis cells/mL faecal homogenate. Faeces samples collected from latrines at badger setts in a region of endemic bovine tuberculosis infection were tested; 78 (18%) of 441 samples tested IMS-LFD assay pos., whereas 140 (32%) tested IMS-qPCR pos. (Kappa agreement -0.009 ± 0.044, p = 0.838). Subsequently, when 130 faeces samples from live captured, or captive, badgers of known infection status (on the basis of StatPak, interferon-γ and/or culture results) were tested, the IMS-LFD assay had higher relative diagnostic specificity (Sp 0.926), but poorer relative diagnostic sensitivity (Se 0.081), than IMS-qPCR (Sp 0.706, Se 0.581) and IMS-culture (Sp 0.794, Se 0.436). The novel IMS-LFD assay, although very specific for M. bovis, has low anal. sensitivity (indicated by the LOD50%) and would only detect badgers shedding high nos. of M. bovis (>104-5 cells/g) in their faeces. The novel LFD would, therefore, have limited value as a non-invasive test for badger TB surveillance purposes but it may have value for alternative veterinary diagnostic applications.
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    Long, G. L.; Winefordner, J. D. Limit of Detection. A Closer Look at the IUPAC Definition. Anal. Chem. 1983, 55, 712A– 724A,  DOI: 10.1021/ac00258a724
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    Limit of detection. A closer look at the IUPAC definition
    Long, Gary L.; Winefordner, J. D.
    Analytical Chemistry (1983), 55 (7), 712A-714A, 716A, 718A, 720A, 722A, 724ACODEN: ANCHAM; ISSN:0003-2700.
    The statistical meaning of detection limit values in trace anal. is examd. in a format consistent with the IUPAC definition. Various methods of calcg. detection limit values are reviewed.
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    International Organization for Standardization. Microbiology of Food and Animal Feeding Stuffs: Horizontal Method for the Detection of Salmonella Spp. Detection of Salmonella Spp. in Animal Faeces and in Environmental Samples from the Primary Production Stage , 2007.
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    Mishra, P.; Ray, S.; Sinha, S.; Das, B.; Khan, M. I.; Behera, S. K.; Yun, S.-I.; Tripathy, S. K.; Mishra, A. Facile Bio-Synthesis of Gold Nanoparticles by Using Extract of Hibiscus Sabdariffa and Evaluation of Its Cytotoxicity against U87 Glioblastoma Cells under Hyperglycemic Condition. Biochem. Eng. J. 2016, 105, 264– 272,  DOI: 10.1016/j.bej.2015.09.021
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    Facile bio-synthesis of gold nanoparticles by using extract of Hibiscus sabdariffa and evaluation of its cytotoxicity against U87 glioblastoma cells under hyperglycemic condition
    Mishra, Pratik; Ray, Sambit; Sinha, Sayantan; Das, Bhaskar; Khan, Md. Imran; Behera, Susant K.; Yun, Soon-Il; Tripathy, Suraj K.; Mishra, Amrita
    Biochemical Engineering Journal (2016), 105 (Part_A), 264-272CODEN: BEJOFV; ISSN:1369-703X. (Elsevier B.V.)
    In the present paper, a facile synthesis of gold nanoparticles is reported with leaf and stem ext. of Hibiscus sabdariffa. Structural features of as synthesized nanoparticles are characterized by UV-vis spectroscopy, XRD, FTIR, and XPS. Morphol. of the above synthesized gold nanoparticles is investigated by electron microscopy. The stability of the nanoparticles is studied in different concns. of glucose which suggested their possible application under hyperglycemic condition. As synthesized nanoparticles has shown selective toxicity towards U87 glioblastoma multiforme cell line under normal and hyperglycemic condition, indicating their potential to be used in the development of value-added products in the biochem. industries. The possible mode of activity of the above nanoparticles has been studied by in vitro mol. techniques.
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    McVey, C.; Logan, N.; Thanh, N. T. K.; Elliott, C.; Cao, C. Unusual Switchable Peroxidase-Mimicking Nanozyme for the Determination of Proteolytic Biomarker. Nano Res. 2019, 12, 509– 516,  DOI: 10.1007/s12274-018-2241-3
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    Unusual switchable peroxidase-mimicking nanozyme for the determination of proteolytic biomarker
    McVey, Claire; Logan, Natasha; Thanh, Nguyen T. K.; Elliott, Christopher; Cao, Cuong
    Nano Research (2019), 12 (3), 509-516CODEN: NRAEB5; ISSN:1998-0000. (Springer GmbH)
    Detection of enzyme biomarkers originating from either bio-fluids or contaminating microorganisms is of utmost importance in clin. diagnostics and food safety. Herein, we present a simple, low-cost and easy-to-use sensing approach based on the switchable peroxidase-mimicking activity of plasmonic gold nanoparticles (AuNPs) that can catalyze for the oxidn. of 3,3',5'5-tetramethylbenzidine (TMB) for the detn. of protease enzyme. The AuNP surface is modified with casein, showing dual functionalities. The first function of the coating mol. is to suppress the intrinsic peroxidase-mimicking activity of AuNPs by up to 77.1%, due to surface shielding effects. Secondly, casein also functions as recognition sites for the enzyme biomarker. In the presence of protease, the enzyme binds to and catalyzes the degrdn. of the coating layer on the AuNP surface, resulting in the recovery of peroxidase-mimicking activity. This is shown visually in the development of a blue colored product (oxidized TMB) or spectroscopically as an increase in absorbance at 370 and 650 nm. This mechanism allows for the detection of protease at 44 ng·mL-1 in 90 min. The nanosensor circumvents issues assocd. with current methods of detection in terms of ease of use, compatibility with point-of-care testing, low-cost prodn. and short anal. time. The sensing approach has also been applied for the detection of protease spiked in ultra-heat treated (UHT) milk and synthetic human urine samples at a limit of detection of 490 and 176 ng·mL-1, resp., showing great potential in clin. diagnostics, food safety and quality control.[Figure not available: see fulltext.].
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    Wang, S.; Chen, W.; Liu, A.-L.; Hong, L.; Deng, H.-H.; Lin, X.-H. Comparison of the Peroxidase-like Activity of Unmodified, Amino-Modified, and Citrate-Capped Gold Nanoparticles. ChemPhysChem 2012, 13, 1199– 1204,  DOI: 10.1002/cphc.201100906
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    Comparison of the Peroxidase-Like Activity of Unmodified, Amino-Modified, and Citrate-Capped Gold Nanoparticles
    Wang, Sheng; Chen, Wei; Liu, Ai-Lin; Hong, Lei; Deng, Hao-Hua; Lin, Xin-Hua
    ChemPhysChem (2012), 13 (5), 1199-1204CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The origin of the peroxidase-like activity of gold nanoparticles and the impact of surface modification were studied. Furthermore, some influencing factors, such as fabrication process, redox property of the modifier, and charge property of the substrate, were studied. Compared to amino-modified or citrate-capped gold nanoparticles, unmodified gold nanoparticles show significantly higher catalytic activity toward peroxidase substrates, i.e., the superficial gold atoms are a contributing factor to the obsd. peroxidase-like activity. The different catalytic activities of amino-modified and citrate-capped gold nanoparticles toward 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) show that the charge characteristics of the nanoparticles and the substrate also play an important role in the catalytic reactions.
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    Arnao, M. B.; Acosta, M.; del Rio, J. A. F.; García-Cánovas, F. Inactivation of Peroxidase by Hydrogen Peroxide and Its Protection by a Reductant Agent. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1990, 1038, 85– 89,  DOI: 10.1016/0167-4838(90)90014-7
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    Inactivation of peroxidase by hydrogen peroxide and its protection by a reductant agent
    Arnao, M. B.; Acosta, M.; Del Rio, J. A.; Garcia-Canovas, F.
    Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1990), 1038 (1), 85-9CODEN: BBAEDZ; ISSN:0167-4838.
    Hydrogen peroxide, the oxidant substrate of peroxidase, is also an inactivating agent of this enzyme. The reductant substrates protect the enzyme from the inactivating process. A reaction mechanism is proposed, in which 2 competitive routes exist for Compd. I of peroxidase; one catalytic and one inactivating. The anal. soln. produced at the end of the reaction supports the proposed mechanism and shows the dependence between the no. of turnovers of the enzyme and the ratio of both substrates.
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    Drozd, M.; Pietrzak, M.; Parzuchowski, P.; Mazurkiewicz-Pawlicka, M.; Malinowska, E. Peroxidase-like Activity of Gold Nanoparticles Stabilized by Hyperbranched Polyglycidol Derivatives over a Wide PH Range. Nanotechnology 2015, 26, 495101,  DOI: 10.1088/0957-4484/26/49/495101
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    Peroxidase-like activity of gold nanoparticles stabilized by hyperbranched polyglycidol derivatives over a wide pH range
    Drozd, Marcin; Pietrzak, Mariusz; Parzuchowski, Pawel; Mazurkiewicz-Pawlicka, Marta; Malinowska, Elzbieta
    Nanotechnology (2015), 26 (49), 495101/1-495101/15CODEN: NNOTER; ISSN:1361-6528. (IOP Publishing Ltd.)
    The aim of this work was to carry out comparative studies on the peroxidase-like activity of gold nanoparticles (AuNPs) stabilized with low mol. wt. hyperbranched polyglycidol (HBPG-OH) and its deriv. modified with maleic acid residues (HBPG-COOH). The influence of the stabilizer to gold precursor ratio on the size and morphol. of nanoparticles obtained was checked, and prepd. nanoparticles were characterized by means of transmission electron microscopy and UV-Vis spectroscopy. The results indicated the divergent effect of increasing the concn. of stabilizers (HBPG-OH or HBPG-COOH) on the size of the nanostructures obtained. The gold nanoparticles obtained were characterized as having intrinsic peroxidase-like activity and the mechanism of catalysis in acidic and alk. mediums was consistent with the std. Michaelis-Menten kinetics, revealing a strong affinity of AuNPs with 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3, 3', 5, 5'-tetramethylbenzidine (TMB), and significantly lower affinity towards phenol. By comparing the kinetic parameters, a negligible effect of polymeric ligand charge on activity against various types of substrates (anionic or cationic) was indicated. The superiority of steric stabilization via the application of tested low-wt. hyperbranched polymers over typical stabilizers in preventing salt-induced aggregation and maintaining high catalytic activity in time was proved. The applied hyperbranched stabilizers provide a good tool for manufg. gold-based nanozymes, which are highly stable and active over a wide pH range.
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    Zheng, C.; Ke, W.; Yin, T.; An, X. Intrinsic Peroxidase-like Activity and the Catalytic Mechanism of Gold@carbon Dots Nanocomposites. RSC Adv. 2016, 6, 35280– 35286,  DOI: 10.1039/c6ra01917j
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    Intrinsic peroxidase-like activity and the catalytic mechanism of gold@carbon dots nanocomposites
    Zheng, Cui; Ke, Wenjing; Yin, Tianixiang; An, Xueqin
    RSC Advances (2016), 6 (42), 35280-35286CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
    Well-dispersed AuNPs@CDs nanocomposites were successfully synthesized from the redn. of chloroauric acid by carbon dots (CDs) at room temp. The as-prepd. AuNPs@CDs were characterized by transmission electron microscopy (TEM), Fourier transform IR spectroscopy (FTIR), Raman spectroscopy and XPS, and the results indicate AuNPs@CDs nanocomposites with a core-shell structure. The AuNPs@CDs are found to possess intrinsic peroxidase-like activity, and the catalytic activity is higher than that of AuNPs. The mechanism of peroxidase-like activity of AuNPs@CDs was investigated by fluorescence spectroscopy, ESR and cyclic voltammetry, and it is found that the mechanism could be ascribed to facilitating the electron transfer between TMB and H2O2.
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    Drozd, M.; Pietrzak, M.; Parzuchowski, P. G.; Malinowska, E. Pitfalls and Capabilities of Various Hydrogen Donors in Evaluation of Peroxidase-like Activity of Gold Nanoparticles. Anal. Bioanal. Chem. 2016, 408, 8505– 8513,  DOI: 10.1007/s00216-016-9976-z
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    Pitfalls and capabilities of various hydrogen donors in evaluation of peroxidase-like activity of gold nanoparticles
    Drozd, Marcin; Pietrzak, Mariusz; Parzuchowski, Pawel G.; Malinowska, Elzbieta
    Analytical and Bioanalytical Chemistry (2016), 408 (29), 8505-8513CODEN: ABCNBP; ISSN:1618-2642. (Springer)
    Catalytic nanomaterials, widely used as substitutes of peroxidase, exhibit unique properties, which are unattainable for native enzymes. However, their activity is usually examd. by means of substrates developed and methods standardized for horseradish peroxidase (HRP). The aim of the presented work was to det. the scope of usefulness of chromogenic substrates for gold nanoparticle (AuNP) activity studies under conditions which significantly extend beyond the activity range of a native HRP. The applicability of chromogens such as 3,3'5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) beyond the typical range of pH, and for the samples of high concn. of hydrogen peroxide was examd. The conducted research confirmed the usefulness of ABTS and TMB in acidic media (pH 2.5-3.5). At the same time, potential interferences from chloride anion, unobservable for HRP-based assays, were indicated. Moreover, a no. of potentially useful hints concerning relations of concn. of substrates and catalyst for arom. amine oxidn. (TMB and OPD) were proposed. By increasing the concn. of chromogens and thanks to assuring the relatively low conversion of the reaction, the stability of TMB and OPD oxidn. product was improved even in acidic media. The comparative studies of H2O2 affinity to the surface of AuNPs in the presence of various hydrogen donors underlined the superiority of phenolic compds. over arom. amines and ABTS in the case of the samples of relatively low H2O2 concn. This work highlights some improvements in the methods of HRP-like activity characterization of NPs. It provides a crit. anal. of the major challenges, which may emerge in a case of bioanal. assays employing the catalytic nanoparticles as labels.
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    Monier, M.; Ayad, D. M.; Wei, Y.; Sarhan, A. A. Immobilization of Horseradish Peroxidase on Modified Chitosan Beads. Int. J. Biol. Macromol. 2010, 46, 324– 330,  DOI: 10.1016/j.ijbiomac.2009.12.018
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    Immobilization of horseradish peroxidase on modified chitosan beads
    Monier, M.; Ayad, D. M.; Wei, Y.; Sarhan, A. A.
    International Journal of Biological Macromolecules (2010), 46 (3), 324-330CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
    A method has been developed to immobilize horseradish peroxidase (HRP) on modified chitosan beads by means of graft copolymn. of polyethylacrylate in presence of potassium persulfate and Mohr's salt redox initiator. The activity of free and immobilized HRP was studied. FTIR spectroscopy and SEM were used to characterize HRP immobilization. The efficiency of the immobilization was investigated by examg. the relative enzymic activity of free enzyme before and after the HRP immobilization. The obtained values were found to reach 98.4%. The results show that the optimum temp. of immobilized HRP was 45 °C, which was identical to that of free enzyme, and the immobilized HRP exhibited a higher relative activity than that of free HRP over 45 °C. The optimal pH for immobilized HRP was 10, which was higher than that of the free HRP (pH 9.0), and the immobilization resulted in stabilization of enzyme over a broader pH range. The apparent kinetic const. value (Km) of immobilized HRP was 3.784 mmol mL-1, which was higher than that of free HRP. On the other hand, the activity of immobilized HRP decreased slowly against time when compared to that of the free HRP, and could retain 65.8% residual activity after 6 consecutive cycles.
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  38. 38
    Ahmad, N.; Sharma, S. Green Synthesis of Silver Nanoparticles Using Extracts of Ananas Comosus. Green Sustainable Chem. 2012, 02, 141– 147,  DOI: 10.4236/gsc.2012.24020
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    Green synthesis of silver nano particles using extracts of Ananas comosus
    Ahmad, Naheed; Sharma, Seema
    Green and Sustainable Chemistry (2012), 2 (4), 141-147, 7 pp.CODEN: GSCRCJ; ISSN:2160-696X. (Scientific Research Publishing, Inc.)
    The biosynthesis of nanoparticles has been proposed as a cost effective and environmental friendly alternative to chem. and phys. methods. Plant mediated synthesis of nanoparticles is a green chem. approach that intercomnects nanotechnol. and plant biotechnol. In the present study, synthesis of silver nanoparticles (AgNPs) or (Green-Silver) has been demonstrated using exts. of Ananas comosus reducing aq. silver nitrate. The AgNPs were characterized by UV-Visible (UV-Vis) Spectrometer, Energy Dispersive X-ray Anal. (EDAX), Selected Area Diffraction Pattern (SAED) and High Resoln. Transmission Electron Microscopy (HRTEM). TEM micrographs showed spherical particles with an av. size of 12 nm. The XRD pattern showed the characteristic Bragg peaks of (111), (200), (220) and (311) facets of the face center cubic (fcc) silver nanoparticles and confirmed that these nanoparticles are cryst. in nature. The different types of antioxidants presented in the pineapple juice synergistically reduce the Ag metal ions, as each antioxidant is unique in terms of its structure and antioxidant function. The reaction process was simple for formation of silver nanoparticles and AgNPs presented in the aq. medium were quite stable, even up to 4 mo of incubation. This work proved the capability of using biomaterial towards the synthesis of silver nanoparticle, by adopting the principles of green chem.
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    Huang, D.; Boxin, O. U.; Prior, R. L. The Chemistry behind Antioxidant Capacity Assays. J. Agric. Food Chem. 2005, 53, 1841– 1856,  DOI: 10.1021/jf030723c
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    The Chemistry behind Antioxidant Capacity Assays
    Huang, Dejian; Ou, Boxin; Prior, Ronald L.
    Journal of Agricultural and Food Chemistry (2005), 53 (6), 1841-1856CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    A review summarizing the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidn. and analyzes the chem. principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decompn. of azo compds. These assays include inhibition of induced low-d. lipoprotein autoxidn., oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the redn. of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concns. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addn., other assays intended to measure a sample's scavenging capacity of biol. relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this anal., it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addn. to these two commonly accepted assays.
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    Everette, J. D.; Bryant, Q. M.; Green, A. M.; Abbey, Y. A.; Wangila, G. W.; Walker, R. B. Thorough Study of Reactivity of Various Compound Classes toward the Folin-Ciocalteu Reagent. J. Agric. Food Chem. 2010, 58, 8139– 8144,  DOI: 10.1021/jf1005935
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    Thorough Study of Reactivity of Various Compound Classes toward the Folin-Ciocalteu Reagent
    Everette, Jace D.; Bryant, Quinton M.; Green, Ashlee M.; Abbey, Yvonne A.; Wangila, Grant W.; Walker, Richard B.
    Journal of Agricultural and Food Chemistry (2010), 58 (14), 8139-8144CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
    A thorough study was done to test the reactivity of the Folin-Ciocalteu (F-C) reagent toward various compd. classes. Over 80 compds. were tested. Compd. classes included phenols, thiols, vitamins, amino acids, proteins, nucleotide bases, unsatd. fatty acids, carbohydrates, org. acids, inorg. ions, metal complexes, aldehydes, and ketones. All phenols, proteins, and thiols tested were reactive toward the reagent. Many vitamin derivs. were also reactive, as were the inorg. ions Fe+2, Mn2+, I-, and SO32-. Other compds. showing reactivity included the nucleotide base guanine and the trioses glyceraldehyde and dihydroxyacetone. Copper complexation enhanced the reactivity of salicylate derivs. toward the reagent, whereas zinc complexation did not. Several amino acids and sugars that were reported to be reactive toward the F-C reagent in earlier studies were found not to be reactive in this study, at least in the concns. used. Reaction kinetics of each compd. with the F-C reagent were also measured. Most compds. tested showed a biphasic kinetic pattern with half-lives under 1 min. Trolox and ascorbic acid displayed a rapid monophasic pattern in which the reaction reached end point within 1 min. In summary, this study has shown that the F-C reagent is significantly reactive toward other compds. besides phenols. As other investigators have suggested, the F-C assay should be seen as a measure of total antioxidant capacity rather than phenolic content. Because phenolics are the most abundant antioxidants in most plants, it gives a rough approxn. of total phenolic content in most cases.
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    Goodarzi, V.; Zamani, H.; Bajuli, L.; Moradshahi, A. Evaluation of Antioxidant Potential and Reduction Capacity of Some Plant Extracts in Silver Nanoparticles’ Synthesis. Mol. Biol. Res. Commun. 2014, 3, 165– 174,  DOI: 10.22099/mbrc.2014.2196
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    Evaluation of antioxidant potential and reduction capacity of some plant extracts in silver nanoparticle synthesis
    Goodarzi, Vahid; Zamani, Hajar; Bajuli, Leila; Moradshahi, Ali
    Molecular Biology Research Communications (2014), 3 (3), 165-174, 10 pp.CODEN: MBRCBT; ISSN:2345-2005. (Shiraz University)
    The green synthesis of metallic nanoparticles is an active research area in nanotechnol. In the present study, antioxidant potential, total reducing capacity and silver nanoparticles (Ag NPs) synthetic potential of methanolic leaf exts. of seven plant species were evaluated and compared. Antioxidant capacity, expressed as μmol Trolox equiv. g-1 DW (μmol TE g-1 DW), ranged from 116.0 to 1.80. The plants Rosmarinus sp. and Zataria Multiflora showed highest antioxidant capacities with IC50 of 1.07 and 1.22 mg ml-1, resp. Total reducing capacity ranged from 7.6 to 0.17 mg gallic acid equiv. to g-1 DW (mg GAE g-1 DW). Plants with high antioxidant potentials also showed higher total reducing capacity. In fact, the order of the plants reducing capacity was similar to that of their antioxidant potential. The same two plant species, i.e., Zataria Multiflora and Rosmarinus sp. with high reducing capacities, showed higher potentials for Ag NPs synthesis. It is concluded that reducing substances in the exts. contribute significantly to the antioxidant potential of the tested plant species and plants with a high reducing capacity are excellent sources for the green synthesis of metallic nanoparticles. In addn., synthetic antioxidants have adverse effects on human health; therefore, to benefit more from the health promoting properties of plant species, evaluating their novel natural antioxidants is recommended.
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    Ramamurthy, C.; Padma, M.; mariya samadanam, I. D.; Mareeswaran, R.; Suyavaran, A.; Kumar, M. S.; Premkumar, K.; Thirunavukkarasu, C. The Extra Cellular Synthesis of Gold and Silver Nanoparticles and Their Free Radical Scavenging and Antibacterial Properties. Colloids Surf., B 2013, 102, 808– 815,  DOI: 10.1016/j.colsurfb.2012.09.025
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    The extra cellular synthesis of gold and silver nanoparticles and their free radical scavenging and antibacterial properties
    Ramamurthy, Ch.; Padma, M.; Samadanam, I. Daisy Mariya; Mareeswaran, R.; Suyavaran, A.; Kumar, M. Suresh; Premkumar, K.; Thirunavukkarasu, C.
    Colloids and Surfaces, B: Biointerfaces (2013), 102 (), 808-815CODEN: CSBBEQ; ISSN:0927-7765. (Elsevier B.V.)
    The bio redn. of chloroauric acid (HAuCl4) and silver nitrate (AgNO3) is achieved extracellularly by the aq. ext. of Solanum torvum (S. torvum) fruit. The nanoparticle formation was screened by UV-visible spectroscopy through color conversion due to surface plasma resonance bands at 560 nm and 430 nm for gold and silver nanoparticles resp. The spherical shapes with smooth surface of gold and silver nanoparticles were analyzed through scanning electron microscope and its presence was confirmed by energy dispersive x-ray spectroscopy (SEM/EDX). The functional groups in the gold and silver salts and the bio interactive functional groups present in the S. torvum ext. were characterized by employing Fourier transform infra-red spectroscopy (FTIR). The biomedical properties of gold and silver nanoparticles were premeditated as free radical scavenging activity and antibacterial static agents. Gold and silver nanoparticles serve as strong hydroxyl, superoxide, nitric oxide and DPPH radical scavengers in contrast to their corresponding metal oxides. The radical quenching properties of gold and silver nanoparticles were found to correlate with in vitro DNA protective effect. The silver nanoparticles show strong zone of inhibition against Escherichia coli, Pseudomonas and Bacillus whereas, gold nanoparticles exhibit fair zone of inhibition. To the authors' knowledge this is the first report that S. torvum ext. can reduce metal acids to nanomaterials.
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    Patra, J. K.; Baek, K.-H. Green Synthesis of Silver Chloride Nanoparticles Using Prunus Persica L. Outer Peel Extract and Investigation of Antibacterial, Anticandidal, Antioxidant Potential. Green Chem. Lett. Rev. 2016, 9, 132– 142,  DOI: 10.1080/17518253.2016.1192692
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    Green synthesis of silver chloride nanoparticles using Prunus persica L. outer peel extract and investigation of antibacterial, anticandidal, antioxidant potential
    Patra, Jayanta Kumar; Baek, Kwang-Hyun
    Green Chemistry Letters and Reviews (2016), 9 (2), 132-142CODEN: GCLRAI; ISSN:1751-7192. (Taylor & Francis Ltd.)
    The present study was conducted to synthesize silver chloride nanoparticles using the aq. ext. of outer peel of peach fruit (Prunus persica L.) and to evaluate its antibacterial activity, synergistic antibacterial and anticandidal potential against five foodborne pathogenic bacteria and five pathogenic Candida species resp. along with its antioxidant potential. The synthesized silver chloride nanoparticles (PE-AgClNPs) were visually confirmed with surface plasmon resonance peak at 440 nm upon UV-Vis spectroscopy anal. Furthermore, the morphol., elemental compn. and crystallinity nature were also characterized. PE-AgClNPs displayed strong antibacterial potentials (9.01-10.83 mm inhibition zone) against foodborne pathogenic bacteria and increased synergistic effect with kanamycin and rifampicin. PE-AgClNPs also displayed strong anticandidal synergistic activity with std. amphotericin b (10.51- 14.01 mm inhibition zones), along with strong free radical scavenging and reducing power. Based on strong antibacterial and antioxidant capacities, PE-AgClNPs are anticipated to have potential applications in the biomedical and food sector industries.
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    Bravo, L. Polyphenols: Chemistry, Dietary Sources, Metabolism, and Nutritional Significance. Nutr. Rev. 1998, 56, 317– 333,  DOI: 10.1111/j.1753-4887.1998.tb01670.x
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    Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance
    Bravo L
    Nutrition reviews (1998), 56 (11), 317-33 ISSN:0029-6643.
    Polyphenols constitute one of the most numerous and ubiquitous groups of plant metabolites and are an integral part of both human and animal diets. Ranging from simple phenolic molecules to highly polymerized compounds with molecular weights of greater than 30,000 Da, the occurrence of this complex group of substances in plant foods is extremely variable. Polyphenols traditionally have been considered antinutrients by animal nutritionists, because of the adverse effect of tannins, one type of polyphenol, on protein digestibility. However, recent interest in food phenolics has increased greatly, owing to their antioxidant capacity (free radical scavenging and metal chelating activities) and their possible beneficial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease, and other pathologies. Much of the literature refers to a single group of plant phenolics, the flavonoids. This review offers an overview of the nutritional effects of the main groups of polyphenolic compounds, including their metabolism, effects on nutrient bioavailability, and antioxidant activity, as well as a brief description of the chemistry of polyphenols and their occurrence in plant foods.
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    Vivek, K.; Mishra, S.; Pradhan, R. C. Physicochemical Characterization and Mass Modelling of Sohiong (Prunus Nepalensis L.) Fruit. J. Food Meas. Char. 2018, 12, 923– 936,  DOI: 10.1007/s11694-017-9708-x
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    Kashyap, P.; Riar, C. S.; Jindal, N. Optimization of Ultrasound Assisted Extraction of Polyphenols from Meghalayan Cherry Fruit (Prunus Nepalensis) Using Response Surface Methodology (RSM) and Artificial Neural Network (ANN) Approach. J. Food Meas. Char. 2021, 15, 119,  DOI: 10.1007/s11694-020-00611-0
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    Swer, T. L.; Mukhim, C.; Bashir, K.; Chauhan, K. Optimization of Enzyme Aided Extraction of Anthocyanins from Prunus Nepalensis L. LWT--Food Sci. Technol. 2018, 91, 382– 390,  DOI: 10.1016/j.lwt.2018.01.043
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    Optimization of enzyme aided extraction of anthocyanins from Prunus nepalensis L.
    Swer, Tanya L.; Mukhim, C.; Bashir, Khalid; Chauhan, Komal
    LWT--Food Science and Technology (2018), 91 (), 382-390CODEN: LSTWB3; ISSN:0023-6438. (Elsevier Ltd.)
    A central composite design was employed to obtain the optimal conditions for enzyme assisted extn. (EAE) of anthocyanins from Prunus nepalensis L. using cellulase. Response surface methodol. was used to evaluate the effects of temp. T (35-50 °C), cellulase enzyme concn. EC (5-15% E/S) and time t (2.50-4.00 h) on five responses anthocyanins recovery (TAC), total phenolic content (TPC), in vitro antioxidant activity (FRAP and DPPH) and total ext. yield (TY). The optimum extn. conditions were found to be T = 45.6 °C, EC = 12.9% E/S and t = 3.98 h resulting in TY = 62.9 ± 1.40%, TAC = 9.89 ± 0.24 mg C3G equiv./g dm, TPC = 8.32 ± 0.18 mg GAE/100 g dm with antioxidant capacity of 28.7 ± 0.12 mmol AAE/100g DM for FRAP and 91.2 ± 0.39% DPPH inhibition (20 g/mL). EAE demonstrated higher anthocyanins recovery compared to conventional solvent extn. process. HPLC anal. showed the presence of cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin. FTIR and SEM anal. were also performed. The study provides an economical alternative for com. extn. of anthocyanins from Sohiong fruit which can be used as a biocolorant for various food products. This offers immense potential for the fruit producers as well as food processors.
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    Kora, A. J.; Rastogi, L. Peroxidase Activity of Biogenic Platinum Nanoparticles: A Colorimetric Probe towards Selective Detection of Mercuric Ions in Water Samples. Sens. Actuators, B 2018, 254, 690– 700,  DOI: 10.1016/j.snb.2017.07.108
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    Peroxidase activity of biogenic platinum nanoparticles: A colorimetric probe towards selective detection of mercuric ions in water samples
    Kora, Aruna Jyothi; Rastogi, Lori
    Sensors and Actuators, B: Chemical (2018), 254 (), 690-700CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
    A biogenic green method for the synthesis of platinum nanoparticles (Pt NP) was developed using non-toxic, renewable, biodegradable plant exudate gum, gum olibanum (Boswellia serrata). The effect of parameters such as concns. of gum (0.1-0.5%) and chloroplatinic acid (0.125-1.0 mM) on nanoparticle synthesis was studied. The resulting nanoparticles exhibited excellent peroxidase activity which catalyzes the oxidn. of the chromogen 3, 3', 5, 5'-tetramethylbenzidine (TMB) to a blue color product, in the presence of hydrogen peroxide. The peroxidase activity of the nanoparticles was selectively inhibited by mercuric ions (Hg2+) due to the formation of amalgam by Hg-Pt specific interaction. Notably, the inhibition was not affected by other metal ions even at a concn. of 5 M. The decrease in oxidized TMB intensity at 652 nm (blue color), upon addn. of mercuric ions was linear in the range of 50-500 nM for MilliQ, tap and ground waters and the resp. limit of quantification values for Hg2+ using the developed method were 16.9, 26 and 47.3 nM. The proposed method was effectively applied for the detn. of Hg2+ in various ground water samples and verified with CVAAS. We envisage that the biogenic Pt NP based colorimetric probe can have promising applications in the screening and field detection of mercuric ions in various water bodies and public drinking water distribution systems.
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    Dauthal, P.; Mukhopadhyay, M. In-Vitro Free Radical Scavenging Activity of Biosynthesized Gold and Silver Nanoparticles Using Prunus Armeniaca (Apricot) Fruit Extract Nanomaterials in Energy, Health and Environment. Guest Editors: Puru Jena, Samy El Shall, Anil Kandalam. J. Nanopart. Res. 2013, 15, 1366,  DOI: 10.1007/s11051-012-1366-7
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    In-vitro free radical scavenging activity of biosynthesized gold and silver nanoparticles using Prunus armeniaca (apricot) fruit extract
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  • Abstract

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    Figure 1

    Figure 1. Characterization of biogenic Au NPs. (A) UV–vis spectrum of biologically synthesized Au NPs using P. nepalensis fruit extract. The inset shows the color formation of the reaction mixture due to the reduction of the gold salt to form Au NPs: (i) gold salt solution, (ii) P. nepalensis fruit extract, (iii) crude Au NPs, and (iv) washed Au NPs. (B) XRD analysis of Au NPs synthesized by P. nepalensis fruit extract.

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    Figure 2

    Figure 2. TEM images. (A) Bright field images (inset: SAED pattern). (B) Dark field image. (C) High-angle annular dark field image. (D) Particle size analysis performed on approximately 100 particles using ImageJ software and (E) EDX analysis of biogenic Au NPs synthesized from P. nepalensis fruit extract.

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    Figure 3

    Figure 3. (A) UV–vis spectroscopic analysis of the catalyzed reaction of TMB/H2O2 in the absence and presence of Au NPs. Figure 3A inset: (i) TMB, (ii) TMB + H2O2, (iii) Au NPs, and (iv) TMB + H2O2 +Au NPs. (B) UV–vis full spectral analysis of the catalyzed reaction of 1 mM TMB/6% H2O2 in the presence of 100 pM Au NPs.

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    Figure 4

    Figure 4. Typical kinetic analysis of TMB oxidation. (A) Kinetic analysis of TMB oxidation by Au NPs in the presence of varying TMB (0–1 mM) and H2O2 concentrations (1–10%). The concentration of the Au nanozyme was fixed at 100 pM. (B) Kinetic analysis of TMB oxidation by the HRP enzyme in the presence of varying TMB (0–1 mM) and H2O2 concentrations (1–10%). Samples were analyzed in triplicate (n = 3), and the standard deviation was deduced from these data. (C) Kinetic analysis of the HRP enzyme along with Au NPs at different pH values (2–12). (D) Kinetic analysis of the HRP enzyme along with Au NPs at different temperatures (20–60 °C). All the absorbance values were measured at 370 nm.

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    Figure 5

    Figure 5. Comparison of the catalytic efficiency of HRP and Au NPs. The reaction was carried out in the presence of 0.1 mM TMB and various H2O2 concentrations for 10 min at RT. Concentrations of the natural enzyme and nanozyme were fixed at 100 pM, and the absorbance was measured at 370 nm.

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    Figure 6

    Figure 6. FTIR spectra of biogenic Au NPs synthesized by using P. nepalensis extract.

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    Figure 7

    Figure 7. (A) UV–vis spectrum of biogenic Au NPs-QUBMA-Bov conjugates in comparison with that of Au NPs only (B) kinetic analysis of the catalyzed reaction of 1 mM TMB/6% (v/v) H2O2 in the presence of 100 pM Au NPs and Au NPs-QUBMA-Bov conjugates.

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    Figure 8

    Figure 8. iELISA sensitivity assay using AuNPs-QUBMA-Bov conjugates to detect M. bovis (A) in a laboratory buffer solution (PBS, pH 7.4, Tween-20 0.05% v/v, PBST) and (B) in a solid cheese matrix solution. (C) iELISA specificity assay using AuNPs-QUBMA-Bov conjugates to detect M. bovis in a laboratory buffer (PBST) solution.

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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlemurjI&md5=0399e29b55c770589c1129e38feb3af4
    3. 3
      Slocik, J. M.; Naik, R. R.; Stone, M. O.; Wright, D. W. Viral Templates for Gold Nanoparticle Synthesis. J. Mater. Chem. 2005, 15, 749– 753,  DOI: 10.1039/b413074j
      3
      Viral templates for gold nanoparticle synthesis
      Slocik, Joseph M.; Naik, Rajesh R.; Stone, Morley O.; Wright, David W.
      Journal of Materials Chemistry (2005), 15 (7), 749-753CODEN: JMACEP; ISSN:0959-9428. (Royal Society of Chemistry)
      Viruses present a confined environment and unique protein surface topol. (i.e. polarity, residue charge, and surface relief) for nanoparticle synthesis and are amenable to mol. biol. manipulations. Consequently, we have examd. the cowpea chlorotic mottle viruses of unmodified SubE (yeast), (HRE)-SubE engineered with interior HRE peptide epitopes (AHHAHHAAD), and wild-type as viral templates for the potentiated redn. and symmetry directed synthesis of gold nanoparticles. In the first approach, the viral capsid actively potentiated the redn. of AuCl4- by electron transfer from surface tyrosine residues resulting in a gold nanoparticle decorated viral surface. Viral redn. appeared to be selective for gold as a collection of metal precursor substrates of Ag+, Pt4+, Pd4+, and an insol. AuI complex were not reduced to zero-valent nanoclusters by virus. Alternatively, the viral capsid provided a template for the symmetry directed synthesis of Au0 nanoparticles from a non-reducible gold precursor.
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    4. 4
      Yang, N.; Weihong, L.; Hao, L. Biosynthesis of Au Nanoparticles Using Agricultural Waste Mango Peel Extract and Its in Vitro Cytotoxic Effect on Two Normal Cells. Mater. Lett. 2014, 134, 67– 70,  DOI: 10.1016/j.matlet.2014.07.025
      4
      Biosynthesis of Au nanoparticles using agricultural waste mango peel extract and its in vitro cytotoxic effect on two normal cells
      Yang, Ning; Li, Wei Hong; Hao, Lin
      Materials Letters (2014), 134 (), 67-70CODEN: MLETDJ; ISSN:0167-577X. (Elsevier B.V.)
      Gold nanoparticles (GNPs) were successfully synthesized with the aid of non toxic ecofriendly biol. material namely mango peel ext. The reaction rate for the synthesis process is relatively higher than other plant exts. The GNPs were characterized using UV-vis spectrum, X-ray diffraction (XRD), Transmission electron microscope (TEM) and Fourier-transform IR spectroscopy (FTIR). The results revealed that the biosynthesized GNPs were in the size range of 6.03±2.77 to 18.01±3.67 nm. The biosynthesized GNPs have no biol. cytotoxicity on African green monkey kidney normal cells (CV-1) and Normal human fetal lung fibroblast cells (WI-38), even at a concn. of 160 μg/mL.
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    5. 5
      Raghunandan, D.; Basavaraja, S.; Mahesh, B.; Balaji, S.; Manjunath, S. Y.; Venkataraman, A. Biosynthesis of Stable Polyshaped Gold Nanoparticles from Microwave-Exposed Aqueous Extracellular Anti-Malignant Guava (Psidium Guajava) Leaf Extract. Nanobiotechnology 2009, 5, 34– 41,  DOI: 10.1007/s12030-009-9030-8
      5
      Biosynthesis of Stable Polyshaped Gold Nanoparticles from Microwave-Exposed Aqueous Extracellular Anti-malignant Guava (Psidium guajava) Leaf Extract
      Raghunandan, D.; Basavaraja, S.; Mahesh, B.; Balaji, S.; Manjunath, S. Y.; Venkataraman, A.
      NanoBiotechnology (2009), 5 (1-4), 34-41CODEN: NANOCG; ISSN:1551-1286. (Humana Press Inc.)
      Addn. of microwave-exposed aq. extracellular anti-malignant guava (Psidium guajava) leaf ext. to the aq. gold chloride soln. yielded stable polyshaped gold nanoparticles of high compn. Microwave-assisted route selected for the prepn. of aq. guava leaf ext. was to suppress the enzymic action. The formation of nanoparticles was understood from the UV-visible and x-ray diffraction studies. The size and shape anal. was done using field emission SEM, TEM, and at. force microscopy. Zeta potential expt. shows that the bio-functionalized gold nanoparticles colloidal soln. obtained as above will maintain its stability even after 30 wk of storage. It is obsd. that the flavonoids which are sepd. during microwave heating of extracellular soln. of the guava leaves are responsible for the biosynthesis of gold nanoparticles.
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    6. 6
      Parashar, V.; Parashar, R.; Sharma, B.; Pandey, A. Parthenium Leaf Extract Mediated Synthesis of Silver Nanoparticles: A Novel Approach towards Weed Utilization. Dig. J. Nanomater. Biostructures 2009, 4, 45– 50
      There is no corresponding record for this reference.
    7. 7
      Singh, A. K.; Talat, M.; Singh, D. P.; Srivastava, O. N. Biosynthesis of Gold and Silver Nanoparticles by Natural Precursor Clove and Their Functionalization with Amine Group. J. Nanopart. Res. 2010, 12, 1667– 1675,  DOI: 10.1007/s11051-009-9835-3
      7
      Biosynthesis of gold and silver nanoparticles by natural precursor clove and their functionalization with amine group
      Singh, Ashwani Kumar; Talat, Mahe; Singh, D. P.; Srivastava, O. N.
      Journal of Nanoparticle Research (2010), 12 (5), 1667-1675CODEN: JNARFA; ISSN:1388-0764. (Springer)
      We report a simple and cost effective way for synthesis of metallic nanoparticles (Au and Ag) using natural precursor clove. Au and Ag nanoparticles have been synthesized by reducing the aq. soln. of AuCl4 and AgNO3 with clove ext. One interesting aspect here is that redn. time is quite small (few minutes instead of hours as compared to other natural precursors). We synthesized gold and silver nanoparticles of different shape and size by varying the ratio of AuCl4 and AgNO3 with respect to clove ext., where the dominant component is eugenol. The evolution of Au and Ag nanoparticles from the redn. of different ratios of AuCl4 and AgNO3 with optimized concn. of the clove ext. has been evaluated through monitoring of surface plasmon behavior as a function of time. The redn. of AuCl4 and AgNO3 by eugenol is because of the inductive effect of methoxy and allyl groups which are present at ortho and para positions of proton releasing -OH group as two electrons are released from one mol. of eugenol. This is followed by the formation of resonating structure of the anionic form of eugenol. The presence of methoxy and allyl groups has been confirmed by FTIR. To the best of our knowledge, use of clove as reducing agent, the consequent very short time (minutes instead of hours and without any scavenger) and the elucidation of mechanism of redn. based on FTIR anal. has not been attempted earlier.
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    8. 8
      Shankar, S. S.; Ahmad, A.; Pasricha, R.; Sastry, M. Bioreduction of Chloroaurate Ions by Geranium Leaves and Its Endophytic Fungus Yields Gold Nanoparticles of Different Shapes. J. Mater. Chem. 2003, 13, 1822– 1826,  DOI: 10.1039/b303808b
      8
      Bioreduction of chloroaurate ions by geranium leaves and its endophytic fungus yields gold nanoparticles of different shapes
      Shankar, S. Shiv; Ahmad, Absar; Pasricha, Renu; Sastry, Murali
      Journal of Materials Chemistry (2003), 13 (7), 1822-1826CODEN: JMACEP; ISSN:0959-9428. (Royal Society of Chemistry)
      Development of biol. inspired exptl. processes for the synthesis of nanoparticles is an important branch of nanotechnol. The use of geranium leaves (Pelargonium graveolens) and its endophytic fungus in the extra-cellular synthesis of gold nanoparticles was investigated. Sterilized geranium leaves and an endophytic fungus (Colletotrichum sp.) growing in the leaves were sep. exposed to aq. chloroaurate ions. In both cases, rapid redn. of the metal ions was obsd. and resulted in the formation of stable gold nanoparticles of variable size. In the case of gold nanoparticles synthesized using geranium leaves, the reducing and capping agents appear to be terpenoids while they are identified to be polypeptides/enzymes in the Colletotrichum sp. case. The biogenic gold nanoparticles synthesized using the fungus were essentially spherical in shape, while the particles grown using the leaves exhibited a variety of shapes that included rods, flat sheets and triangles. While the exact reasons for shape variability are not clear at this stage, the possibility of achieving nanoparticle shape control in a host leaf-fungus system is considered.
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    9. 9
      Dubey, S. P.; Lahtinen, M.; Sillanpää, M. Green Synthesis and Characterizations of Silver and Gold Nanoparticles Using Leaf Extract of Rosa Rugosa. Colloids Surf., A 2010, 364, 34– 41,  DOI: 10.1016/j.colsurfa.2010.04.023
      9
      Green synthesis and characterizations of silver and gold nanoparticles using leaf extract of Rosa rugosa
      Dubey, Shashi Prabha; Lahtinen, Manu; Sillanpaa, Mika
      Colloids and Surfaces, A: Physicochemical and Engineering Aspects (2010), 364 (1-3), 34-41CODEN: CPEAEH; ISSN:0927-7757. (Elsevier B.V.)
      Metal nanostructures have unusual physicochem. properties and biol. activities compared to their bulk parent materials. Thus in recent years a no. of phys., chem. and biol. techniques were applied for the development of metal nanoparticles (NP). Here we have synthesized silver and gold nanoparticles (AgNPs and AuNPs) by using leaves exts. of Rosa rugosa. Surface plasmon resonance spectra for silver and gold are obtained at 451 and 578 nm with brown yellow and pink-red color, resp. AgNPs and AuNPs vary in size according to different leaves ext. and metal concn. used for the synthesis. Different instrumental techniques were applied to characterize the synthesized AgNPs and AuNPs viz. UV-vis, TEM, XRD, FTIR, Zetasizer and EDX. The synthesized NPs were mostly spherical (in case of AgNPs) with some triangular and hexagonal (esp. in case of AuNPs) in shapes with an av. size of 12 and 11 nm for AgNPs and AuNPs, resp.
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    10. 10
      Tai, Y.; Chang, L.-W.; Tran, N. T. T.; Tsai, Y.-C.; Fang, J.-Y. One-Step Synthesis of Highly Biocompatible Multi-Shaped Gold Nanostructures with Fruit Extract. IET Nanobiotechnol. 2011, 5, 52– 59,  DOI: 10.1049/iet-nbt.2010.0028
      10
      One-step synthesis of highly biocompatible multi-shaped gold nanostructures with fruit extract
      Tai, Y.; Tran, N. T. T.; Tsai, Y.-C.; Fang, J.-Y.; Chang, L.-W.
      IET Nanobiotechnology (2011), 5 (2), 52-59CODEN: INEACX; ISSN:1751-8741. (Institution of Engineering and Technology)
      In this study, the authors demonstrate the synthesis of various gold nanostructures through a one-step, green and complete bio-modulation approach. Nanoparticles were successfully synthesized by the addn. of gold aq. soln. to fruit exts., including orange, papaya, peach or lemon. The particles were of various shapes and sizes with high abundance, such as sphere, marigold, triangle and hexagon. The biocompatibility of the presented gold nanostructures was examd.; hemolysis tests revealed a non-toxicity result in blood cell uptake of such gold nanostructures. This study opens the exciting possibility of synthesizing various multi-shaped nanoparticles through a simple and green approach, as well as paving the way for future bio-applications.
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    11. 11
      Das, J.; Velusamy, P. Catalytic Reduction of Methylene Blue Using Biogenic Gold Nanoparticles from Sesbania Grandiflora L. J. Taiwan Inst. Chem. Eng. 2014, 45, 2280– 2285,  DOI: 10.1016/j.jtice.2014.04.005
      11
      Catalytic reduction of methylene blue using biogenic gold nanoparticles from Sesbania grandiflora L
      Das, J.; Velusamy, P.
      Journal of the Taiwan Institute of Chemical Engineers (2014), 45 (5), 2280-2285CODEN: JTICA8; ISSN:1876-1070. (Elsevier B.V.)
      In the present investigation, we report the synthesis of gold nanoparticles (AuNPs) using leaf ext. of Sesbania grandiflora. The processes of nucleation and growth of AuNPs were followed by monitoring the absorption spectra during the reaction. UV-vis spectrum of the aq. medium contg. AuNPs showed a peak at around 534 nm. FE-SEM and TEM micrograph anal. of the AuNPs indicated that they were predominantly spherical, well-dispersed within 7-34 nm. The synthesized AuNPs are obsd. to have an excellent catalytic activity on the redn. of methylene blue by S. grandiflora ext. which is confirmed by the decrease in absorbance max. values of methylene blue (MB) with respect to time using UV-vis spectrophotometer and is attributed as an effective catalyst for degrading chem. dyes.
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    12. 12
      Lee, K. X.; Shameli, K.; Miyake, M.; Kuwano, N.; Bt Ahmad Khairudin, N. B.; Bt Mohamad, S. E.; Yew, Y. P. Green Synthesis of Gold Nanoparticles Using Aqueous Extract of Garcinia Mangostana Fruit Peels. J. Nanomater. 2016, 2016, 8489094,  DOI: 10.1155/2016/8489094
      There is no corresponding record for this reference.
    13. 13
      Dauthal, P.; Mukhopadhyay, M. Prunus Domestica Fruit Extract-Mediated Synthesis of Gold Nanoparticles and Its Catalytic Activity for 4-Nitrophenol Reduction. Ind. Eng. Chem. Res. 2012, 51, 13014– 13020,  DOI: 10.1021/ie300369g
      13
      Prunus domestica Fruit Extract-Mediated Synthesis of Gold Nanoparticles and Its Catalytic Activity for 4-Nitrophenol Reduction
      Dauthal, Preeti; Mukhopadhyay, Mausumi
      Industrial & Engineering Chemistry Research (2012), 51 (40), 13014-13020CODEN: IECRED; ISSN:0888-5885. (American Chemical Society)
      Gold nanoparticles (Au-NPs) were synthesized at room temp. using Prunus domestica (plum) fruit ext. as reducing agent. The UV-visible absorption spectrum showed a characteristic optical absorption peak of Au-NPs at 543 nm. The X-ray diffraction pattern suggested the formation and crystallinity of Au-NPs. Spherical Au-NPs synthesized with an av. particle size of 20 ± 6 nm were confirmed by transmission electron microscopy. Fourier transform IR spectroscopy anal. supported the role of water-sol. polyols and amino acids of plum fruit ext. for bioredn. and stabilization of Au-NPs. The catalytic activity of Au-NPs was investigated for 4-nitrophenol (4-NP) redn. using UV-visible absorption spectroscopy. Biosynthesized Au-NPs showed a dose-dependent catalytic activity. Catalytic redn. followed pseudo-first-order kinetics with respect to 4-NP.
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    14. 14
      Kumar, V.; Bano, D.; Singh, D. K.; Mohan, S.; Singh, V. K.; Hasan, S. H. Size-Dependent Synthesis of Gold Nanoparticles and Their Peroxidase-Like Activity for the Colorimetric Detection of Glutathione from Human Blood Serum. ACS Sustainable Chem. Eng. 2018, 6, 7662– 7675,  DOI: 10.1021/acssuschemeng.8b00503
      14
      Size-Dependent Synthesis of Gold Nanoparticles and Their Peroxidase-Like Activity for the Colorimetric Detection of Glutathione from Human Blood Serum
      Kumar, Vijay; Bano, Daraksha; Singh, Devendra K.; Mohan, Sweta; Singh, Vikas Kumar; Hasan, Syed Hadi
      ACS Sustainable Chemistry & Engineering (2018), 6 (6), 7662-7675CODEN: ASCECG; ISSN:2168-0485. (American Chemical Society)
      An eco-friendly and economic efficient approach has been developed for the photoinduced synthesis of more stable AuNPs using an aq. ext. of Croton bonplandianum (AEC) as a reducing and capping agent. The reaction mixt. of AEC and HAuCl4.xH2O, when exposed to sunlight turned purple which primarily confirmed the biosynthesis of AuNPs. The biosynthesis was monitored using UV-vis spectroscopy which exhibited a sharp SPR band at 530 nm after 16 min of sunlight exposure. The parameters affecting the synthesis of AuNPs such as sunlight exposure, AEC inoculum dose, and HAuCl4.xH2O concn. were also optimized. The HR-TEM study revealed that, as the metal ion concns. increased, the av. size and anisotropic nature of the AuNPs increased. The x-ray diffraction pattern of AuNPs synthesized confirmed the formation of face-centered cubic crystal lattice of metallic gold. The involvement of polyphenolics in the synthesis of AuNPs was confirmed by comparing the FTIR anal. of pure tannic acid, AEC, and pre- and postannealed AuNPs. The XPS anal. corroborated the presence of two individual peaks attributed to the Au 4f7/2 and Au 4f5/2 binding energies which corresponded to the presence of metallic gold. The AuNPs thus obtained showed peroxidase-like mimicking activity which catalyzed the oxidn. of TMB to oxTMB with the development of blue color and absorption spectra at 652 nm. However, the presence of GSH caused further redn. of oxTMB. This detection expt. showed an excellent linear relationship between 1 and 40 μM with a limit of detection of 0.013 μM. In addn. to this, the significant recovery of GSH from human blood serum advocated that the developed system was simple and sensitive for the real sample anal.
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    15. 15
      Li, R. S.; Liu, H.; Chen, B. B.; Zhang, H. Z.; Huang, C. Z.; Wang, J. Stable Gold Nanoparticles as a Novel Peroxidase Mimic for Colorimetric Detection of Cysteine. Anal. Methods 2016, 8, 2494– 2501,  DOI: 10.1039/c6ay00367b
      15
      Stable gold nanoparticles as a novel peroxidase mimic for colorimetric detection of cysteine
      Li, Rong Sheng; Liu, Hai; Chen, Bin Bin; Zhang, Hong Zhi; Huang, Cheng Zhi; Wang, Jian
      Analytical Methods (2016), 8 (11), 2494-2501CODEN: AMNEGX; ISSN:1759-9679. (Royal Society of Chemistry)
      In this work, a novel method for colorimetric detection of cysteine was proposed based on the inhibition of peroxidase-like behavior of AuNPs. Kiwi juice prepd. gold nanoparticles presented strong catalytic activity, which could catalyze H2O2-TMB to generate a blue product, resulting in strong absorption at 650 nm. In HAc-NaAc (pH 4.0) buffer, cysteine induced the aggregation of gold nanoparticles through covalent Au-S bonds and electrostatic as well as hydrogen bonding, leading to the redn. of the catalytic activity of gold nanoparticles with a weakened blue color product and a lower absorbance. Under optimal conditions, the concns. of cysteine were proportional to the degree of reduced absorbance. This approach offers a new, simple, sensitive and selective assay for cysteine.
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    16. 16
      Il Kim, M.; Park, C. Y.; Seo, J. M.; Kang, K. S.; Park, K. S.; Kang, J.; Hong, K. S.; Choi, Y.; Lee, S. Y.; Park, J. P.; Park, H. G.; Park, T. J. J. In Situ Biosynthesis of a Metal Nanoparticle Encapsulated in Alginate Gel for Imageable Drug-Delivery System. ACS Appl. Mater. Interfaces 2021, 13, 36697– 36708,  DOI: 10.1021/acsami.1c02286
      16
      In Situ Biosynthesis of a Metal Nanoparticle Encapsulated in Alginate Gel for Imageable Drug-Delivery System
      Il Kim, Moon; Park, Chan Yeong; Seo, Ji Min; Kang, Kyoung Suk; Park, Ki Soo; Kang, Jongeun; Hong, Kwan Soo; Choi, Yoojin; Lee, Sang Yup; Park, Jong Pil; Park, Hyun Gyu; Park, Tae Jung
      ACS Applied Materials & Interfaces (2021), 13 (31), 36697-36708CODEN: AAMICK; ISSN:1944-8244. (American Chemical Society)
      Development of drug-delivery systems that allow simultaneous in vivo imaging has gained much interest. We report a novel strategy to encapsulate metal nanoparticles (NPs) within alginate gel for in vivo imaging. The cell lysate of recombinant Escherichia coli strain, expressing Arabidopsis thaliana phytochelatin synthase and Pseudomonas putida metallothionein genes, was encapsulated within the alginate gel. Incubation of alginate gel with metal ion precursors followed by UV irradn. resulted in the synthesis of high concns. of metal NPs, such as Au, Ag, CdSe, and EuSe NPs, within the gel. The alginate gel with metal NPs was used as a drug-delivery system by further co-encapsulating doxorubicin and rifampicin, the release of which was made to be pH-dependent. This system can be conveniently and safely used for in vitro and in vivo bioimaging, enabled by the metal NPs formed within the gel matrix without using toxic reducing reagents or surfactants.
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    17. 17
      Agrahar-Murugkar, D.; Subbulakshmi, G. Nutritive Values of Wild Edible Fruits, Berries, Nuts, Roots and Spices Consumed by the Khasi Tribes of India. Ecol. Food Nutr. 2005, 44, 207– 223,  DOI: 10.1080/03670240590953025
      There is no corresponding record for this reference.
    18. 18
      Chaudhuri, D.; Ghate, N. B.; Panja, S.; Das, A.; Mandal, N. Wild Edible Fruit of Prunus Nepalensis Ser. (Steud), a Potential Source of Antioxidants, Ameliorates Iron Overload-Induced Hepatotoxicity and Liver Fibrosis in Mice. PLoS One 2015, 10, e0144280  DOI: 10.1371/journal.pone.0144280
      18
      Wild edible fruit of Prunus nepalensis Ser. (Steud), a potential source of antioxidants, ameliorates iron overload-induced hepatotoxicity and liver fibrosis in mice
      Chaudhuri, Dipankar; Ghate, Nikhil Baban; Das, Sourav Panja Abhishek; Manda, Nripendranath
      PLoS One (2015), 10 (12), e0144280/1-e0144280/20CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      The antioxidant and restoration potentials of hepatic injury by Prunus nepalensis Ser. (Steud), a wild fruit plant from the Northeastern region of India, were investigated. The fruit ext. (PNME) exhibited excellent antioxidant and reducing properties and also scavenged the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (IC50 = 30.92 ± 0.40 μg/mL). PNME demonstrated promising scavenging potency, as assessed by the scavenging of different reactive oxygen and nitrogen species. Moreover, the ext. revealed an exceptional iron chelation capacity with an IC50 of 25.64 ± 0.60 μg/mL. The ext. induced significant improvement of hepatic injury and liver fibrosis against iron overload induced hepatotoxicity in mice in a dose-dependent manner, and this effect was supported by different histopathol. studies. The phytochem. constitutions and their identification by HPLC confirmed the presence of purpurin, tannic acid, Me gallate, reserpine, gallic acid, ascorbic acid, catechin and rutin. The identified compds. were investigated for their individual radical scavenging and iron chelation activity; some compds. exhibited excellent radical scavenging and iron chelation properties, but most were toxic towards normal cells (WI-38). On the other hand, crude PNME was found to be completely nontoxic to normal cells, suggesting its feasibility as a safe oral drug. The above study suggests that different phytochems. in PNME contributed to its free radical scavenging and iron chelation activity; however, further studies are required to det. the pathway in which PNME acts to treat iron-overload diseases.
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    19. 19
      Lou-Franco, J.; Das, B.; Elliott, C.; Cao, C. Gold Nanozymes: From Concept to Biomedical Applications. Nano-Micro Lett. 2021, 13, 10,  DOI: 10.1007/s40820-020-00532-z
      There is no corresponding record for this reference.
    20. 20
      Wang, X.; Hu, Y.; Wei, H. Nanozymes in Bionanotechnology: From Sensing to Therapeutics and Beyond. Inorg. Chem. Front. 2016, 3, 41– 60,  DOI: 10.1039/c5qi00240k
      20
      Nanozymes in bionanotechnology: from sensing to therapeutics and beyond
      Wang, Xiaoyu; Hu, Yihui; Wei, Hui
      Inorganic Chemistry Frontiers (2016), 3 (1), 41-60CODEN: ICFNAW; ISSN:2052-1553. (Royal Society of Chemistry)
      In the past few decades, researchers have developed lots of artificial enzymes with various materials to mimic the structures and functions of natural enzymes. Recently, nanozymes, nanomaterials with enzyme-like characteristics, are emerging as novel artificial enzymes, and attracting researchers' enormous interest. Remarkable advances have been made in the area of nanozymes due to their unique properties compared with natural enzymes and classic artificial enzymes. Until now, lots of nanomaterials have been studied to mimic various natural enzymes for wide applications. To highlight the recent progress of nanozymes (esp. in bionanotechnol.), here we discuss the diverse applications of nanozymes, which range from sensing, imaging, and therapeutics, to logic gates, pollutant removal, water treatment, etc. Finally, we address the current challenges facing nanozyme research as well as possible directions to fulfill their great potential in future.
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    21. 21
      Das, B.; Franco, J. L.; Logan, N.; Balasubramanian, P.; Kim, M. I.; Cao, C. Nanozymes in Point-of-Care Diagnosis: An Emerging Futuristic Approach for Biosensing. Nano-Micro Letters 2021, 13, 193,  DOI: 10.1007/s40820-021-00717-0
      21
      Nanozymes in point-of-care diagnosis: an emerging futuristic approach for biosensing
      Das, Bhaskar; Franco, Javier Lou; Logan, Natasha; Balasubramanian, Paramasivan; Kim, Moon Il; Cao, Cuong
      Nano-Micro Letters (2021), 13 (), 193CODEN: NLAEBV; ISSN:2150-5551. (Nano-Micro Letters)
      Nanomaterial-based artifcial enzymes (or nanozymes) have attracted great attention in the past few years owing to their capability not only to mimic functionality but also to overcome the inherent drawbacks of the natural enzymes. Numerous advantages of nanozymes such as diverse enzyme-mimicking activities, low cost, high stability, robustness, unique surface chem., and ease of surface tunability and biocompatibility have allowed their integration in a wide range of biosensing applications. Several metal, metal oxide, metal-org. framework-based nanozymes have been exploited for the development of biosensing systems, which present the potential for point-of-care anal. To highlight recent progress in the feld, in this review, more than 260 research articles are discussed systematically with suitable recent examples, elucidating the role of nanozymes to reinforce, miniaturize, and improve the performance of point-of-care diagnostics addressing the ASSURED (afordable, sensitive, specifc, user-friendly, rapid and robust, equipment-free and deliverable to the end user) criteria formulated by World Health Organization. The review reveals that many biosensing strategies such as electrochem., colorimetric, fuorescent, and immunol. sensors required to achieve the ASSURED stds. can be implemented by using enzyme-mimicking activities of nanomaterials as signal producing components. However, basic system functionality is still lacking. Since the enzyme-mimicking properties of the nanomaterials are dictated by their size, shape, compn., surface charge, surface chem. as well as external parameters such as pH or temp., these factors play a crucial role in the design and function of nanozyme-based point-of-care diagnostics. Therefore, it requires a deliberate exertion to integrate various parameters for truly ASSURED solns. to be realized. This review also discusses possible limitations and research gaps to provide readers a brief scenario of the emerging role of nanozymes in state-of-the-art POC diagnosis system development for futuristic biosensing applications.
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    22. 22
      Taylor, G. M.; Worth, D. R.; Palmer, S.; Jahans, K.; Hewinson, R. G. Rapid Detection of Mycobacterium Bovis DNA in Cattle Lymph Nodes with Visible Lesions Using PCR. BMC Vet. Res. 2007, 3, 12,  DOI: 10.1186/1746-6148-3-12
      22
      Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR
      Taylor G Michael; Worth Danny R; Palmer Si; Jahans Keith; Hewinson R Glyn
      BMC veterinary research (2007), 3 (), 12 ISSN:.
      BACKGROUND: We have evaluated a sensitive screening assay for Mycobacterium tuberculosis (MTB) complex organisms and a specific assay for detecting Mycobacterium bovis DNA in lymph nodes taken from cattle with evidence of bovine tuberculosis. Underlying these series of experiments was the need for a versatile DNA extraction protocol which could handle tissue samples and with the potential for automation.The target for the screening assay was the multi-copy insertion element IS1081, present in 6 copies in the MTB complex. For confirmation of M. bovis we used primers flanking a specific deletion in the genome of M. bovis known as region of difference 4 (RD4). The sensitivity and specificity of these PCRs has been tested on genomic DNA from MTB complex reference strains, mycobacteria other than tuberculosis (MOTT), spiked samples and on clinical material. RESULTS: The minimum detection limits of the IS1081 method was < I genome copy and for the RD4 PCR was 5 genome copies. Both methods can be readily adapted for quantitative PCR with the use of SYBR Green intercalating dye on the RotorGene 3000 platform (Corbett Research). Initial testing of field samples of bovine lymph nodes with visible lesions (VL, n = 109) highlighted two shortfalls of the molecular approach. Firstly, comparison of IS1081 PCR with the "gold standard" of culture showed a sensitivity of approximately 70%. The sensitivity of the RD4 PCR method was 50%. Secondly, the success rate of spoligotyping applied directly to clinical material was 51% compared with cultures. A series of further experiments indicated that the discrepancy between sensitivity of detection found with purified mycobacterial DNA and direct testing of field samples was due to limited mycobacterial DNA recovery from tissue homogenates rather than PCR inhibition. The resilient mycobacterial cell wall, the presence of tissue debris and the paucibacillary nature of some cattle VL tissue may all contribute to this observation. Any of these factors may restrict application of other more discriminant typing methods.A simple means of increasing the efficiency of mycobacterial DNA recovery was assessed using a further pool of 95 cattle VL. Following modification of the extraction protocol, detection rate with the IS1081 and RD4 methods increased to 91% and 59% respectively. CONCLUSION: The IS1081 PCR is a realistic screening method for rapid identification of positive cases but the sensitivity of single copy methods, like RD4 and also of spoligotyping will need to be improved to make these applicable for direct testing of tissue extracts.
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    23. 23
      Bolaños, C. A. D.; Paula, C. L. d.; Guerra, S. T.; Franco, M. M. J.; Ribeiro, M. G. Diagnosis of Mycobacteria in Bovine Milk: An Overview. Rev. Inst. Med. Trop. Sao Paulo 2017, 59, e40  DOI: 10.1590/S1678-9946201759040
      23
      Diagnosis of mycobacteria in bovine milk: an overview
      Bolanos, Carmen Alicia Daza; Lechinski de Paula, Carolina; Guerra, Simony Trevizan; Franco, Marilia Masello Junqueira; Ribeiro, Marcio Garcia
      Revista do Instituto de Medicina Tropical de Sao Paulo (2017), 59 (), e40/1-e40/13CODEN: RMTSAE; ISSN:1678-9946. (Instituto de Medicina Tropical de Sao Paulo)
      Tuberculosis remains as the world's biggest threat. In 2014, human tuberculosis ranked as a major infectious disease by the first time, overcoming HIV death rates. Bovine tuberculosis is a chronic disease of global distribution that affects animals and can be transmitted to humans by the consumption of raw milk, representing a serious public health concern. Despite the efforts of different countries to control and eradicate bovine tuberculosis, the high neg. economic impact on meat and milk prodn. chains remains, given the decreased prodn. efficiency (approx. 25%), the high no. of condemned carcasses, and increased animal culling rates. This scenario has motivated the establishment of official programs based on regulations and diagnostic procedures. Although Mycobacterium tuberculosis and Mycobacterium bovis are the major pathogenic species to humans and bovines, resp., nontuberculous mycobacteria within the Mycobacterium genus have become increasingly important in recent decades due to human infections, including the ones that occur in immunocompetent people. Diagnosis of mycobacteria can be performed by microbiol. culture from tissue samples (lymph nodes, lungs) and secretions (sputum, milk). In general, these pathogens demand special nutrient requirements for isolation/growth, and the use of selective and rich culture media. Indeed, within these genera, mycobacteria are classified as either fast- or slow-growth microorganisms. Regarding the latter ones, incubation times can vary from 45 to 90 days. Although microbiol. culture is still considered the gold std. method for diagnosis, mol. approaches have been increasingly used. We describe here an overview of the diagnosis of Mycobacterium species in bovine milk.
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    24. 24
      Dean, A. S.; Forcella, S.; Olea-Popelka, F.; Idrissi, A. E.; Glaziou, P.; Benyahia, A.; Mumford, E.; Erlacher-Vindel, E.; Gifford, G.; Lubroth, J.; Raviglione, M.; Fujiwara, P. A Roadmap for Zoonotic Tuberculosis: A One Health Approach to Ending Tuberculosis. Lancet Infect. Dis. 2018, 18, 137– 138,  DOI: 10.1016/S1473-3099(18)30013-6
      24
      A roadmap for zoonotic tuberculosis: a One Health approach to ending tuberculosis
      Dean Anna S; Forcella Simona; Olea-Popelka Francisco; Idrissi Ahmed El; Lubroth Juan; Glaziou Philippe; Raviglione Mario; Benyahia Amina; Mumford Elizabeth; Erlacher-Vindel Elisabeth; Gifford Glen; Fujiwara Paula
      The Lancet. Infectious diseases (2018), 18 (2), 137-138 ISSN:.
      There is no expanded citation for this reference.
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    25. 25
      Ji, T.; Muenker, M. C.; Papineni, R. V. L.; Harder, J. W.; Vizard, D. L.; Mclaughlin, W. E. Increased Sensitivity in Antigen Detection with Fluorescent Latex Nanosphere-IgG Antibody Conjugates. Bioconjugate Chem. 2010, 21, 427– 435,  DOI: 10.1021/bc900295v
      25
      Increased Sensitivity in Antigen Detection with Fluorescent Latex Nanosphere-IgG Antibody Conjugates
      Ji, Tao; Muenker, M. Catherine; Papineni, Rao V. L.; Harder, John W.; Vizard, Douglas L.; McLaughlin, William E.
      Bioconjugate Chemistry (2010), 21 (3), 427-435CODEN: BCCHES; ISSN:1043-1802. (American Chemical Society)
      IgG antibodies were conjugated to Kodak X-Sight nanospheres to develop fluorescent-labeled antibodies using two different synthetic routes: one involving the DTT redn. method, and the other involving Traut's Reagent modification method. These two methods result in different conjugation efficiencies and different performances in antigen detection. Western blotting shows that the nanosphere-IgG antibody conjugates synthesized using the DTT redn. method are more immunospecific than the conjugates synthesized using Traut's Reagent modification method. In addn., the conjugates synthesized using DTT redn. also show higher antigen detection sensitivity than other com. available fluorescent-IgG antibody conjugates, including Alexa Fluor, Qdot, and CyDye conjugates.
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    26. 26
      Stewart, L. D.; McNair, J.; McCallan, L.; Thompson, S.; Kulakov, L. A.; Grant, I. R. Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium Bovis. J. Clin. Microbiol. 2012, 50, 1598– 1605,  DOI: 10.1128/JCM.05747-11
      26
      Production and evaluation of antibodies and phage display-derived peptide ligands for immunomagnetic separation of Mycobacterium bovis
      Stewart, Linda D.; McNair, James; McCallan, Lyanne; Thompson, Suzan; Kulakov, Leonid A.; Grant, Irene R.
      Journal of Clinical Microbiology (2012), 50 (5), 1598-1605CODEN: JCMIDW; ISSN:0095-1137. (American Society for Microbiology)
      This study describes the development and optimization of an immunomagnetic sepn. (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extd. surface antigens of such cells were used to produce M. bovis-specific polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-specific peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concn. range, 10 to 105 CFU/mL) was evaluated by IMS combined with an M. bovis-specific touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic labs. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrixes, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/mL of lymph node tissue homogenate for IMS-PCR and IMS-culture, resp.). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.
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    27. 27
      Stewart, L. D.; Tort, N.; Meakin, P.; Argudo, J. M.; Nzuma, R.; Reid, N.; Delahay, R. J.; Ashford, R.; Montgomery, W. I.; Grant, I. R. Development of a Novel Immunochromatographic Lateral Flow Assay Specific for Mycobacterium Bovis Cells and Its Application in Combination with Immunomagnetic Separation to Test Badger Faeces. BMC Vet. Res. 2017, 13, 131,  DOI: 10.1186/s12917-017-1048-x
      27
      Development of a novel immunochromatographic lateral flow assay specific for Mycobacterium bovis cells and its application in combination with immunomagnetic separation to test badger faeces
      Stewart, Linda D.; Tort, Nuria; Meakin, Paul; Argudo, Jose M.; Nzuma, Ruramayi; Reid, Neil; Delahay, Richard J.; Ashford, Roland; Montgomery, W. Ian; Grant, Irene R.
      BMC Veterinary Research (2017), 13 (), 131/1-131/12CODEN: BVRMA9; ISSN:1746-6148. (BioMed Central Ltd.)
      The European badger is an important wildlife reservoir of Mycobacterium bovis implicated in the spread of bovine tuberculosis in the United Kingdom and Ireland. Infected badgers are known to shed M. bovis in their urine and faeces, which may contaminate the environment. To aid bovine tuberculosis control efforts novel diagnostic tests for detecting infected and shedding badgers are needed. We proposed development of a novel, rapid immunochromatog. lateral flow device (LFD) as a non-invasive test to detect M. bovis cells in badger faeces. Its application in combination with immunomagnetic sepn. (IMS) to detect Mycobacterium bovis cells in badger faeces is reported here. A novel prototype LFD for M. bovis cells was successfully developed, with unique specificity for M. bovis and a limit of detection 50% (LOD50%) of 1.7× 104M. bovis cells/mL. When IMS was employed to selectively capture and conc. M. bovis cells from badger faeces prior to LFD testing, the LOD50% of the IMS-LFD assay was 2.8 × 105M. bovis cells/mL faecal homogenate. Faeces samples collected from latrines at badger setts in a region of endemic bovine tuberculosis infection were tested; 78 (18%) of 441 samples tested IMS-LFD assay pos., whereas 140 (32%) tested IMS-qPCR pos. (Kappa agreement -0.009 ± 0.044, p = 0.838). Subsequently, when 130 faeces samples from live captured, or captive, badgers of known infection status (on the basis of StatPak, interferon-γ and/or culture results) were tested, the IMS-LFD assay had higher relative diagnostic specificity (Sp 0.926), but poorer relative diagnostic sensitivity (Se 0.081), than IMS-qPCR (Sp 0.706, Se 0.581) and IMS-culture (Sp 0.794, Se 0.436). The novel IMS-LFD assay, although very specific for M. bovis, has low anal. sensitivity (indicated by the LOD50%) and would only detect badgers shedding high nos. of M. bovis (>104-5 cells/g) in their faeces. The novel LFD would, therefore, have limited value as a non-invasive test for badger TB surveillance purposes but it may have value for alternative veterinary diagnostic applications.
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    28. 28
      Long, G. L.; Winefordner, J. D. Limit of Detection. A Closer Look at the IUPAC Definition. Anal. Chem. 1983, 55, 712A– 724A,  DOI: 10.1021/ac00258a724
      28
      Limit of detection. A closer look at the IUPAC definition
      Long, Gary L.; Winefordner, J. D.
      Analytical Chemistry (1983), 55 (7), 712A-714A, 716A, 718A, 720A, 722A, 724ACODEN: ANCHAM; ISSN:0003-2700.
      The statistical meaning of detection limit values in trace anal. is examd. in a format consistent with the IUPAC definition. Various methods of calcg. detection limit values are reviewed.
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    29. 29
      International Organization for Standardization. Microbiology of Food and Animal Feeding Stuffs: Horizontal Method for the Detection of Salmonella Spp. Detection of Salmonella Spp. in Animal Faeces and in Environmental Samples from the Primary Production Stage , 2007.
      There is no corresponding record for this reference.
    30. 30
      Mishra, P.; Ray, S.; Sinha, S.; Das, B.; Khan, M. I.; Behera, S. K.; Yun, S.-I.; Tripathy, S. K.; Mishra, A. Facile Bio-Synthesis of Gold Nanoparticles by Using Extract of Hibiscus Sabdariffa and Evaluation of Its Cytotoxicity against U87 Glioblastoma Cells under Hyperglycemic Condition. Biochem. Eng. J. 2016, 105, 264– 272,  DOI: 10.1016/j.bej.2015.09.021
      30
      Facile bio-synthesis of gold nanoparticles by using extract of Hibiscus sabdariffa and evaluation of its cytotoxicity against U87 glioblastoma cells under hyperglycemic condition
      Mishra, Pratik; Ray, Sambit; Sinha, Sayantan; Das, Bhaskar; Khan, Md. Imran; Behera, Susant K.; Yun, Soon-Il; Tripathy, Suraj K.; Mishra, Amrita
      Biochemical Engineering Journal (2016), 105 (Part_A), 264-272CODEN: BEJOFV; ISSN:1369-703X. (Elsevier B.V.)
      In the present paper, a facile synthesis of gold nanoparticles is reported with leaf and stem ext. of Hibiscus sabdariffa. Structural features of as synthesized nanoparticles are characterized by UV-vis spectroscopy, XRD, FTIR, and XPS. Morphol. of the above synthesized gold nanoparticles is investigated by electron microscopy. The stability of the nanoparticles is studied in different concns. of glucose which suggested their possible application under hyperglycemic condition. As synthesized nanoparticles has shown selective toxicity towards U87 glioblastoma multiforme cell line under normal and hyperglycemic condition, indicating their potential to be used in the development of value-added products in the biochem. industries. The possible mode of activity of the above nanoparticles has been studied by in vitro mol. techniques.
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    31. 31
      McVey, C.; Logan, N.; Thanh, N. T. K.; Elliott, C.; Cao, C. Unusual Switchable Peroxidase-Mimicking Nanozyme for the Determination of Proteolytic Biomarker. Nano Res. 2019, 12, 509– 516,  DOI: 10.1007/s12274-018-2241-3
      31
      Unusual switchable peroxidase-mimicking nanozyme for the determination of proteolytic biomarker
      McVey, Claire; Logan, Natasha; Thanh, Nguyen T. K.; Elliott, Christopher; Cao, Cuong
      Nano Research (2019), 12 (3), 509-516CODEN: NRAEB5; ISSN:1998-0000. (Springer GmbH)
      Detection of enzyme biomarkers originating from either bio-fluids or contaminating microorganisms is of utmost importance in clin. diagnostics and food safety. Herein, we present a simple, low-cost and easy-to-use sensing approach based on the switchable peroxidase-mimicking activity of plasmonic gold nanoparticles (AuNPs) that can catalyze for the oxidn. of 3,3',5'5-tetramethylbenzidine (TMB) for the detn. of protease enzyme. The AuNP surface is modified with casein, showing dual functionalities. The first function of the coating mol. is to suppress the intrinsic peroxidase-mimicking activity of AuNPs by up to 77.1%, due to surface shielding effects. Secondly, casein also functions as recognition sites for the enzyme biomarker. In the presence of protease, the enzyme binds to and catalyzes the degrdn. of the coating layer on the AuNP surface, resulting in the recovery of peroxidase-mimicking activity. This is shown visually in the development of a blue colored product (oxidized TMB) or spectroscopically as an increase in absorbance at 370 and 650 nm. This mechanism allows for the detection of protease at 44 ng·mL-1 in 90 min. The nanosensor circumvents issues assocd. with current methods of detection in terms of ease of use, compatibility with point-of-care testing, low-cost prodn. and short anal. time. The sensing approach has also been applied for the detection of protease spiked in ultra-heat treated (UHT) milk and synthetic human urine samples at a limit of detection of 490 and 176 ng·mL-1, resp., showing great potential in clin. diagnostics, food safety and quality control.[Figure not available: see fulltext.].
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    32. 32
      Wang, S.; Chen, W.; Liu, A.-L.; Hong, L.; Deng, H.-H.; Lin, X.-H. Comparison of the Peroxidase-like Activity of Unmodified, Amino-Modified, and Citrate-Capped Gold Nanoparticles. ChemPhysChem 2012, 13, 1199– 1204,  DOI: 10.1002/cphc.201100906
      32
      Comparison of the Peroxidase-Like Activity of Unmodified, Amino-Modified, and Citrate-Capped Gold Nanoparticles
      Wang, Sheng; Chen, Wei; Liu, Ai-Lin; Hong, Lei; Deng, Hao-Hua; Lin, Xin-Hua
      ChemPhysChem (2012), 13 (5), 1199-1204CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The origin of the peroxidase-like activity of gold nanoparticles and the impact of surface modification were studied. Furthermore, some influencing factors, such as fabrication process, redox property of the modifier, and charge property of the substrate, were studied. Compared to amino-modified or citrate-capped gold nanoparticles, unmodified gold nanoparticles show significantly higher catalytic activity toward peroxidase substrates, i.e., the superficial gold atoms are a contributing factor to the obsd. peroxidase-like activity. The different catalytic activities of amino-modified and citrate-capped gold nanoparticles toward 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) show that the charge characteristics of the nanoparticles and the substrate also play an important role in the catalytic reactions.
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    33. 33
      Arnao, M. B.; Acosta, M.; del Rio, J. A. F.; García-Cánovas, F. Inactivation of Peroxidase by Hydrogen Peroxide and Its Protection by a Reductant Agent. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1990, 1038, 85– 89,  DOI: 10.1016/0167-4838(90)90014-7
      33
      Inactivation of peroxidase by hydrogen peroxide and its protection by a reductant agent
      Arnao, M. B.; Acosta, M.; Del Rio, J. A.; Garcia-Canovas, F.
      Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology (1990), 1038 (1), 85-9CODEN: BBAEDZ; ISSN:0167-4838.
      Hydrogen peroxide, the oxidant substrate of peroxidase, is also an inactivating agent of this enzyme. The reductant substrates protect the enzyme from the inactivating process. A reaction mechanism is proposed, in which 2 competitive routes exist for Compd. I of peroxidase; one catalytic and one inactivating. The anal. soln. produced at the end of the reaction supports the proposed mechanism and shows the dependence between the no. of turnovers of the enzyme and the ratio of both substrates.
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    34. 34
      Drozd, M.; Pietrzak, M.; Parzuchowski, P.; Mazurkiewicz-Pawlicka, M.; Malinowska, E. Peroxidase-like Activity of Gold Nanoparticles Stabilized by Hyperbranched Polyglycidol Derivatives over a Wide PH Range. Nanotechnology 2015, 26, 495101,  DOI: 10.1088/0957-4484/26/49/495101
      34
      Peroxidase-like activity of gold nanoparticles stabilized by hyperbranched polyglycidol derivatives over a wide pH range
      Drozd, Marcin; Pietrzak, Mariusz; Parzuchowski, Pawel; Mazurkiewicz-Pawlicka, Marta; Malinowska, Elzbieta
      Nanotechnology (2015), 26 (49), 495101/1-495101/15CODEN: NNOTER; ISSN:1361-6528. (IOP Publishing Ltd.)
      The aim of this work was to carry out comparative studies on the peroxidase-like activity of gold nanoparticles (AuNPs) stabilized with low mol. wt. hyperbranched polyglycidol (HBPG-OH) and its deriv. modified with maleic acid residues (HBPG-COOH). The influence of the stabilizer to gold precursor ratio on the size and morphol. of nanoparticles obtained was checked, and prepd. nanoparticles were characterized by means of transmission electron microscopy and UV-Vis spectroscopy. The results indicated the divergent effect of increasing the concn. of stabilizers (HBPG-OH or HBPG-COOH) on the size of the nanostructures obtained. The gold nanoparticles obtained were characterized as having intrinsic peroxidase-like activity and the mechanism of catalysis in acidic and alk. mediums was consistent with the std. Michaelis-Menten kinetics, revealing a strong affinity of AuNPs with 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3, 3', 5, 5'-tetramethylbenzidine (TMB), and significantly lower affinity towards phenol. By comparing the kinetic parameters, a negligible effect of polymeric ligand charge on activity against various types of substrates (anionic or cationic) was indicated. The superiority of steric stabilization via the application of tested low-wt. hyperbranched polymers over typical stabilizers in preventing salt-induced aggregation and maintaining high catalytic activity in time was proved. The applied hyperbranched stabilizers provide a good tool for manufg. gold-based nanozymes, which are highly stable and active over a wide pH range.
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    35. 35
      Zheng, C.; Ke, W.; Yin, T.; An, X. Intrinsic Peroxidase-like Activity and the Catalytic Mechanism of Gold@carbon Dots Nanocomposites. RSC Adv. 2016, 6, 35280– 35286,  DOI: 10.1039/c6ra01917j
      35
      Intrinsic peroxidase-like activity and the catalytic mechanism of gold@carbon dots nanocomposites
      Zheng, Cui; Ke, Wenjing; Yin, Tianixiang; An, Xueqin
      RSC Advances (2016), 6 (42), 35280-35286CODEN: RSCACL; ISSN:2046-2069. (Royal Society of Chemistry)
      Well-dispersed AuNPs@CDs nanocomposites were successfully synthesized from the redn. of chloroauric acid by carbon dots (CDs) at room temp. The as-prepd. AuNPs@CDs were characterized by transmission electron microscopy (TEM), Fourier transform IR spectroscopy (FTIR), Raman spectroscopy and XPS, and the results indicate AuNPs@CDs nanocomposites with a core-shell structure. The AuNPs@CDs are found to possess intrinsic peroxidase-like activity, and the catalytic activity is higher than that of AuNPs. The mechanism of peroxidase-like activity of AuNPs@CDs was investigated by fluorescence spectroscopy, ESR and cyclic voltammetry, and it is found that the mechanism could be ascribed to facilitating the electron transfer between TMB and H2O2.
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    36. 36
      Drozd, M.; Pietrzak, M.; Parzuchowski, P. G.; Malinowska, E. Pitfalls and Capabilities of Various Hydrogen Donors in Evaluation of Peroxidase-like Activity of Gold Nanoparticles. Anal. Bioanal. Chem. 2016, 408, 8505– 8513,  DOI: 10.1007/s00216-016-9976-z
      36
      Pitfalls and capabilities of various hydrogen donors in evaluation of peroxidase-like activity of gold nanoparticles
      Drozd, Marcin; Pietrzak, Mariusz; Parzuchowski, Pawel G.; Malinowska, Elzbieta
      Analytical and Bioanalytical Chemistry (2016), 408 (29), 8505-8513CODEN: ABCNBP; ISSN:1618-2642. (Springer)
      Catalytic nanomaterials, widely used as substitutes of peroxidase, exhibit unique properties, which are unattainable for native enzymes. However, their activity is usually examd. by means of substrates developed and methods standardized for horseradish peroxidase (HRP). The aim of the presented work was to det. the scope of usefulness of chromogenic substrates for gold nanoparticle (AuNP) activity studies under conditions which significantly extend beyond the activity range of a native HRP. The applicability of chromogens such as 3,3'5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) beyond the typical range of pH, and for the samples of high concn. of hydrogen peroxide was examd. The conducted research confirmed the usefulness of ABTS and TMB in acidic media (pH 2.5-3.5). At the same time, potential interferences from chloride anion, unobservable for HRP-based assays, were indicated. Moreover, a no. of potentially useful hints concerning relations of concn. of substrates and catalyst for arom. amine oxidn. (TMB and OPD) were proposed. By increasing the concn. of chromogens and thanks to assuring the relatively low conversion of the reaction, the stability of TMB and OPD oxidn. product was improved even in acidic media. The comparative studies of H2O2 affinity to the surface of AuNPs in the presence of various hydrogen donors underlined the superiority of phenolic compds. over arom. amines and ABTS in the case of the samples of relatively low H2O2 concn. This work highlights some improvements in the methods of HRP-like activity characterization of NPs. It provides a crit. anal. of the major challenges, which may emerge in a case of bioanal. assays employing the catalytic nanoparticles as labels.
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    37. 37
      Monier, M.; Ayad, D. M.; Wei, Y.; Sarhan, A. A. Immobilization of Horseradish Peroxidase on Modified Chitosan Beads. Int. J. Biol. Macromol. 2010, 46, 324– 330,  DOI: 10.1016/j.ijbiomac.2009.12.018
      37
      Immobilization of horseradish peroxidase on modified chitosan beads
      Monier, M.; Ayad, D. M.; Wei, Y.; Sarhan, A. A.
      International Journal of Biological Macromolecules (2010), 46 (3), 324-330CODEN: IJBMDR; ISSN:0141-8130. (Elsevier B.V.)
      A method has been developed to immobilize horseradish peroxidase (HRP) on modified chitosan beads by means of graft copolymn. of polyethylacrylate in presence of potassium persulfate and Mohr's salt redox initiator. The activity of free and immobilized HRP was studied. FTIR spectroscopy and SEM were used to characterize HRP immobilization. The efficiency of the immobilization was investigated by examg. the relative enzymic activity of free enzyme before and after the HRP immobilization. The obtained values were found to reach 98.4%. The results show that the optimum temp. of immobilized HRP was 45 °C, which was identical to that of free enzyme, and the immobilized HRP exhibited a higher relative activity than that of free HRP over 45 °C. The optimal pH for immobilized HRP was 10, which was higher than that of the free HRP (pH 9.0), and the immobilization resulted in stabilization of enzyme over a broader pH range. The apparent kinetic const. value (Km) of immobilized HRP was 3.784 mmol mL-1, which was higher than that of free HRP. On the other hand, the activity of immobilized HRP decreased slowly against time when compared to that of the free HRP, and could retain 65.8% residual activity after 6 consecutive cycles.
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    38. 38
      Ahmad, N.; Sharma, S. Green Synthesis of Silver Nanoparticles Using Extracts of Ananas Comosus. Green Sustainable Chem. 2012, 02, 141– 147,  DOI: 10.4236/gsc.2012.24020
      38
      Green synthesis of silver nano particles using extracts of Ananas comosus
      Ahmad, Naheed; Sharma, Seema
      Green and Sustainable Chemistry (2012), 2 (4), 141-147, 7 pp.CODEN: GSCRCJ; ISSN:2160-696X. (Scientific Research Publishing, Inc.)
      The biosynthesis of nanoparticles has been proposed as a cost effective and environmental friendly alternative to chem. and phys. methods. Plant mediated synthesis of nanoparticles is a green chem. approach that intercomnects nanotechnol. and plant biotechnol. In the present study, synthesis of silver nanoparticles (AgNPs) or (Green-Silver) has been demonstrated using exts. of Ananas comosus reducing aq. silver nitrate. The AgNPs were characterized by UV-Visible (UV-Vis) Spectrometer, Energy Dispersive X-ray Anal. (EDAX), Selected Area Diffraction Pattern (SAED) and High Resoln. Transmission Electron Microscopy (HRTEM). TEM micrographs showed spherical particles with an av. size of 12 nm. The XRD pattern showed the characteristic Bragg peaks of (111), (200), (220) and (311) facets of the face center cubic (fcc) silver nanoparticles and confirmed that these nanoparticles are cryst. in nature. The different types of antioxidants presented in the pineapple juice synergistically reduce the Ag metal ions, as each antioxidant is unique in terms of its structure and antioxidant function. The reaction process was simple for formation of silver nanoparticles and AgNPs presented in the aq. medium were quite stable, even up to 4 mo of incubation. This work proved the capability of using biomaterial towards the synthesis of silver nanoparticle, by adopting the principles of green chem.
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    39. 39
      Huang, D.; Boxin, O. U.; Prior, R. L. The Chemistry behind Antioxidant Capacity Assays. J. Agric. Food Chem. 2005, 53, 1841– 1856,  DOI: 10.1021/jf030723c
      39
      The Chemistry behind Antioxidant Capacity Assays
      Huang, Dejian; Ou, Boxin; Prior, Ronald L.
      Journal of Agricultural and Food Chemistry (2005), 53 (6), 1841-1856CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      A review summarizing the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidn. and analyzes the chem. principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decompn. of azo compds. These assays include inhibition of induced low-d. lipoprotein autoxidn., oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the redn. of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concns. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addn., other assays intended to measure a sample's scavenging capacity of biol. relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this anal., it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addn. to these two commonly accepted assays.
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    40. 40
      Everette, J. D.; Bryant, Q. M.; Green, A. M.; Abbey, Y. A.; Wangila, G. W.; Walker, R. B. Thorough Study of Reactivity of Various Compound Classes toward the Folin-Ciocalteu Reagent. J. Agric. Food Chem. 2010, 58, 8139– 8144,  DOI: 10.1021/jf1005935
      40
      Thorough Study of Reactivity of Various Compound Classes toward the Folin-Ciocalteu Reagent
      Everette, Jace D.; Bryant, Quinton M.; Green, Ashlee M.; Abbey, Yvonne A.; Wangila, Grant W.; Walker, Richard B.
      Journal of Agricultural and Food Chemistry (2010), 58 (14), 8139-8144CODEN: JAFCAU; ISSN:0021-8561. (American Chemical Society)
      A thorough study was done to test the reactivity of the Folin-Ciocalteu (F-C) reagent toward various compd. classes. Over 80 compds. were tested. Compd. classes included phenols, thiols, vitamins, amino acids, proteins, nucleotide bases, unsatd. fatty acids, carbohydrates, org. acids, inorg. ions, metal complexes, aldehydes, and ketones. All phenols, proteins, and thiols tested were reactive toward the reagent. Many vitamin derivs. were also reactive, as were the inorg. ions Fe+2, Mn2+, I-, and SO32-. Other compds. showing reactivity included the nucleotide base guanine and the trioses glyceraldehyde and dihydroxyacetone. Copper complexation enhanced the reactivity of salicylate derivs. toward the reagent, whereas zinc complexation did not. Several amino acids and sugars that were reported to be reactive toward the F-C reagent in earlier studies were found not to be reactive in this study, at least in the concns. used. Reaction kinetics of each compd. with the F-C reagent were also measured. Most compds. tested showed a biphasic kinetic pattern with half-lives under 1 min. Trolox and ascorbic acid displayed a rapid monophasic pattern in which the reaction reached end point within 1 min. In summary, this study has shown that the F-C reagent is significantly reactive toward other compds. besides phenols. As other investigators have suggested, the F-C assay should be seen as a measure of total antioxidant capacity rather than phenolic content. Because phenolics are the most abundant antioxidants in most plants, it gives a rough approxn. of total phenolic content in most cases.
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    41. 41
      Goodarzi, V.; Zamani, H.; Bajuli, L.; Moradshahi, A. Evaluation of Antioxidant Potential and Reduction Capacity of Some Plant Extracts in Silver Nanoparticles’ Synthesis. Mol. Biol. Res. Commun. 2014, 3, 165– 174,  DOI: 10.22099/mbrc.2014.2196
      41
      Evaluation of antioxidant potential and reduction capacity of some plant extracts in silver nanoparticle synthesis
      Goodarzi, Vahid; Zamani, Hajar; Bajuli, Leila; Moradshahi, Ali
      Molecular Biology Research Communications (2014), 3 (3), 165-174, 10 pp.CODEN: MBRCBT; ISSN:2345-2005. (Shiraz University)
      The green synthesis of metallic nanoparticles is an active research area in nanotechnol. In the present study, antioxidant potential, total reducing capacity and silver nanoparticles (Ag NPs) synthetic potential of methanolic leaf exts. of seven plant species were evaluated and compared. Antioxidant capacity, expressed as μmol Trolox equiv. g-1 DW (μmol TE g-1 DW), ranged from 116.0 to 1.80. The plants Rosmarinus sp. and Zataria Multiflora showed highest antioxidant capacities with IC50 of 1.07 and 1.22 mg ml-1, resp. Total reducing capacity ranged from 7.6 to 0.17 mg gallic acid equiv. to g-1 DW (mg GAE g-1 DW). Plants with high antioxidant potentials also showed higher total reducing capacity. In fact, the order of the plants reducing capacity was similar to that of their antioxidant potential. The same two plant species, i.e., Zataria Multiflora and Rosmarinus sp. with high reducing capacities, showed higher potentials for Ag NPs synthesis. It is concluded that reducing substances in the exts. contribute significantly to the antioxidant potential of the tested plant species and plants with a high reducing capacity are excellent sources for the green synthesis of metallic nanoparticles. In addn., synthetic antioxidants have adverse effects on human health; therefore, to benefit more from the health promoting properties of plant species, evaluating their novel natural antioxidants is recommended.
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    42. 42
      Ramamurthy, C.; Padma, M.; mariya samadanam, I. D.; Mareeswaran, R.; Suyavaran, A.; Kumar, M. S.; Premkumar, K.; Thirunavukkarasu, C. The Extra Cellular Synthesis of Gold and Silver Nanoparticles and Their Free Radical Scavenging and Antibacterial Properties. Colloids Surf., B 2013, 102, 808– 815,  DOI: 10.1016/j.colsurfb.2012.09.025
      42
      The extra cellular synthesis of gold and silver nanoparticles and their free radical scavenging and antibacterial properties
      Ramamurthy, Ch.; Padma, M.; Samadanam, I. Daisy Mariya; Mareeswaran, R.; Suyavaran, A.; Kumar, M. Suresh; Premkumar, K.; Thirunavukkarasu, C.
      Colloids and Surfaces, B: Biointerfaces (2013), 102 (), 808-815CODEN: CSBBEQ; ISSN:0927-7765. (Elsevier B.V.)
      The bio redn. of chloroauric acid (HAuCl4) and silver nitrate (AgNO3) is achieved extracellularly by the aq. ext. of Solanum torvum (S. torvum) fruit. The nanoparticle formation was screened by UV-visible spectroscopy through color conversion due to surface plasma resonance bands at 560 nm and 430 nm for gold and silver nanoparticles resp. The spherical shapes with smooth surface of gold and silver nanoparticles were analyzed through scanning electron microscope and its presence was confirmed by energy dispersive x-ray spectroscopy (SEM/EDX). The functional groups in the gold and silver salts and the bio interactive functional groups present in the S. torvum ext. were characterized by employing Fourier transform infra-red spectroscopy (FTIR). The biomedical properties of gold and silver nanoparticles were premeditated as free radical scavenging activity and antibacterial static agents. Gold and silver nanoparticles serve as strong hydroxyl, superoxide, nitric oxide and DPPH radical scavengers in contrast to their corresponding metal oxides. The radical quenching properties of gold and silver nanoparticles were found to correlate with in vitro DNA protective effect. The silver nanoparticles show strong zone of inhibition against Escherichia coli, Pseudomonas and Bacillus whereas, gold nanoparticles exhibit fair zone of inhibition. To the authors' knowledge this is the first report that S. torvum ext. can reduce metal acids to nanomaterials.
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    43. 43
      Patra, J. K.; Baek, K.-H. Green Synthesis of Silver Chloride Nanoparticles Using Prunus Persica L. Outer Peel Extract and Investigation of Antibacterial, Anticandidal, Antioxidant Potential. Green Chem. Lett. Rev. 2016, 9, 132– 142,  DOI: 10.1080/17518253.2016.1192692
      43
      Green synthesis of silver chloride nanoparticles using Prunus persica L. outer peel extract and investigation of antibacterial, anticandidal, antioxidant potential
      Patra, Jayanta Kumar; Baek, Kwang-Hyun
      Green Chemistry Letters and Reviews (2016), 9 (2), 132-142CODEN: GCLRAI; ISSN:1751-7192. (Taylor & Francis Ltd.)
      The present study was conducted to synthesize silver chloride nanoparticles using the aq. ext. of outer peel of peach fruit (Prunus persica L.) and to evaluate its antibacterial activity, synergistic antibacterial and anticandidal potential against five foodborne pathogenic bacteria and five pathogenic Candida species resp. along with its antioxidant potential. The synthesized silver chloride nanoparticles (PE-AgClNPs) were visually confirmed with surface plasmon resonance peak at 440 nm upon UV-Vis spectroscopy anal. Furthermore, the morphol., elemental compn. and crystallinity nature were also characterized. PE-AgClNPs displayed strong antibacterial potentials (9.01-10.83 mm inhibition zone) against foodborne pathogenic bacteria and increased synergistic effect with kanamycin and rifampicin. PE-AgClNPs also displayed strong anticandidal synergistic activity with std. amphotericin b (10.51- 14.01 mm inhibition zones), along with strong free radical scavenging and reducing power. Based on strong antibacterial and antioxidant capacities, PE-AgClNPs are anticipated to have potential applications in the biomedical and food sector industries.
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    44. 44
      Bravo, L. Polyphenols: Chemistry, Dietary Sources, Metabolism, and Nutritional Significance. Nutr. Rev. 1998, 56, 317– 333,  DOI: 10.1111/j.1753-4887.1998.tb01670.x
      44
      Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance
      Bravo L
      Nutrition reviews (1998), 56 (11), 317-33 ISSN:0029-6643.
      Polyphenols constitute one of the most numerous and ubiquitous groups of plant metabolites and are an integral part of both human and animal diets. Ranging from simple phenolic molecules to highly polymerized compounds with molecular weights of greater than 30,000 Da, the occurrence of this complex group of substances in plant foods is extremely variable. Polyphenols traditionally have been considered antinutrients by animal nutritionists, because of the adverse effect of tannins, one type of polyphenol, on protein digestibility. However, recent interest in food phenolics has increased greatly, owing to their antioxidant capacity (free radical scavenging and metal chelating activities) and their possible beneficial implications in human health, such as in the treatment and prevention of cancer, cardiovascular disease, and other pathologies. Much of the literature refers to a single group of plant phenolics, the flavonoids. This review offers an overview of the nutritional effects of the main groups of polyphenolic compounds, including their metabolism, effects on nutrient bioavailability, and antioxidant activity, as well as a brief description of the chemistry of polyphenols and their occurrence in plant foods.
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    45. 45
      Vivek, K.; Mishra, S.; Pradhan, R. C. Physicochemical Characterization and Mass Modelling of Sohiong (Prunus Nepalensis L.) Fruit. J. Food Meas. Char. 2018, 12, 923– 936,  DOI: 10.1007/s11694-017-9708-x
      There is no corresponding record for this reference.
    46. 46
      Kashyap, P.; Riar, C. S.; Jindal, N. Optimization of Ultrasound Assisted Extraction of Polyphenols from Meghalayan Cherry Fruit (Prunus Nepalensis) Using Response Surface Methodology (RSM) and Artificial Neural Network (ANN) Approach. J. Food Meas. Char. 2021, 15, 119,  DOI: 10.1007/s11694-020-00611-0
      There is no corresponding record for this reference.
    47. 47
      Swer, T. L.; Mukhim, C.; Bashir, K.; Chauhan, K. Optimization of Enzyme Aided Extraction of Anthocyanins from Prunus Nepalensis L. LWT--Food Sci. Technol. 2018, 91, 382– 390,  DOI: 10.1016/j.lwt.2018.01.043
      47
      Optimization of enzyme aided extraction of anthocyanins from Prunus nepalensis L.
      Swer, Tanya L.; Mukhim, C.; Bashir, Khalid; Chauhan, Komal
      LWT--Food Science and Technology (2018), 91 (), 382-390CODEN: LSTWB3; ISSN:0023-6438. (Elsevier Ltd.)
      A central composite design was employed to obtain the optimal conditions for enzyme assisted extn. (EAE) of anthocyanins from Prunus nepalensis L. using cellulase. Response surface methodol. was used to evaluate the effects of temp. T (35-50 °C), cellulase enzyme concn. EC (5-15% E/S) and time t (2.50-4.00 h) on five responses anthocyanins recovery (TAC), total phenolic content (TPC), in vitro antioxidant activity (FRAP and DPPH) and total ext. yield (TY). The optimum extn. conditions were found to be T = 45.6 °C, EC = 12.9% E/S and t = 3.98 h resulting in TY = 62.9 ± 1.40%, TAC = 9.89 ± 0.24 mg C3G equiv./g dm, TPC = 8.32 ± 0.18 mg GAE/100 g dm with antioxidant capacity of 28.7 ± 0.12 mmol AAE/100g DM for FRAP and 91.2 ± 0.39% DPPH inhibition (20 g/mL). EAE demonstrated higher anthocyanins recovery compared to conventional solvent extn. process. HPLC anal. showed the presence of cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin. FTIR and SEM anal. were also performed. The study provides an economical alternative for com. extn. of anthocyanins from Sohiong fruit which can be used as a biocolorant for various food products. This offers immense potential for the fruit producers as well as food processors.
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    48. 48
      Kora, A. J.; Rastogi, L. Peroxidase Activity of Biogenic Platinum Nanoparticles: A Colorimetric Probe towards Selective Detection of Mercuric Ions in Water Samples. Sens. Actuators, B 2018, 254, 690– 700,  DOI: 10.1016/j.snb.2017.07.108
      48
      Peroxidase activity of biogenic platinum nanoparticles: A colorimetric probe towards selective detection of mercuric ions in water samples
      Kora, Aruna Jyothi; Rastogi, Lori
      Sensors and Actuators, B: Chemical (2018), 254 (), 690-700CODEN: SABCEB; ISSN:0925-4005. (Elsevier B.V.)
      A biogenic green method for the synthesis of platinum nanoparticles (Pt NP) was developed using non-toxic, renewable, biodegradable plant exudate gum, gum olibanum (Boswellia serrata). The effect of parameters such as concns. of gum (0.1-0.5%) and chloroplatinic acid (0.125-1.0 mM) on nanoparticle synthesis was studied. The resulting nanoparticles exhibited excellent peroxidase activity which catalyzes the oxidn. of the chromogen 3, 3', 5, 5'-tetramethylbenzidine (TMB) to a blue color product, in the presence of hydrogen peroxide. The peroxidase activity of the nanoparticles was selectively inhibited by mercuric ions (Hg2+) due to the formation of amalgam by Hg-Pt specific interaction. Notably, the inhibition was not affected by other metal ions even at a concn. of 5 M. The decrease in oxidized TMB intensity at 652 nm (blue color), upon addn. of mercuric ions was linear in the range of 50-500 nM for MilliQ, tap and ground waters and the resp. limit of quantification values for Hg2+ using the developed method were 16.9, 26 and 47.3 nM. The proposed method was effectively applied for the detn. of Hg2+ in various ground water samples and verified with CVAAS. We envisage that the biogenic Pt NP based colorimetric probe can have promising applications in the screening and field detection of mercuric ions in various water bodies and public drinking water distribution systems.
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    49. 49
      Dauthal, P.; Mukhopadhyay, M. In-Vitro Free Radical Scavenging Activity of Biosynthesized Gold and Silver Nanoparticles Using Prunus Armeniaca (Apricot) Fruit Extract Nanomaterials in Energy, Health and Environment. Guest Editors: Puru Jena, Samy El Shall, Anil Kandalam. J. Nanopart. Res. 2013, 15, 1366,  DOI: 10.1007/s11051-012-1366-7
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      In-vitro free radical scavenging activity of biosynthesized gold and silver nanoparticles using Prunus armeniaca (apricot) fruit extract
      Dauthal, Preeti; Mukhopadhyay, Mausumi
      Journal of Nanoparticle Research (2013), 15 (1), 1366/1-1366/11, 11 pp.CODEN: JNARFA; ISSN:1388-0764. (Springer)
      In-vitro free radical scavenging activity of biosynthesized gold (Au-NPs) and silver (Ag-NPs) nanoparticles was investigated in the present study. Natural precursor Prunus armeniaca (apricot) fruit ext. was used as a reducing agent for the nanoparticle synthesis. The free radical scavenging activity of the nanoparticles were obsd. by modified 1,1'-diphynyl-2-picrylhydrazyl, DPPH and 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), ABTS assay. The synthesized nanoparticles were characterized by UV-Visible spectroscopy, dynamic light scattering, transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive spectroscopy, and Fourier transform IR spectroscopy (FTIR). Appearance of optical absorption peak at 537 nm (2.20 keV) and 435 nm (3 keV) within 0.08 and 0.5 h of reaction time was confirmed the presence of metallic Au and Ag nanoclusters, resp. Nearly spherical nanoparticles with majority of particle below 20 nm (TEM) for both Au-NPs and Ag-NPs were synthesized. XRD pattern confirmed the existence of pure nanocryst. Au-NPs while few addnl. peaks in the vicinity of fcc silver-speculated crystn. of metalloproteins of fruit ext. on the surface of the Ag-NPs and vice versa. FTIR spectra was supported the role of amino acids of protein/enzymes of fruit ext. for synthesis and stabilization of nanoparticles. Dose-dependent scavenging activity was obsd. for Au-NPs and Ag-NPs in both DPPH and ABTS in-vitro assay. 50% scavenging activity for DPPH were 11.27 and 16.18 mg and for ABTS 3.40 and 7.12 mg with Au-NPs and Ag-NPs, resp.
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    50. 50
      Alara, O. R.; Abdurahman, N. H.; Ukaegbu, C. I.; Azhari, N. H. Vernonia Cinerea Leaves as the Source of Phenolic Compounds, Antioxidants, and Anti-Diabetic Activity Using Microwave-Assisted Extraction Technique. Ind. Crops Prod. 2018, 122, 533– 544,  DOI: 10.1016/j.indcrop.2018.06.034
      50
      Vernonia cinerea leaves as the source of phenolic compounds, antioxidants, and anti-diabetic activity using microwave-assisted extraction technique
      Alara, Oluwaseun Ruth; Abdurahman, Nour Hamid; Ukaegbu, Chinonso Ishmael; Azhari, Nour Hamid
      Industrial Crops and Products (2018), 122 (), 533-544CODEN: ICRDEW; ISSN:0926-6690. (Elsevier B.V.)
      Diabetes mellitus is a metabolic disorder disease emanating from the inability of body's response to high glucose levels in the blood. However, the side-effects from consuming synthetic drugs have reduced the effectiveness, thus, the potential of purple fleabane (Vernonia cinerea) leaf was investigated using microwave-assisted extn. (MAE) in this study. The influences of extn. factors such as irradn. time, microwave power level, feed/solvent, and ethanol concn. on the recovery yields of polyphenol and flavonoids were as well studied. Thirty exptl. runs using a face-centered central composite design (FCCCD) with six center points were employed and optimal yields were obtained by generating quadratic models based on response surface methodol. The obtained optimal MAE conditions were as follows: irradn. time, 2 min; microwave power level, 444 W; feed/solvent, 1:14 g/mL; and ethanol concn., 47% vol./vol. There was an insignificant difference between the predicted and exptl. data (p > 0.05), showing good predictive models for the extn. of phenolic compds. from V. cinerea leaves using MAE. Also, the results confirmed that MAE can recover higher phenolic compds., antioxidant, and anti-diabetic activities from V. cinerea leaves in shorter extn. time. In addn., the phenolic compds. in V. cinerea leaf ext. were identified using liq. chromatog.-mass spectrometry quadrupole time of flight (LC-MS-Q-TOF) and Fourier transform IR spectroscopy (FTIR). The exts. showed higher anti-diabetic activity using alpha-amylase and alpha-glycosidase assays which suggested its exploration in pharmaceutical and functional food industries.
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    51. 51
      Ovais, M.; Khalil, A. T.; Islam, N. U.; Ahmad, I.; Ayaz, M.; Saravanan, M.; Shinwari, Z. K.; Mukherjee, S. Role of Plant Phytochemicals and Microbial Enzymes in Biosynthesis of Metallic Nanoparticles. Appl. Microbiol. Biotechnol. 2018, 102, 6799– 6814,  DOI: 10.1007/s00253-018-9146-7
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      Role of plant phytochemicals and microbial enzymes in biosynthesis of metallic nanoparticles
      Ovais, Muhammad; Khalil, Ali Talha; Islam, Nazar Ul; Ahmad, Irshad; Ayaz, Muhamamd; Saravanan, Muthupandian; Shinwari, Zabta Khan; Mukherjee, Sudip
      Applied Microbiology and Biotechnology (2018), 102 (16), 6799-6814CODEN: AMBIDG; ISSN:0175-7598. (Springer)
      A review. Metal-based nanoparticles have gained tremendous popularity because of their interesting phys., biol., optical, and magnetic properties. These nanoparticles can be synthesized using a variety of different phys., chem., and biol. techniques. The biol. means are largely preferred as it provides an environmentally benign, green, and cost-effective route for the biosynthesis of nanoparticles. These bioresources can act as a scaffold, thereby playing the role of reducing as well as capping agents in the biosynthesis of nanoparticles. Medicinal plants tend to have a complex phytochem. constituent such as alcs., phenols, terpenes, alkaloids, saponins, and proteins, while microbes have key enzymes which can act as reducing as well as stabilizing agent for NP synthesis. However, the mechanism of biosynthesis is still highly debatable. Herein, the present review is directed to give an updated comprehensive overview towards the mechanistic aspects in the biosynthesis of nanoparticles via plants and microbes. Various biosynthetic pathways of secondary metabolites in plants and key enzyme prodn. in microbes have been discussed in detail, along with the underlying mechanisms for biogenic NP synthesis.
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    52. 52
      Wang, X.; Mei, Z.; Wang, Y.; Tang, L. Gold Nanorod Biochip Functionalization by Antibody Thiolation. Talanta 2015, 136, 1– 8,  DOI: 10.1016/j.talanta.2014.11.023
      52
      Gold nanorod biochip functionalization by antibody thiolation
      Wang, Xuefeng; Mei, Zhong; Wang, Yanyan; Tang, Liang
      Talanta (2015), 136 (), 1-8CODEN: TLNTA2; ISSN:0039-9140. (Elsevier B.V.)
      Conjugation of biomols. on gold nanorod (GNR) surfaces is the basis for successful applications in biosensing, imaging, and drug delivery. Current functionalization methods are often problematic, involving multi-step nanoparticle modification to replace surfactant bilayer, delicate nanoparticle protection during surfactant exchange, and material loss due to inevitable aggregation. Instead of intensive surface modification of GNRs, we describe herein a facile method to functionalize gold nanorod surfaces via covalent Au-S bonds by thiolating receptors. The resulting GNR-bioconjugates showed superior dispersion and stability in buffer for months without morphol. change and aggregation. ELISA tests confirmed the high biofunctionality of the thiolated anti-IgG moieties immobilized on the GNR surfaces. Furthermore, this simple method facilitated a straightforward functionalization of GNR assembly on glass substrate to construct a specific biochip, which can detect human IgG targets in a label-free fashion with high sensitivity and specificity. Compared to electropolymeric coating to functionalize the GNR, our method exhibited a five-fold enhancement in the spectral sensitivity to refractive index change caused by the target binding. This universal GNR bioconjugation method can be extended to bind different proteins and antibodies for development of biosensors or drug delivery.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhvVertL0%253D&md5=15560b0dbaf084b21512a744355c2ad7
    53. 53
      Wang, X.; Mei, Z.; Wang, Y.; Tang, L. Comparison of Four Methods for the Biofunctionalization of Gold Nanorods by the Introduction of Sulfhydryl Groups to Antibodies. Beilstein J. Nanotechnol. 2017, 8, 372– 380,  DOI: 10.3762/bjnano.8.39
      53
      Comparison of four methods for the biofunctionalization of gold nanorods by the introduction of sulfhydryl groups to antibodies
      Wang, Xuefeng; Mei, Zhong; Wang, Yanyan; Tang, Liang
      Beilstein Journal of Nanotechnology (2017), 8 (), 372-380CODEN: BJNEAH; ISSN:2190-4286. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
      Introducing sulfhydryl groups to biomols. to functionalize gold nanorods (GNRs) is an attractive method that involves the creation of a strong Au-S bond. Previously, we developed a facile method to functionalize GNR surfaces by thiolating antibodies using Traut's reagent. In the current study, we evaluated several methods for the introduction of thiol groups onto the surface of GNRs by using Traut's reagent, dithiotreitol (DTT), dithiolarom. PEG6-CONHNH , and thiol-polyethylene glycolamine (SHPEG-NH) combined with EDC reaction. We showed that the four above-mentioned thiolation methods can efficiently functionalize GNRs and simplify the functionalization procedures. The formed GNR-bioconjugates showed superior stability without compromising the biol. activity. The GNR nanochip prepd. with these four thiolated antibodies can detect human IgG targets with specificity. However, SH-PEG-NH combined with EDC reaction may affect the amt. of functionalized GNRs because of the efficiency of thiol moiety linkage to antibodies, thereby affecting the sensitivity of the GNR sensor. The introduction of a thiol group to antibodies by using Traut's reagent, DTT, and PEG6-CONHNH allowed for direct immobilization onto the GNR surface, improved the efficacy of functionalized GNRs, and increased the sensitivity in response to target detection as a biosensor. Given that PEG6-CONHNH modification requires glycosylated biomols., Traut's reagent and DTT thiolation are recommended as universal applications of GNR biofunctionalization and can be easily extended to other sensing applications based on other gold nanostructures or new biomols.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpslejsLk%253D&md5=7502733f116e9df264b6ab7a8c3910a1
    54. 54
      Li, W.; Fan, G.-C.; Gao, F.; Cui, Y.; Wang, W.; Luo, X. High-Activity Fe 3 O 4 Nanozyme as Signal Amplifier: A Simple, Low-Cost but Efficient Strategy for Ultrasensitive Photoelectrochemical Immunoassay. Biosens. Bioelectron. 2019, 127, 64– 71,  DOI: 10.1016/j.bios.2018.11.043
      54
      High-activity Fe3O4 nanozyme as signal amplifier: A simple, low-cost but efficient strategy for ultrasensitive photoelectrochemical immunoassay
      Li, Wenshi; Fan, Gao-Chao; Gao, Fengxian; Cui, Yige; Wang, Wei; Luo, Xiliang
      Biosensors & Bioelectronics (2019), 127 (), 64-71CODEN: BBIOE4; ISSN:0956-5663. (Elsevier B.V.)
      Sensitive but with simple, inexpensive detection of disease-related biomarkers in real biol. samples is of quite necessity for early diagnosis and disease surveillance. In the proposal, to illustrate and describe the PEC platform, prostate-specific antigen (PSA, Ag) was used as a target model. Specifically, ZnO nanorods (ZnO-NRs) grown vertically on a bare indium-tin oxide (ITO) electrode was deposited with ZnIn2S4 nanocrystals, producing ZnIn2S4/ZnO-NRs/ITO photoelectrode as the PEC matrix to modify capture PSA antibody (Ab1). Histidine-modified Fe3O4(his-Fe3O4) nanozyme as signal amplifier was linked with signal PSA antibody (Ab2) to form his-Fe3O4@Ab2 conjugate, and was anchored through specific sandwich immunoreaction. His-Fe3O4 nanozyme acted as a peroxidase to induce the generation of the insol. and insulating pptn., resulting in an evident decrease in the photocurrent signal. On account of combined effects of high catalytic of the his-Fe3O4 nanozyme and excellent PEC properties of the ZnIn2S4/ZnO-NRs/ITO photoelectrode,ultralow detection limit of 18 fg/mL for target Ag detection was achieved. High-activity his-Fe3O4 nanozyme has substituted natural enzyme as signal amplifier, simplicity and low cost of the PEC immunoassay was realized.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXis1Srt7vK&md5=d5c7e0fad08160a510ac8ed700103fbc
    55. 55
      Ma, X.; Song, S.; Kim, S.; Kwon, M.-s.; Lee, H.; Park, W.; Sim, S. J. Single Gold-Bridged Nanoprobes for Identification of Single Point DNA Mutations. Nat. Commun. 2019, 10, 836,  DOI: 10.1038/s41467-019-08769-y
      55
      Single gold-bridged nanoprobes for identification of single point DNA mutations
      Ma Xingyi; Song Sojin; Kim Soohyun; Sim Sang Jun; Kwon Mi-Sun; Lee Hyunsook; Park Wounjhang
      Nature communications (2019), 10 (1), 836 ISSN:.
      Consensus ranking of protein affinity to identify point mutations has not been established. Therefore, analytical techniques that can detect subtle variations without interfering with native biomolecular interactions are required. Here we report a rapid method to identify point mutations by a single nanoparticle sensing system. DNA-directed gold crystallization forms rod-like nanoparticles with bridges based on structural design. The nanoparticles enhance Rayleigh light scattering, achieving high refractive-index sensitivity, and enable the system to monitor even a small number of protein-DNA binding events without interference. Analysis of the binding affinity can compile an atlas to distinguish the potential of various point mutations recognized by MutS protein. We use the atlas to analyze the presence and type of single point mutations in BRCA1 from samples of human breast and ovarian cancer cell lines. The strategy of synthesis-by-design of plasmonic nanoparticles for sensors enables direct identification of subtle biomolecular binding distortions and genetic alterations.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3cfltlGruw%253D%253D&md5=07b50a923008909ac490339dfc3540ca
    56. 56
      Lee, J. U.; Kim, W. H.; Lee, H. S.; Park, K. H.; Sim, S. J. Quantitative and Specific Detection of Exosomal MiRNAs for Accurate Diagnosis of Breast Cancer Using a Surface-Enhanced Raman Scattering Sensor Based on Plasmonic Head-Flocked Gold Nanopillars. Small 2019, 15, 1804968,  DOI: 10.1002/smll.201804968
      There is no corresponding record for this reference.
    57. 57
      Young, J. S.; Gormley, E.; Wellington, E. M. H. Molecular Detection of Mycobacterium Bovis and Mycobacterium Bovis BCG (Pasteur) in Soil. Appl. Environ. Microbiol. 2005, 71, 1946– 1952,  DOI: 10.1128/AEM.71.4.1946-1952.2005
      57
      Molecular detection of Mycobacterium bovis and Mycobacterium bovis BCG (Pasteur) in soil
      Young, Jamie S.; Gormley, Eamonn; Wellington, Elizabeth M. H.
      Applied and Environmental Microbiology (2005), 71 (4), 1946-1952CODEN: AEMIDF; ISSN:0099-2240. (American Society for Microbiology)
      PCR primers specific for the Mycobacterium tuberculosis complex were used to detect the presence of Mycobacterium bovis BCG (Pasteur) in soil microcosms and Mycobacterium bovis in environmental samples taken from a farm in Ireland with a history of bovine tuberculosis. M. bovis genes were detected in soil at 4 and 21 mo after possible contamination. Gene levels were found in the range of 1 × 103 to 3.6 × 103 gene copies g of soil-1, depending on the sampling area. Areas around badger setts had the highest levels of detectable genes and were shown to have the highest levels of gene persistence. M. bovis-specific 16S rRNA sequences were detected, providing evidence of the presence of viable cells in Irish soils. Studies of DNA turnover in soil microcosms proved that dead cells of M. bovis BCG did not persist beyond 10 days. Further microcosm expts. revealed that M. bovis BCG survival was optimal at 37°C with moist soil (-20 kPa; 30% [vol/wt]). This study provides clear evidence that M. bovis can persist in the farm environment outside of its hosts and that climatic factors influence survival rates.
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    58. 58
      Cezar, R. D. S.; Lucena-Silva, N.; Borges, J. M.; Santana, V. L. A.; Pinheiro Junior, J. W. Detection of Mycobacterium Bovis in Artisanal Cheese in the State of Pernambuco, Brazil. Int. J. Mycobact. 2016, 5, 269– 272,  DOI: 10.1016/j.ijmyco.2016.04.007
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.2c00180.

    • Optimization study of the biogenic synthesis process of Au NPs; chemical analysis of P. nepalensis extract; free radical scavenging activity and antioxidant potential of P. nepalensis fruit extract; reducing power assay; DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) scavenging ability; determination of the total phenolic content; determination of the total flavonoid content; characterization of peroxidase-mimicking activity of biogenic Au NPs; understanding the reaction mechanism of TMB oxidation catalyzed by peroxidase-mimicking biogenic Au NPs; determining the role of P. nepalensis fruit extract in the catalysis of TMB oxidation; additional results and discussion: optimization study, FESEM analysis, and characterization of the peroxidase-mimicking activity of biogenic Au NPs; UV–vis spectra of optimization study of Au NP synthesis using P. nepalensis extract; FESEM image of Au NPs; zeta potential analysis of the biogenic Au NPs; reaction mechanism of the TMB oxidation process; UV–vis absorption spectra of the reaction kinetics of TMB oxidation; role of P. nepalensis fruit extract in the TMB oxidation process; GC–MS analysis of the P. nepalensis fruit extract; UV–vis absorption spectra (370 nm) of the reaction kinetics of TMB oxidation comparative analysis; Michaelis–Menten kinetic parameters calculation; GC–MS analysis of P. nepalensis extract; and comparison between different biosensing assays for the detection of bacteria (PDF)


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