Chemometrics has the potential to embolden microfluidics to become that enabling technology for so long sought after. In this Feature article, we describe a historical perspective on microfluidics and its current challenges, a perspective on chemometric methods including response surface methodology (RSM), and how a combination of artificial neural network with experimental design (ANN-ED) have demonstrated promise in addressing basic microfluidic problems.

Monoclonal antibodies (mAbs) represent an important and rapidly growing class of biotherapeutics. Correct folding of a mAb is critical for drug efficacy, while misfolding can impact safety by eliciting unwanted immune or other off-target responses. Robust methods are therefore needed for the precise measurement of mAb structure for drug quality assessment and comparability. To date, the perception in the field has been that NMR could not be applied practically to mAbs due to the size (∼150 kDa) and complexity of these molecules, as well as the insensitivity of the method. The feasibility of applying NMR methods to stable isotope-labeled, protease-cleaved, mAb domains (Fab and Fc) has been demonstrated from both E. coli and Chinese hamster ovaries (CHO) cell expression platforms; however, isotopic labeling is not typically available when analyzing drug products. Here, we address the issue of feasibility of NMR-based mapping of mAb structure by demonstrating for the first time the application of a 2D ¹³C NMR methyl fingerprint method for structural mapping of an intact mAb at natural isotopic abundance. Further, we show that 2D ¹³C NMR spectra of protease-cleaved Fc and Fab fragments can provide accurate reporters on the domain structures that can be mapped directly to the intact mAb. Through combined use of rapid acquisition and nonuniform sampling techniques, we show that these Fab and Fc fingerprint spectra can be rapidly acquired in as short as approximately 30 min.

The selective concentration of the contents in a microdroplet using spontaneous emulsification was proposed and demonstrated in a microfluidic channel. Aqueous microdroplets having a 40-μm diameter, in octane containing 100 mM of Span 80, shrank to 10 μm within 10 min with nanodroplet formation at the interface of the microdroplets. The microdroplets’ contents either stayed in the microdroplet or partitioned into the nanodroplets, depending on their properties. The size and the hydrophobicity of the contents are two parameters that determine concentration/separation. In addition, this method was applied to a bound complex and free ligand (B/F) separation to demonstrate its applicability to biochemical analyses. Here we report the separation of water-soluble molecules in microdroplets for the first time. This method is expected to enhance the flexibility of the design of droplet analytical processes and widen their applicability.

The fabrication and use of a multifunctional electrochemical probe incorporating two independent carbon working electrodes and two electrolyte-filled barrels, equipped with quasi-reference counter electrodes (QRCEs), in the end of a tapered micrometer-scale pipet is described. This “quad-probe” (4-channel probe) was fabricated by depositing carbon pyrolytically into two diagonally opposite barrels of a laser-pulled quartz quadruple-barrelled pipet. After filling the open channels with electrolyte solution, a meniscus forms at the end of the probe and covers the two working electrodes. The two carbon electrodes can be used to drive local electrochemical reactions within the meniscus while a bias between the QRCEs in the electrolyte channels provides an ion conductance signal that is used to control and position the meniscus on a surface of interest. When brought into contact with a surface, localized high resolution amperometric imaging can be achieved with the two carbon working electrodes with a spatial resolution defined by the meniscus contact area. The substrate can be an insulating material or (semi)conductor, but herein, we focus mainly on conducting substrates that can be connected as a third working electrode. Studies using both aqueous and ionic liquid electrolytes in the probe, together with gold and individual single walled carbon nanotube samples, demonstrate the utility of the technique. Substrate generation-dual tip collection measurements are shown to be characterized by high collection efficiencies (approaching 100%). This hybrid configuration of scanning electrochemical microscopy (SECM) and scanning electrochemical cell microscopy (SECCM) should be powerful for future applications in electrode mapping, as well as in studies of insulating materials as demonstrated by transient spot redox-titration measurements at an electrostatically charged Teflon surface and at a pristine calcite surface, where a functionalized probe is used to follow the immediate pH change due to dissolution.

In washing-free electrochemical detection, various redox and reactive species cause significant interference. To minimize this interference, we report a washing-free electrochemical immunosensor using flavin adenine dinucleotide (FAD)-dependent glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphate (GP) as an enzyme label and its substrate, respectively, because the reaction of FAD-dependent dehydrogenases with dissolved O₂ is slow and the level of GP preexisting in blood is low (<0.1 mM). A combination of a low electrocatalytic indium–tin oxide (ITO) electrode and fast electron-mediating Ru(NH₃)₆³⁺ is employed to obtain a high signal-to-background ratio via proximity-dependent electron mediation of Ru(NH₃)₆³⁺ between the ITO electrode and the GPDH label. Electrochemical oxidation of GPDH-generated Ru(NH₃)₆²⁺ is performed at 0.05 V vs Ag/AgCl, at which point the electrochemical interference is very low. When a washing-free immunosensor is applied to cardiac troponin I detection in human serum, the calculated detection limit is approximately 10 pg/mL, indicating that the immunosensor is very sensitive in spite of the use of washing-free detection with a short detection period (10 min for incubation and 100 s for electrochemical measurement). The low-interference washing-free electrochemical immunosensor shows good promise for fast and simple point-of-care testing.

Significant challenges are present in antibiotic drug discovery and development. One of these is the number of efficient approaches Gram-negative bacteria have developed to avoid intracellular accumulation of drugs and other cell-toxic species. In order to better understand these processes and correlate in vitro enzyme inhibition to whole cell activity, a better assay to evaluate a key factor, intracellular accumulation of the drug, is urgently needed. Here, we describe a unique liquid chromatography (LC)-mass spectrometry (MS) approach to measure the amount of cellular uptake of antibiotics by Gram-negative bacteria. This method, which measures the change of extracellular drug concentration, was evaluated by comparing the relative uptake of linezolid by Escherichia coli wild-type versus an efflux pump deficient strain. A higher dosage of the drug showed a higher accumulation in these bacteria in a dosing range of 5–50 ng/mL. The Escherichia coli efflux pump deficient strain had a higher accumulation of the drug than the wild-type strain as predicted. The approach was further validated by determining the relative meropenem uptake by Pseudomonas aeruginosa wild-type versus a mutant strain lacking multiple porins. These studies show great promise of being applied within antibiotic drug discovery, as a universal tool to aid in the search for compounds that can easily penetrate bacterial cells.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which primarily infects macrophages and Schwann cells, affecting skin and peripheral nerves. Clinically, the most common form of identification is through the observation of anesthetic lesions on skin; however, up to 30% of infected patients may not present this clinical manifestation. Currently, the gold standard diagnostic test for leprosy is based on skin lesion biopsy, which is invasive and presents low sensibility for suspect cases. Therefore, the development of a fast, sensible and noninvasive method that identifies infected patients would be helpful for assertive diagnosis. The aim of this work was to identify lipid markers in leprosy patients directly from skin imprints, using a mass spectrometric analytical strategy. For skin imprint samples, a 1 cm² silica plate was gently pressed against the skin of patients or healthy volunteers. Imprinted silica lipids were extracted and submitted to direct-infusion electrospray ionization high-resolution mass spectrometry (ESI-HRMS). All samples were differentiated using a lipidomics-based data workup employing multivariate data analysis, which helped electing different lipid markers, for example, mycobacterial mycolic acids, inflammatory and apoptotic molecules were identified as leprosy patients’ markers. Otherwise, phospholipids and gangliosides were pointed as healthy volunteers’ skin lipid markers, according to normal skin composition. Results indicate that silica plate skin imprinting associated with ESI-HRMS is a promising fast and sensible leprosy diagnostic method. With a prompt leprosy diagnosis, an early and effective treatment could be feasible and thus the chain of leprosy transmission could be abbreviated.

A two-dimensional separation system is presented combining scanning temperature gradient focusing (TGF) and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) in a PDMS/glass microfluidic chip. Denatured proteins are first focused and separated in a 15 mm long channel via TGF with a temperature range of 16–47 °C and a pressure scanning rate of −0.5 Pa/s and then further separated via SDS-PAGE in a 25 mm long channel. A side channel is designed at the intersection between the two dimensions to continuously inject SDS into the gel, allowing SDS molecules to be compiled within the focused bands. Separation experiments are performed using several fluorescently labeled proteins with single point detection. Experimental results show a dramatic improvement in peak capacity over one-dimensional separation techniques.

The current rectification factor can be tailored by changing the degree of asymmetry between the fluid baths on opposite sides of a nanocapillary membrane (NCM). A symmetric device with symmetric fluid baths connected to opposite sides of the NCM did not rectify ionic current; while a NCM connected between fluid baths with a 32-fold difference in cross-sectional area produced a rectification factor of 75. The data suggests that the primary mechanism for the current rectification is the change in cross-sectional area of the fluid baths and the polarity dependent propagation of the enriched and depleted concentration polarization (CP) zones into these regions. An additional contribution to the increasing rectification factor with increasing bath asymmetry appears to be a result of electroconvection in the macropore, with inside diameters (IDs) of 625 and 850-μm. Power spectral density (PSD) analysis reveals chaotic oscillations that are consistent with electroconvection in the I-t data of the 625 and 850-μm ID macropore devices. In the ON state, current rectification keeps ionic transport toward the NCM high, increasing the speed of processes like sample enrichment. A simple means is provided to fabricate fluidic diodes with tailored current rectification factors.

Metabolomics experiments are inevitably subject to a component of unwanted variation, due to factors such as batch effects, long runs of samples, and confounding biological variation. Although the removal of this unwanted variation is a vital step in the analysis of metabolomics data, it is considered a gray area in which there is a recognized need to develop a better understanding of the procedures and statistical methods required to achieve statistically relevant optimal biological outcomes. In this paper, we discuss the causes of unwanted variation in metabolomics experiments, review commonly used metabolomics approaches for handling this unwanted variation, and present a statistical approach for the removal of unwanted variation to obtain normalized metabolomics data. The advantages and performance of the approach relative to several widely used metabolomics normalization approaches are illustrated through two metabolomics studies, and recommendations are provided for choosing and assessing the most suitable normalization method for a given metabolomics experiment. Software for the approach is made freely available.

Carbon stationary phases have been widely used in HPLC due to their unique selectivity and high stability. Amorphous carbon as a stationary phase has at least two sites of interaction with analytes: basal-plane and edge-plane carbon sites. The polarity and adsorptivity of the two sites are different. In this work, the edge-plane carbon stationary phase is prepared by surface-directed liquid crystal assembly. Specific precursor polymers form discotic liquid crystal phases during the pyrolysis process. By using silica as the substrate to align the discotic liquid crystal, edge-plane carbon surfaces were formed. Similar efficiencies as observed for Hypercarb were observed in chromatograms. The column efficiency was studied as a function of linear flow rate. A minimum reduced plate height of 6 was observed in these studies. To evaluate the performance of the homogeneous edge-plane carbon stationary phase, linear solvation energy relationships were used to compare these ordered carbon surfaces to commercially available carbon stationary phases, including Hypercarb. Reversed-phase separations of nucleosides, nucleotides, and amino acids and derivatives were demonstrated using the ordered carbon surfaces, respectively. The column batch-to-batch reproducibility was also evaluated. The retention times for the analytes were reproducible within 1–6% depending on the analyte.

Throughout the fields of biomedical imaging, materials analysis, and routine chemical analysis, it is desirable to have a toolkit of molecules that can allow noninvasive/remote chemical sensing with minimal sample preparation. Here, we describe the photophysical properties involved in photoacoustic (PA) measurements and present a detailed analysis of the requirements and complications involved in PA sensing. We report the use of nitrazine yellow (NY) as a well-behaved PA pH reporter molecule. Both the basic and acidic forms of NY are photoacoustically well-behaved and allow for rapid and noninvasive measurement of pH in either transparent or turbid media. We also find that the serum protein-bound form of NY is photoacoustically well-behaved and should permit applications in noninvasive 3D imaging (e.g., the lymphatic system).

Hydrogen polysulfides (H₂S_n, n > 1), derived from hydrogen sulfide (H₂S), have attracted increasing attention in biochemical research, which may perform as the actual signaling molecules during cell signaling processes. Because of the closed biological and chemical relationship between H₂S and H₂S_n, it is of great value to develop sensitive and specific techniques to distinguish the intracellular level of H₂S_n. To improve the understanding of the physiological and pathological roles played by H₂S_n, we now develop a specific fluorescent probe Mito-ss for capturing H₂S_n in cells and in vivo. When triggered by H₂S_n, Mito-ss replies a turn-on fluorescence signal and exhibits a higher selectivity toward H₂S_n than other abundant competing biothiols, such as glutathione, cysteine and H₂S. The probe Mito-ss can also be applied to visual H₂S_n in living cells, as well as in vivo, providing a potentially powerful approach for probing H₂S_n in biological systems.

It has been observed recently that the resonant Raman scattering (RRS) peak of an X-ray spectrum contains information about the chemical environment of the irradiated matter. This information is extracted with complex processing of the spectrum data. Principal component analysis (PCA) is a statistical multivariate technique that allows exploring the variance–covariance structure of a set of data, through a few linear combinations of the original variables. This methodology can be applied to obtain information from RRS spectra. To analyze its potentiality, several measurements of different oxides in surface nanolayers were measured in total reflection conditions using synchrotron radiation. Multivariate analysis techniques, in particular, PCA, were used to obtain the information encrypted in the RRS peak, and to establish a new methodology, simpler and more accurate. The results show that multivariate analysis techniques are suitable for the analysis of this kind of spectra, foreseeing its application in future research.

Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in the United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS-based approaches cannot detect all GSH conjugates present in complex mixtures, especially those present in extracts of botanical dietary supplements. To overcome these limitations, a fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advanced knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The general new method was then used to test the licorice dietary supplement Glycyrrhiza glabra, which was found to form multiple GSH conjugates upon metabolic activation. Among the GSH conjugates found in the licorice assay were conjugates with isoliquiritigenin and glabridin, which is an irreversible inhibitor of cytochrome P450 enzymes.

Indoor air pollution became a recent concern found to be oftentimes worse than outdoor air quality. We developed a tool that is cheap and simple and enables continuous monitoring of air toxicity. It is a biosensor with both a nondisposable (monitor) and disposable (calcium alginate pads with immobilized bacteria) elements. Various parameters to enhance its signal have been tested (including the effect of the pad’s orientation, it’s exposure to either temperature or time with the air toxicant analyte, and various concentrations thereof). Lastly, the sensor has demonstrated its ability to sense the presence of chemicals in a real, indoor environment. This is the first step in the creation of a sensitive and simple operative tool that may be used in different indoor environments.

In this work, three pieces of historical on-site glass windows dated from the 13th to 16th century and one archeological sample (8th century) showing Mn-rich brown spots at their surface or subsurface have been characterized by optical microscopy and Scanning Electron Microscopy coupled with Energy Dispersive X-ray spectroscopy. The oxidation state of Mn as well as the Mn environment in the alteration phase have been characterized by X-ray absorption spectroscopy at the Mn K-edge. Results show that the oxidation state of Mn and therefore the nature of the alteration phase varies according to the sample considered and is correlated with the extent of the brown alteration. The larger the brown areas the more oxidized the Mn. However, by contrast with literature, the samples presenting the more extended brown areas are not similar to pyrolusite and contain Mn mainly under a (+III) oxidation state.

Maintaining the correct folding of cellular proteins is essential for preserving cellular homeostasis. Protein dishomeostasis, aberrant protein folding, and protein aggregation are indeed involved in several diseases including cancer, aging-associated, and neurodegenerative disorders. Accumulation of protein aggregates can also be induced from a variety of stressful conditions, such as temperature increase or oxidative stress. In this work, we monitored by Fourier transform-infrared (FT-IR) microspectroscopy the response of live breast cancer MCF-7 and mammary breast adenocarcinoma MDA-MB 231 cell lines to severe heat-shock (HS), caused by the rise of the cellular medium temperature from 37 ± 0.5 °C to 42 ± 0.5 °C. Through the study of the time-evolution of the second derivatives of the spectra and by the 2D correlation analysis of FT-IR absorbance data, we were able to identify a common sudden heat-shock response (HSR) among the two cell lines. The hyperfluidization of mammalian cell membranes, the transient increment of the signal lipids, as well as the alteration of proteome profile were all monitored within the first 40 min of stress application, while the persistent intracellular accumulation of extended β-folded protein aggregates was detected after 40 min up to 2 h. As a whole, this paper offers a further prove of the diagnostic capabilities of FT-IR microspectroscopy for monitoring in real-time the biochemical rearrangements undergone by live cells upon external stimulation.

Mitochondrial reactive oxygen species (ROS) and pH fluctuations are closely correlated with mitochondrial dysfunctions, which are implicated in various human diseases including neurodegenerative disorders and cancers. Simultaneously monitoring the changes of ROS and pH of mitochondria remains a major challenge in the mitochondrial biology. In this study, we develop a novel mitochondria-targeted fluorescent nanosensor for real-time imaging of the fluctuations of hydrogen peroxide (H₂O₂) and pH in living cells. The fluorescence probes for detecting pH and H₂O₂ were loaded in the small-sized mesoporous silica nanoparticles (MSN). Then the polyethylenimine was attached to cap the pores of MSN, the triphenylphosphonium was further modified to target mitochondria in living cells. Confocal fluorescence imaging indicated that the nanosensor could effectively target mitochondria and successfully achieved real-time imaging of mitochondrial H₂O₂ and pH fluctuations in living cells. Notably, this is a single nanosensing system that is capable of visualizing multiple subcellular analytes at the same time and position by multicolor fluorescence imaging. The current approach can provide a promising tool to investigate the interplaying roles of various subcellular analytes in living cells.

We describe the construction and characterization of a novel membrane designed to allow proteins separated by gel electrophoresis (SDS-PAGE) to be detected as peptides by mass spectrometry in an efficient and comprehensive manner. The key attribute of the membrane is a bifunctional design that allows for the digestion of protein(s) and retention of the resulting peptides with minimal lateral diffusion. Silane chemistries are used to differentially treat the opposing surfaces of a glass filter paper to enable this unique capability.

A novel hydrophilic fluorescence temperature probe (PNDP) based on polarity-sensitive triarylboron compound (DPTB) and PNIPAM is designed and synthesized. In order to overcome the shortcomings of the single-intensity-based sensing mechanism and obtain more robust signals, ratiometric readout is achieved by designing an efficient FRET system (PNDP-NR) between DPTB and Nile Red (NR). PNDP-NR possesses some excellent features, including wide temperature range, good linear relationship, high temperature resolution, excellent reversibility, and stability. Within a sensing temperature range of 30–55 °C, the fluorescence color of PNDP-NR experiences significant change from red to green-blue. PNDP-NR is also introduced into NIH/3T3 cells to sense the temperature at the single-cell level. It gave excellent photostability and low cytotoxicity in vivo.

Instrumental neutron activation analysis with both relative and k₀ standardization was used in four experienced laboratories to determine element mass fractions in single-wall carbon nanotube certified reference material (CRM) SWCNT-1. Results obtained were evaluated using the National Institute of Standards and Technology (NIST) “Type B On Bias” approach and yielded consensus values in agreement with National Research Council Canada (NRCC) certified values for Fe, Co, Ni, and Mo and provided mass fraction values for 13 additional elements, namely, Na, Mg, Al, K, Ca, Ti, V, Cr, Mn, Br, La, W, and Au. In addition, prompt γ neutron activation analysis was employed to determine mass fractions of H, B, Co, Ni, and Mo. Results of this work provide a basis for the establishment of reference values of element mass fractions in CRM SWCNT-1, thus expanding its usability for more accurate characterization and benchmarking of similar nanotechnology materials.

Digital PCR (dPCR) offers absolute quantification through the limiting dilution of template nucleic acid molecules and has the potential to offer high reproducibility. However, the robustness of dPCR has yet to be evaluated using complex genomes to compare different dPCR methods and platforms. We used DNA templates from the pathogen Mycobacterium tuberculosis to evaluate the impact of template type, master mixes, primer pairs and, crucially, extraction methods on dPCR performance. Performance was compared between the chip (BioMark) and droplet (QX100) formats. In the absence of any external calibration, dPCR measurements were generally consistent within ∼2-fold between different master mixes and primers. Template DNA integrity could influence dPCR performance: high molecular weight gDNA resulted in underperformance of one master mix, while restriction digestion of a low molecular weight sample also caused underestimation. Good concordance (≤1.5-fold difference) was observed between chip and droplet formats. Platform precision was in agreement with predicted Poisson error based on partition number, but this was a minor component (<10%) of the total variance when extraction was included. dPCR offers a robust reproducible method for DNA measurement; however, as a predominant source of error, the process of DNA extraction will need to be controlled with suitable calibrators to maximize agreement between laboratories.

A key requirement of electrospray ionization (ESI) and other techniques facilitating ionization at elevated pressures is the efficient transport of free gas-phase ions into the high vacuum region of the mass spectrometer. Radio frequency (RF) multipole ion guides that allow for collisional cooling are one of the most popular means of achieving this. However, their performance is highly dependent on several experimental factors, including pressure and various electrode potentials along the ion path. To experimentally visualize these effects, we have employed a position-sensitive detector at the exit of a quadrupole mass spectrometer (QMS) instrument operated in RF only mode that employs an RF only octopole as a collisional cooling ion guide. This allows the spatial distribution of the ions, and its dependence on experimentally determined conditions, to be directly visualized at the exit of the quadrupole. This investigation provides a detailed insight into the ion dynamics occurring inside multipole ion guides. This knowledge can directly be applied to instrument development and to improve the ion transmission efficiency and, thus, sensitivity. Numerical simulations using custom-developed trajectory simulation software are compared and contrasted with the experimental observations.

Carbohydrate arrays are potentially one of the most attractive tools to study carbohydrate-based interactions. This paper describes a new analytical platform that exploits metal-enhanced fluorescence for the sensitive and selective screening of carbohydrate-lectin interactions. The chip consists of a glass slide covered with gold nanostructures, postcoated with a thin layer of amorphous silicon–carbon alloy (a-Si_0.8C_0.2:H). An immobilization strategy based on the formation of a covalent bond between propargyl-terminated glycans and surface-linked azide groups was used to attach various glycans at varying surface densities onto the interface and to fabricate a carbohydrate array via efficient local “click” chemistry strategy. The specific association of the new interface with fluorescently labeled lectins was assessed by fluorescence imaging and an excellent selectivity to specific proteins was achieved. Optimization of the surface architecture and the plasmonic transducer resulted in an enhancement of the fluorescence intensity by 1 order of magnitude, when compared to the corresponding substrate devoid of gold nanostructures. The limit of detection (LOD) of such microarrays is in the picomolar range, making it a promising system for development in pharmaceutical or biomedical applications.

Many mobility studies (IMS) of electrospray ions with charge states z reduced to unity have shown a singular ability to analyze large protein complexes and viruses, though with wide mobility peaks (fwhm ∼ 20%). Here we confirm that this limitation arises primarily when early charge reduction precedes drop evaporation (suppressing secondary atomization by the usual sequence of many Coulomb explosions). By drying before neutralizing, we achieve a protein fwhm of ∼3.7%. A positively biased electrospraying capillary is coaxial with a cylindrical charge-reduction (CR) chamber coated with radioactive Ni-63 (10 mCi) that fills the CR chamber with a bipolar ionic atmosphere. A screen interposed between the spraying capillary and the CR chamber limits penetration of the neutralizing anions into the electrospray (ES) chamber, precluding destabilization of the ES tip, even when brought very close to the grid to enhance ion transmission. As ES cations cross the grid, driven by their own space charge, they recombine with CR ions reducing their charge state as well as space charge dispersion. The setup is tested with the protein ovalbumin (M_W ∼ 44.3 kDa) and its clusters up to the tetramer, by analyzing the charge-reduced ions with a differential mobility analyzer (DMA). At gas sample flow rates of ∼1 L/min, the dominant peaks are singly charged (z = 1). They are widened by clustering of involatile solution impurities, depending on spray quality and solution cleanness, with fwhm as small as 3.7% achieved in desalted and acidified solutions. When using sharp nanospray capillaries, the grid may be removed, resulting in ∼2-fold increased ion transmission. In the absence of the grid, however, spray stability and quality are often compromised, even with capillary tip diameters as small as 30 μm.

We fabricated a multifunctional theragnostic agent Ag-Sgc8-FAM for apoptosis-based cancer therapy and fluorescence-enhanced cell imaging. For cancer therapy, aptamers Sgc8 and TDO5 acted as recognizing molecules to bind CCRF-CEM and Ramos cells specifically. It was found that aptamer–silver conjugates (Ag-Sgc8, Ag-TDO5) could be internalized into cells by receptor-mediated endocytosis, inducing specific apoptosis of CCRF-CEM and Ramos cells. The apoptosis of cells depended on the concentration of aptamer–silver conjugates, as well as the incubation time between cells and aptamer-silver conjugates. The apoptotic effects on CCRF-CEM and Ramos cells were different. Annexin V/PI staining, AO/PI staining, MTT assays and ROS (reactive oxygen species) detection demonstrated the specific apoptosis of CCRF-CEM and Ramos cells. For fluorescence-enhanced cell imaging, Ag-Sgc8-FAM was prepared. Compared to Sgc8-FAM molecules, Ag-Sgc8-FAM was an excellent imaging agent as numerous Sgc8-FAM molecules were enriched on the surface of AgNPs for multiple binding with CCRF-CEM cells and signal amplification. Moreover, AgNPs could increase the fluorescence intensity of FAM by metal-enhanced fluorescence (MEF) effect. Therefore, aptamer–silver conjugates can be potential theragnostic agents for inducing specific apoptosis of cells and achieving cells imaging in real time.

Flow reactors, denuders, and sampling tubes are essential tools for many applications in analytical and physical chemistry and engineering. We derive a new method for determining radial diffusion effects and the penetration or transmission of gas molecules and aerosol particles through cylindrical tubes under laminar flow conditions using explicit analytical equations. In contrast to the traditional Brown method [Brown, R. L. J. Res. Natl. Bur. Stand. (U. S.) 1978, 83, 1−8] and CKD method (Cooney, D. O.; Kim, S. S.; Davis, E. J. Chem. Eng. Sci. 1974, 29, 1731−1738), the new approximation developed in this study (known as the KPS method) does not require interpolation or numerical techniques. The KPS method agrees well with the CKD method under all experimental conditions and also with the Brown method at low Sherwood numbers. At high Sherwood numbers corresponding to high uptake on the wall, flow entry effects become relevant and are considered in the KPS and CKD methods but not in the Brown method. The practical applicability of the KPS method is demonstrated by analysis of measurement data from experimental studies of rapid OH, intermediate NO₃, and slow O₃ uptake on various organic substrates. The KPS method also allows determination of the penetration of aerosol particles through a tube, using a single equation to cover both the limiting cases of high and low deposition described by Gormley and Kennedy (Proc. R. Ir. Acad., Sect. A. 1949, 52A, 163−169). We demonstrate that the treatment of gas and particle diffusion converges in the KPS method, thus facilitating prediction of diffusional loss and penetration of gases and particles, analysis of chemical kinetics data, and design of fluid reactors, denuders, and sampling lines.

This work describes the first multiple spiking isotope dilution procedure for organic compounds using ¹³C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine–creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled ¹³C analogues (¹³C₁-creatinine and ¹³C₂-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine–creatinine interconversion during LC-MS/MS and gas chromatography–mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography–isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine–creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors derived from the sample preparation step.

A paper test card has been engineered to perform an iodometric titration, an application that requires storage and mixing on demand of several mutually incompatible reagents. The titration is activated when a user applies a test solution to the test card: the dried reagents are reconstituted and combined through a surface-tension-enabled mixing (STEM) mechanism. The device quantifies 0.8–15 ppm of iodine atoms from iodate in aqueous solutions. This is useful, for example, to quantify iodine levels in fortified salt. A blinded internal laboratory validation established the accuracy as 1.4 ppm I and the precision as 0.9 ppm I when the test card was read by newly trained users. Using computer software to process images, the accuracy and precision both improved to 0.9 ppm I. The paper card can also detect substandard β lactam antibiotics using an iodometric back-titration. When used to quantify amoxicillin, good distinction is achieved between solutions that differ by 0.15 mg/mL over a working range of 0–0.9 mg/mL. The test card was designed to meet the World Health Organization ASSURED criteria for use in low resource settings, where laboratory-based analytical procedures are often not available.

Point-of-care (POC) testing has become widely used in clinical analysis because of its speed and portability; however, POC tools, such as lateral flow assays, suffer from low specificity, unclear readouts, and susceptibility to environmental and user errors. Herein, we report an ELISA-based competitive volumetric bar-chart chip (CV-chip) that eliminates these limitations. The CV-chip displays the readout in the form of ink bar charts based on direct competition between gases generated by the sample and the internal control. By employing a “competition mode”, this platform decreases the potential influence of background resulting from environmental factors and provides visually clear positive or negative results without the requirement of calibration. In addition, the on-chip comparison enables the device to distinguish imperceptible differences (less than 1.3-fold) in human chorionic gonadotropin (hCG) concentrations that are near the cutoff value for pregnancy (∼1.4 ng/mL). We also utilized the ELISA-based CV-chip to successfully detect biomarkers from cancer cells. As a proof-of-concept application in a clinical setting, the CV-chip was employed to evaluate the status of drugs of abuse in 18 patients. For six different drugs, zero false-positive and very few false-negative (<2%) results were reported in more than 100 tests. This new ELISA platform offers a clinical diagnostics tool that is portable and easy to use, and provides improved clarity and sensitivity due to the inclusion of a real-time internal control.

A theoretical model is proposed to describe the steady-state average limiting current associated with a single redox moiety (ox or red) trapped in a closed generator-collector system along with excess supporting electrolyte. By “closed” we mean that neither solvent nor solutes can enter or leave the system. The potential difference, E_OE – E_RE, between the oxidizing electrode (OE) and the reducing electrode (RE) is maintained constant with the values of E_OE and E_RE chosen so that the operative faradaic electrode processes are very fast, i.e., red = ox + n_ETe^– (k_ox = ∞) at the OE and ox + n_ETe^– = red (k_red = ∞) at the RE. Because there is only a single redox moiety the faradaic process occurs at only one electrode at a time while current at the other electrode is purely capacitive (we refer to this as capacitive coupling). We propose that a two-step process is required to transfer n_ETq_e coulombs (q_e is the absolute value of the elemental electronic charge). The first step is associated with diffusion (approximated as a random walk) of a single red moiety to the OE where it is oxidized to ox with a concomitant transfer of q_step1 (= n_ETq_e/(1 + A_OEC_OE/A_REC_RE)) coulombs; the second step is associated with the diffusion (random walk) of the newly formed single ox moiety to the RE with the concomitant transfer of q_step2 (= n_ETq_e/(1 + A_REC_RE/A_OEC_OE)) coulombs (A_RE,A_OE andC_RE,C_OEare the areas (cm²) and differential capacitances (farads cm^–2) of the corresponding electrodes). The total charge transferred in the two steps is n_ETq_e(= q_step1 + q_step2). Transport of the redox moiety from one electrode to the other is accomplished by a random walk. The probability density function (pdf) and cumulative density function (CDF) for the duration of a full redox cycle are presented as the analytical solution to a 1-dimensional bounded random-walk problem (confirmed by numerical simulation). These show that t_full, the average time for the full redox cycle (step 1 + step 2), is equal to L²/D where L is the intraelectrode distance and D is the diffusion coefficient. The average steady-state limiting current is shown to be described by the familiar expression for a generator-collector system: i_lim = (q_step1 + q_step2)/t_full = n_ETq_e/t_full = n_ETq_eD/L².

The bioproduction of recombinant monoclonal antibodies results in complex mixtures of a main isoform and numerous macro- and microvariants. Monoclonal antibodies therefore present different levels of heterogeneities ranging from primary sequence variants to post-translational modifications. Among these heterogeneities, the truncation and fragmentation of the primary amino-acid sequence result in shorter or cleaved polypeptide chains while the incomplete processing of the signal peptide produces N-terminal elongated polypeptide chains. Here, we present an in-gel protein N-terminal chemical derivatization method using (N-succinimidyloxycarbonylmethyl)-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This chemical tag enhances the detection by mass spectrometry of the N-terminal positions of proteins and allows their unambiguous assignment without altering the identification of internal digestion peptides. This method adds just one step to the classical peptide mapping workflow. Using this in-gel N-TOP (N-terminal oriented proteomics) strategy, the N-terminal sequence heterogeneities of several monoclonal antibodies, among which are minor unexpected proteoforms, were successfully detected and characterized.

Shell-isolated gold nanoparticles (SHINs) were employed to record shell-isolated nanoparticle-enhanced Raman spectra (SHINERS) of a passive layer formed at a gold surface during gold leaching from thiosulfate solutions. The (3-aminopropyl)triethoxysilane (APTES) and a sodium silicate solution were used to coat gold nanoparticles with a protective silica layer. This protective silica layer prevented interactions between the thiosulfate electrolyte and the gold core of the SHINs when the SHINs-modified gold electrode was immersed into the thiosulfate lixiviant. The SHINERS spectra of the passive layer, formed from thiosulfate decomposition, contained bands indicative of hydrolyzed APTES. We have demonstrated how to exploit the presence of these APTES bands as an internal standard to compensate for fluctuations of the surface enhancement of the electric field of the photon. We have also developed a procedure that allows for removal of the interfering APTES bands from the SHINERS spectra. These methodological advancements have enabled us to identify the species forming the passive layer and to determine that the formation of elemental sulfur, cyclo-S₈, and polymeric sulfur chains is responsible for inhibition of gold dissolution in oxygen rich thiosulfate solutions.

Glutamine is one of the most abundant metabolites in blood and is a precursor as well as end product central to numerous important metabolic pathways. A number of surprising and unexpected roles for glutamine, including cancer cell glutamine addiction discovered recently, stress the importance of accurate analysis of glutamine concentrations for understanding its role in health and numerous diseases. Utilizing a recently developed NMR approach that offers access to an unprecedented number of quantifiable blood metabolites, we have identified a surprising glutamine cyclization to pyroglutamic acid that occurs during protein removal. Intact, ultrafiltered and protein precipitated samples from the same pool of human serum were comprehensively investigated using ¹H NMR spectroscopy at 800 MHz to detect and quantitatively evaluate the phenomenon. Interestingly, although glutamine cyclization occurs in both ultrafiltered and protein precipitated serum, the cyclization was not detected in intact serum. Strikingly, due to cyclization, the apparent serum glutamine level drops by up to 75% and, concomitantly, the pyroglutamic acid level increases proportionately. Further, virtually under identical conditions, the magnitude of cyclization is vastly different for different portions of samples from the same pool of human serum. However, the sum of glutamine and pyroglutamic acid concentrations in each sample remains the same for all portions. These unexpected findings indicate the importance of considering the sum of apparent glutamine and pyroglutamic acid levels, obtained from the contemporary analytical methods, as the actual blood glutamine level for biomarker discovery and biological interpretations.

In this paper, we report on the identification of batches of analgesic paracetamol (acetaminophen) tablets using nitrogen-14 nuclear quadrupole resonance spectroscopy (¹⁴N NQR). The high sensitivity of NQR to the electron charge distribution surrounding the quadrupolar nucleus enables the unique characterization of the crystal structure of the material. Two hypothesis were tested on batches of the same brand: the within the same batch variability and the difference between batches that varied in terms of their batch number and expiry date. The multivariate analysis of variance (MANOVA) did not provide any within-batches variations, indicating the natural deviation of a medicine manufactured under the same conditions. Alternatively, the statistical analysis revealed a significant discrimination between the different batches of paracetamol tablets. Therefore, the NQR signal is an indicator of factors that influence the physical and chemical integrity of the material. Those factors might be the aging of the medicine, the manufacturing, or storage conditions. The results of this study illustrate the potential of NQR as promising technique in applications such as detection and authentication of counterfeit medicines.

Comprehensive two-dimensional (2D) gas chromatography coupled with time-of-flight mass spectrometry (GC × GC–TOFMS) is a versatile instrumental platform capable of collecting highly informative, yet highly complex, chemical data for a variety of samples. Fisher-ratio (F-ratio) analysis applied to the supervised comparison of sample classes algorithmically reduces complex GC × GC–TOFMS data sets to find class distinguishing chemical features. F-ratio analysis, using a tile-based algorithm, significantly reduces the adverse effects of chromatographic misalignment and spurious covariance of the detected signal, enhancing the discovery of true positives while simultaneously reducing the likelihood of detecting false positives. Herein, we report a study using tile-based F-ratio analysis whereby four non-native analytes were spiked into diesel fuel at several concentrations ranging from 0 to 100 ppm. Spike level comparisons were performed in two regimes: comparing the spiked samples to the nonspiked fuel matrix and to each other at relative concentration factors of two. Redundant hits were algorithmically removed by refocusing the tiled results onto the original high resolution pixel level data. To objectively limit the tile-based F-ratio results to only features which are statistically likely to be true positives, we developed a combinatorial technique using null class comparisons, called null distribution analysis, by which we determined a statistically defensible F-ratio cutoff for the analysis of the hit list. After applying null distribution analysis, spiked analytes were reliably discovered at ∼1 to ∼10 ppm (∼5 to ∼50 pg using a 200:1 split), depending upon the degree of mass spectral selectivity and 2D chromatographic resolution, with minimal occurrence of false positives. To place the relevance of this work among other methods in this field, results are compared to those for pixel and peak table-based approaches.

The study of arctic or subarctic indigenous skin clothing material, known for its design and ability to keep the body warm, provides information about the tanning materials and techniques. The study also provides clues about the culture that created it, since tanning processes are often specific to certain indigenous groups. Untreated skin samples and samples treated with willow (Salix sp) bark extract and cod liver oil are compared in this study using both MRI and unilateral NMR techniques. The two types of samples show different proton spatial distributions and different relaxation times, which may also provide information about the tanning technique and aging behavior.

We report on a silver decahedral nanoparticles (Ag₁₀NPs)-based FRET (fluorescence resonance energy transfer) sensor for target cell imaging. Fluorophores-functionalized aptamers (Sgc8-FITC) were bound with Ag₁₀NPs via the SH group on the aptamer to form Ag₁₀-Sgc8-FITC. Then, quencher-carrying strands (BHQ-1) were hybridized with Sgc8-FITC to form a Ag₁₀NPs-based FRET sensor (Ag₁₀-Sgc8-F/Q). The sensor interacted with membrane protein tyrosine kinase-7 (PTK-7) on the CCRF-CEM (CCL-119, T-cell line, human acute lymphoblastic leukemia) cell surface to attain fluorescence imaging of CCRF-CEM cells. The addition of CCRF-CEM cells resulted in many sensors binding with cells membrane and the displacement of BHQ-1, thus disrupting the FRET effect and the enhanced fluorescence intensity of FITC. It was found that Ag₁₀NPs largely enhanced the fluorescence intensity of FITC. The results also showed that the Ag₁₀NPs-based FRET sensor (Ag₁₀-Sgc8-F/Q) was not only superior to the bare FRET sensor (Sgc8-F/Q) and sensor Ag-Sgc8-F/Q but also highly sensitive and specific for CCRF-CEM cells imaging.

Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis–Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery.

The integration of diagnostic and therapeutic functions in a single system holds great promise to enhance the theranostic efficacy and prevent the under- or overtreatment. Herein, a folate receptor-targeted and cathepsin B-activatable nanoprobe is designed for background-free cancer imaging and selective therapy. The nanoprobe is prepared by noncovalently assembling phospholipid-poly(ethylene oxide) modified folate and photosensitizer-labeled peptide on the surface of graphene oxide. After selective uptake of the nanoprobe into lysosome of cancer cells via folate receptor-mediated endocytosis, the peptide can be cleaved to release the photosensitizer in the presence of cancer-associated cathepsin B, which leads to 18-fold fluorescence enhancement for cancer discrimination and specific detection of intracellular cathepsin B. Under irradiation, the released photosensitizer induces the formation of cytotoxic singlet oxygen for triggering photosensitive lysosomal cell death. After lysosomal destruction, the lighted photosensitizer diffuses from lysosome into cytoplasm, which provides a visible method for in situ monitoring of therapeutic efficacy. The nanoprobe exhibits negligible dark toxicity and high phototoxicity with the cell mortality rate of 0.06% and 72.1%, respectively, and the latter is specific to folate receptor-positive cancer cells. Therefore, this work provides a simple but powerful protocol with great potential in precise cancer imaging, therapy, and therapeutic monitoring.

Small, robust, sensitive electrodes are desired for in vivo neurotransmitter measurements. Carbon nanopipettes have been previously manufactured and used for single-cell drug delivery and electrophysiological measurements. Here, a modified fabrication procedure was developed to produce batches of solid carbon nanopipette electrodes (CNPEs) with ∼250 nm diameter tips, and controllable lengths of exposed carbon, ranging from 5 to 175 μm. The electrochemical properties of CNPEs were characterized with fast-scan cyclic voltammetry (FSCV) for the first time. CNPEs were used to detect the electroactive neurotransmitters dopamine, serotonin, and octopamine. CNPEs were significantly more sensitive for serotonin detection than traditional carbon-fiber microelectrodes (CFMEs). Similar to CFMEs, CNPEs have a linear response for dopamine concentrations ranging from 0.1 to 10 μM and a limit of detection of 25 ± 5 nM. Recordings with CNPEs were stable for over 3 h when the applied triangle waveform was scanned between −0.4 and +1.3 V vs Ag/AgCl/Cl^– at 400 V/s. CNPEs were used to detect endogenous dopamine release in Drosophila larvae using optogenetics, which verified the utility of CNPEs for in vivo neuroscience studies. CNPEs are advantageous because they are 1 order of magnitude smaller in diameter than typical CFMEs and have a sharp, tunable geometry that facilitates penetration and implantation for localized measurements in distinct regions of small organisms, such as the Drosophila brain.

Porous anodized aluminum oxide (pAAO) is a nanostructured material, which due to its optical properties lends itself to the design of optical biosensors where interactions in the pores of this material are transduced into interferometric reflectance shifts. In this study, a pAAO-based biosensor was developed as a biosensing platform to detect proteinase K, an enzyme which is a readily available model system for the proteinase produced by Pseudomonas aeruginosa. The pAAO pore walls are decorated by means of the layer-by-layer (LbL) deposition technique using poly(sodium-4-styrenesulfonate) and poly-l-lysine as negatively and positively charged polyelectrolytes, respectively. Interferometric reflectance spectroscopy utilized to observe the optical properties of pAAO during LbL deposition shows that the deposition of the polyelectrolyte onto the pore walls increases the net refractive index, thus red-shifting the effective optical thickness (EOT). Upon incubation with proteinase K, a conspicuous blue shift of the EOT is observed, which is attributed to the destabilization of the LbL film upon enzymatic degradation of the poly-l-lysine components. This result is confirmed by scanning electron microscopy results. Finally, as a proof-of-principle, we demonstrate the ability of the label-free pAAO-based biosensing platform to detect the presence of the proteinase K in human wound fluid, highlighting the potential for detection of bacterial infections in chronic wounds.

A novel strategy is introduced that combines high-resolution mass spectrometry (MS) with NMR for the identification of unknown components in complex metabolite mixtures encountered in metabolomics. The approach first identifies the chemical formulas of the mixture components from accurate masses by MS and then generates all feasible structures (structural manifold) that are consistent with these chemical formulas. Next, NMR spectra of each member of the structural manifold are predicted and compared with the experimental NMR spectra in order to identify the molecular structures that match the information obtained from both the MS and NMR techniques. This combined MS/NMR approach was applied to Escherichia coli extract, where the approach correctly identified a wide range of different types of metabolites, including amino acids, nucleic acids, polyamines, nucleosides, and carbohydrate conjugates. This makes this approach, which is termed SUMMIT MS/NMR, well suited for high-throughput applications for the discovery of new metabolites in biological and biomedical mixtures, overcoming the need of experimental MS and NMR metabolite databases.

Two types of plasmonic silver- and gold-coated grating biosensor chips (plasmonic chip) were applied in the detection of α-fetoprotein (AFP) with a sandwich imunoassay and surface plasmon field-enhanced fluorescence. On the plasmonic chip, unlabeled marker in the sandwich immunoassay was first quantitatively detected over a wide range between 10^–12 and 10^–8 g/mL. The affinity constants between AFP and anti-AFP antibody, which were obtained by fitting the experimental data to the Langmuir isotherm adsorption curve, were 1 × 10⁸ g^–1 mL regardless of the kind of metal in the plasmonic chips. Although the fluorescence intensity on the silver plasmonic chip was 5 times larger than that on the gold plasmonic chip, the limit of detection (LOD) was on the order of 10^–11 g/mL and not improved with a silver plasmonic chip. Herein, we used a new setup that generated less dispersions of both the fluorescence intensity for nonspecific adsorption and the background (optical blank) signal and improved the LOD of AFP to 4 pg/mL (55 fM) with the silver plasmonic chip. With the highly sensitive detection in the sandwich immunoassay, the development of a plasmonic chip for clinical diagnosis by a blood test is promising.

In this work, we investigated the interactions between graphene oxide (GO) and conjugated polyelectrolytes (CPEs) with different backbone and side chain structures. By studying the mechanism of fluorescence quenching of CPEs by GO, we find that the charge and the molecular structure of CPEs play important roles for GO–CPEs interactions. Among them, electrostatic interaction, π–π interaction, and cation−π bonding are dominant driving forces. By using a cationic P2, we have developed a sensitive homogeneous sensor for DNA and RNA detection with a detection limit of 50 pM DNA and RNA, which increased the sensitivity by 40-fold as compared to GO-free CPE-based sensors. This GO-assisted CPE sensing strategy is also generic and shows a high potential for biosensor designs based on aptamers, proteins, peptides, and other biological probes.

We report a high-resolution, traceable method to quantify number concentrations and dimensional properties of nanosheet graphene oxide (N-GO) colloids using electrospray-differential mobility analysis (ES-DMA). Transmission electron microscopy (TEM) was employed orthogonally to provide complementary data and imagery of N-GOs. Results show that the equivalent mobility sizes, size distributions, and number concentrations of N-GOs were able to be successfully measured by ES-DMA. Colloidal stability and filtration efficiency of N-GOs were shown to be effectively characterized based on the change of size distributions and number concentrations. Through the use of an analytical model, the DMA data were able to be converted into lateral size distributions, showing the average lateral size of N-GOs was ∼32 nm with an estimated thickness ∼0.8 nm. This prototype study demonstrates the proof of concept of using ES-DMA to quantitatively characterize N-GOs and provides traceability for applications involving the formulation of N-GOs.

Nature owns remarkable capabilities in sensing target molecules, while the artificial biosensor lags far behind nature. Inspired by nature, we devise a new sensing platform that can specifically bind the molecules and synchronously initiate a specific signal response. We rationally designed a type of bipolar probe that is comprised of a hydrophilic DNA part and a hydrophobic conjugated polymer (CP) unit. In aqueous solution, they can form micelles with a hydrophobic CP core and a hydrophilic DNA shell. The aggregation-caused quenching suppresses the fluorescence of CP. Adding telomerase, the hydropathical profile of the bipolar probes is drastically regulated that results in the collapse of micelles and liberates fluorescence simultaneously. The probe has been used in both mimic systems and real urine samples (38 samples). We achieve sensitive and specific detection of telomerase and obtain clearly classification for normal people and cancer patients. It can also be used in a signal off sensor that is used to detect mercury ions.

High-throughput, quantitative, and rapid microfluidic-based separations has been a long-sought goal for applications in proteomics, genomics, biomarker discovery, and clinical diagnostics. Using droplet-interfaced microchip electrophoresis (MCE) techniques, we have developed a novel parallel MCE platform, based on the concept of combining the Slipchip principle with a newly developed “Gelchip”. The platform consists of two plastic plates, with droplet wells on one plate and separation channels with preloaded/cured gel in the other. A single relative movement of one plate enables generation and then loading of multiple sample droplets in parallel into the separation channels, allowing electrophoretic separation of biomolecules in the droplets in parallel and with high-throughput. As proof of concept, we demonstrated the separation of 30 sub-nL sample droplets containing fluorescent dyes or DNA fragments.

There is great interest in the development of integrated tools allowing for miniaturized sample processing, including solid phase extraction (SPE). We introduce a new format for microfluidic SPE relying on C18-functionalized magnetic beads that can be manipulated in droplets in a digital microfluidic platform. This format provides the opportunity to tune the amount (and potentially the type) of stationary phase on-the-fly, and allows the removal of beads after the extraction (to enable other operations in same device-space), maintaining device reconfigurability. Using the new method, we employed a design of experiments (DOE) operation to enable automated on-chip optimization of elution solvent composition for reversed phase SPE of a model system. Further, conditions were selected to enable on-chip fractionation of multiple analytes. Finally, the method was demonstrated to be useful for online cleanup of extracts from dried blood spot (DBS) samples. We anticipate this combination of features will prove useful for separating a wide range of analytes, from small molecules to peptides, from complex matrices.

A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins: botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5, while the C-terminus of the heavy chain (H_C) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity toward SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.

A method utilizing competitive adsorption between polyethylene glycols (PEGs) and labeled protein to nanoparticles was developed for the determination of PEG molecular weight (MW) in a microtiter plate format. Two mix-and-measure systems, time-resolved luminescence resonance energy transfer (TR-LRET) with donor europium(III) polystyrene nanoparticles and acceptor-labeled protein and quenching with quencher gold nanoparticles and fluorescently labeled protein were compared for their performance. MW is estimated from the PEG MW dependent changes in the competitive adsorption properties, which are presented as the luminescence signal vs PEG mass concentration. The curves obtained with the TR-LRET system overlapped for PEGs larger than 400 g/mol providing no information on MW. Distinctly different curves were obtained with the quenching system enabling the assessment of PEG MW within a broad dynamic range. The data was processed with and without prior knowledge of the PEG concentration to measure PEGs over a MW range from 62 to 35 000 g/mol. The demonstration of the measurement independent of the PEG concentration suggests that the estimation of MW is possible with quenching nanoparticle system for neutrally charged and relatively hydrophilic polymeric molecules widening the applicability of the simple and cost-effective nanoparticle-based methods.

N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli l-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-d-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an l-amino acid acylase and its subsequent oxidation by an FAD-dependent l-amino acid oxidase (l-AAO). Cofactor recycling of the l-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-d-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.

A calcium fluoride (CaF₂) plate was exposed to pulsed laser irradiations inducing surface morphological and ionization changes on its surface. More precisely surface damages mainly correspond to intrinsic defects. Electron paramagnetic resonance (EPR) hyperspectral imaging is a powerful technique able to characterize the defects formed on the CaF₂ surface. Indeed, EPR hyperspectral images provide spatial and spectral information about the sample studied. In fact, these images possess a great potential to obtain accurate and reliable knowledge about the chemical composition and the distribution of the component due to the presence of the spatial aspect. However, the complexity of such hyperspectral data sets imposes the use of advanced chemometric tools to extract valuable information on the considered physicochemical system. Therefore, Multivariate Curve Resolution–Alternating Least Squares (MCR-ALS) is proposed to identify and locate the different constituents in the images. The originality of this work is that it reports on the application of MCR-ALS, for the first time, on electron paramagnetic resonance (EPR) imaging data sets that will furnish the distribution maps and the spectral signatures of all components present in the sample. The results show the identification of different intrinsic defects on a CaF₂ sample from the sole information in the raw image measurements and, therefore, confirm the potential of this methodology and the important role of spatial information contained in the image.

The sensitive identification of single nucleotide polymorphisms becomes increasingly important for disease diagnosis, prevention, and practical applicability of pharmacogenomics. Herein, we propose a simple, highly selective, label-free single nucleotide polymorphisms (SNPs) sensing device by electrochemically monitoring the diffusion flux of ferricyanide probe across probe DNA/morpholino duplex functionalized nanochannels of porous anodic alumina. When perfectly matched or mismatched target DNA flows through the nanochannels modified with probe DNA/morpholino duplex, it competes for the probe DNA from morpholino, resulting in a change of the surface charges. Thus, the diffusion flux of negatively charged electroactive probe ferricyanide is modulated since it is sensitive to the surface charge due to the electrostatic interactions in electric double layer-merged nanochannels. Monitoring of the change in diffusion flux of probe enables us to detect not only a single base or two base mismatched sequence but also the specific location of the mismatched base. As is demonstrated, SNPs in the PML/RARα fusion gene, known as a biomarker of acute promyelocytic leukemia (APL), have been successfully detected.

Solid-state dye-sensitized solar cells (sDSCs) are devoid of such issues as electrolyte evaporation or leakage and electrode corrosion, which are typical for traditional liquid electrolyte-based DSCs. Poly(3,4-ethylenedioxythiophene) (PEDOT) is one of the most popular and efficient p-type conducting polymers that are used in sDSCs as a solid-state hole-transporting material. The most convenient way to deposit this insoluble polymer into the dye-sensitized mesoporous working electrode is in situ photoelectrochemical polymerization. Apparently, the structure and the physicochemical properties of the generated conducting polymer, which determine the photovoltaic performance of the corresponding solar cell, can be significantly affected by the preparation conditions. Therefore, a simple and fast analytical method that can reveal information on polymer chain length, possible chemical modifications, and impurities is strongly required for the rapid development of efficient solar energy-converting devices. In this contribution, we applied matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for the analysis of PEDOT directly on sDSCs. It was found that the PEDOT generated in aqueous micellar medium possesses relatively shorter polymeric chains than the PEDOT deposited from an organic medium. Furthermore, the micellar electrolyte promotes a transformation of one of the thiophene terminal units to thiophenone. The introduction of a carbonyl group into the PEDOT molecule impedes the growth of the polymer chain and reduces the conductivity of the final polymer film. Both the simplicity of sample preparation (only application of the organic matrix onto the solar cell is needed) and the rapidity of analysis hold the promise of making MALDI MS an essential tool for the physicochemical characterization of conducting polymer-based sDSCs.

We report the use of multifunctional dendrimer-entrapped gold nanoparticles (Au DENPs) loaded with gadolinium (Gd) chelator/Gd(III) complexes and surface-modified with thiolated cyclo(Arg-Gly-Asp-Phe-Lys(mpa)) (RGD) peptide for targeted dual-mode computed tomography (CT)/magnetic resonance (MR) imaging of small tumors. In this study, amine-terminated generation 5 poly(amidoamine) dendrimers were used as a nanoplatform to be covalently modified with Gd chelator, RGD via a polyethylene glycol (PEG) spacer, and PEG monomethyl ether. Then the multifunctional dendrimers were used as templates to entrap gold nanoparticles, followed by chelating Gd(III) ions and acetylation of the remaining dendrimer terminal amines. The thus-formed multifunctional Au DENPs (in short, Gd–Au DENPs-RGD) were characterized via different techniques. We show that the multifunctional Au DENPs with a Au core size of 3.8 nm are water-dispersible, stable under different pH (5–8) and temperature conditions (4–50 °C), and noncytotoxic at a Au concentration up to 100 μM. With the displayed X-ray attenuation property and the r₁ relaxivity (2.643 mM^–1 s^–1), the developed Gd–Au DENPs-RGD are able to be used as a dual-mode nanoprobe for targeted CT/MR imaging of an α_vβ₃ integrin-overexpressing xenografted small tumor model in vivo via RGD-mediated active targeting pathway. The developed multifunctional Gd–Au DENPs-RGD may be used as a promising dual-mode nanoprobe for targeted CT/MR imaging of different types of α_vβ₃ integrin-overexpressing cancer.

A sensitive electrochemical sensor is designed for DNA detection based on mimetic catalysis of metal–organic framework (MOF) and allosteric switch of hairpin DNA. The functional MOFs are synthesized as signal probes by a one-pot encapsulation of iron(III) meso-5,10,15,20-tetrakis(4-carboxyphenyl) porphyrin chloride (FeTCPP) into a prototypal MOF, HKUST-1(Cu), and sequentially conjugated with streptavidin (SA) as a recognition element. The resulting FeTCPP@MOF composites can mimetically catalyze the oxidation of o-phenylenediamine (o-PD) to 2,2′-diaminoazobenzene, which is a good electrochemical indicator for signal readout. The presence of target DNA introduces the allosteric switch of hairpin DNA to form SA aptamer, and thus, FeTCPP@MOF-SA probe is brought on the electrode surface via the specific recognition between SA and the corresponding aptamer, resulting in the enhancement of electrochemical signal. The “signal-on” electrochemical sensor can detect target DNA down to 0.48 fM with the linear range of 10 fM to 10 nM. Moreover, the MOF-based electrochemical sensor exhibits acceptable selectivity against even a single mismatched DNA and good feasibility in complex serum matrixes. This strategy opens up a new direction of porphyrin-functionalized MOF for signal transduction in electrochemical biosensing.

We report the first assessment of the plasmonic biosensing capabilities of capping agent-free gold nanostars. Capping agent removal was carried out using aqueous solutions of sodium borohydride, which yielded a refractive index sensitivity of 474 nm/RIU for the polyvinylpyrrolidone (PVP)-free nanostars compared with 98 nm/RIU for PVP-coated gold nanostars. Following PVP removal, biotinylated thiol and streptavidin protein were added to the nanostars, which resulted in red shifts as large as 51 nm and a limit of detection as low as 0.1 pM. Refractive index-based sensing of prostate specific antigen (PSA) both in buffer and serum was then carried out and was shown to yield shifts as large as 127 nm and have a limit of detection of 100 pM in serum. Last, a sandwich assay involving PSA was developed to aggregate the nanostars together for greater sensitivity. The sandwich assay did, indeed, give shifts close to 200 nm and was capable of detecting 10^–17 M PSA in serum. The greatly increased sensitivity and amenability to functionalization of PVP-free gold nanostars should prove useful in applications ranging from catalysis to drug delivery.

The effectiveness of the supercharging reagents m-nitrobenzyl alcohol (m-NBA) and propylene carbonate at producing highly charged protein ions in electrospray ionization is compared. Addition of 5% m-NBA or 15% propylene carbonate increases the average charge of three proteins by ∼21% or ∼23%, respectively, when these ions are formed from denaturing solutions (water/methanol/acetic acid). These results indicate that both reagents are nearly equally effective at supercharging when used at their optimum concentrations. A narrowing of the charge state distribution occurs with both reagents, although this effect is greater for propylene carbonate. Focusing the ion signal into fewer charge states has the advantage of improving sensitivity. The maximum charge state of ubiquitin formed with propylene carbonate is 21+, four charges higher than previously reported. Up to nearly 30% of all residues in a protein can be charged, and the collisional cross sections of the most highly charged ions of both ubiquitin and cytochrome c formed with these supercharging reagents were measured for the first time and found to be similar to those calculated for theoretical highly extended, linear or near-linear conformations. Under native supercharging conditions, m-NBA is significantly more effective at producing high charge states than propylene carbonate.

Mesenchymal stem cells (MSC) have the ability to self-renew and differentiate into multiple cell types valuable for clinical treatment of rheumatic pathologies. To study the chondrogenic potential of MSC and identify the conditions that recreate the native cartilage environment, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) for label-free detection of cell-type- and environmental-condition-specific molecular profiles. We observed that coculture of human MSC and chondrocytes under standard culture conditions leads to improved extracellular matrix (ECM) deposition. In marked contrast, this effect was lost under low oxygen tension. This improved extracellular matrix deposition was associated with a significant decrease in lipids and in particular cholesterol under low oxygen tension as revealed by TOF-SIMS coupled to principal component analysis and discriminant analysis. We furthermore demonstrate that the higher cholesterol levels under normoxia might regulate fibroblast growth factor 1 (FGF-1) gene expression which was previously implemented in increased ECM production in the cocultures. In conclusion, our study shows an unexpected role of lipids as orchestrators of chondrogenesis in response to oxygen tension which is, at least in part, mediated through FGF-1.

Gasotransmitters including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S) have attracted more and more attention in the past decades due to their unique signaling and functions. However, as a fundamental issue in the investigations of gasotransmitters, the cell membrane permeability and release behavior of them is controversial in reports because of the lack of an efficient approach to determine gasotransmitters released out of and remaining in the same cells simultaneously. To solve such problem, taking NO as representative, a robust and facile strategy has been reported based on a completely water-soluble fluorescent probe and a commercially available capillary electrophoresis system. A specially designed boron-dipyrromethene (BODIPY)-based fluorescent probe with two sulfonate groups, disodium 2,6-disulfonate-1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl) difluoroboradiaza-s-indance (TMDSDAB), has been developed. As a turn-on fluorescent probe, TMDSDAB can react with NO promptly in aqueous media, and 540-fold enhancement of fluorescence is obtained. Using TMDSDAB, the trapping and quantification of NO released out of and remaining in the same single cell was achieved by capillary electrophoresis with laser-induced fluorescence detection. The limit of detection is 0.5 nM for NO. The proposed method has been applied to estimate the release behavior of single macrophages, and the results indicated that the cell membrane should be a barrier to NO diffusion.

We have developed a new category of sensor for measurement of the ²⁴⁰Pu/²³⁹Pu mass ratio from aqueous solution samples with advantages over existing methods. Aqueous solution plutonium samples were evaporated and encapsulated inside of a gold foil absorber, and a superconducting transition-edge-sensor microcalorimeter detector was used to measure the total reaction energy (Q-value) of nuclear decays via heat generated when the energy is thermalized. Since all of the decay energy is contained in the absorber, we measure a single spectral peak for each isotope, resulting in a simple spectral analysis problem with minimal peak overlap. We found that mechanical kneading of the absorber dramatically improves spectral quality by reducing the size of radioactive inclusions within the absorber to scales below 50 nm such that decay products primarily interact with atoms of the host material. Due to the low noise performance of the microcalorimeter detector, energy resolution values of 1 keV fwhm (full width at half-maximum) at 5.5 MeV have been achieved, an order of magnitude improvement over α-spectroscopy with conventional silicon detectors. We measured the ²⁴⁰Pu/²³⁹Pu mass ratio of two samples and confirmed the results by comparison to mass spectrometry values. These results have implications for future measurements of trace samples of nuclear material.

RNA-cleaving DNAzymes provide a unique platform for developing biosensors. However, a majority of the work has been performed in clean buffer solutions, while the activity of some important DNAzymes in biological sample matrices is still under debate. Two RNA-cleaving DNAzymes (17E and 10-23) are the most widely used. In this work, we carefully studied a few key aspects of the 17E DNAzyme in human blood serum, including hybridization, cleavage activity, and degradation kinetics. Since direct fluorescence monitoring is difficult due to the opacity of serum, denaturing and nondenaturing gel electrophoresis were combined for studying the interaction between serum proteins and DNAzymes. The 17E DNAzyme retains its activity in 90% human blood serum with a cleavage rate of 0.04 min^–1, which is similar to that in the PBS buffer (0.06 min^–1) with a similar ionic strength. The activity in serum can be accelerated to 0.3 min^–1 with an additional 10 mM Ca²⁺. As compared to 17E, the 10-23 DNAzyme produces negligible cleavage in serum. Degradation of both the substrate and the DNAzyme strand is very slow in serum, especially at room temperature. Degradation occurs mainly at the fluorophore label (linked to DNA via an amide bond) instead of the DNA phosphodiester bonds. Serum proteins can bind more tightly to the 17E DNAzyme complex than to the single-stranded substrate or enzyme. The 17E DNAzyme hybridizes extremely fast in serum. With this understanding, the detection of DNA using the 17E DNAzyme is demonstrated in serum.

Quantitative proteomics studies of yeast that use metabolic labeling with amino acids rely on auxotrophic mutations of one or more genes on the amino acid biosynthesis pathways. These mutations affect yeast metabolism and preclude the study of some biological processes. Overcoming this limitation, it has recently been described that proteins in a yeast prototrophic strain can also be metabolically labeled with heavy amino acids. However, the temporal profiles of label incorporation under the different phases of the prototroph’s growth have not been examined. Labeling trajectories are important in the study of protein turnover and dynamics, in which label incorporation into proteins is monitored across many time points. Here we monitored protein labeling trajectories for 48 h after a pulse with heavy lysine in a yeast prototrophic strain and compared them with those of a lysine auxotrophic yeast. Labeling was successful in prototroph yeast during exponential growth phase but not in stationary phase. Furthermore, we were able to determine the half-lives of more than 1700 proteins during exponential phase of growth with high accuracy and reproducibility. We found a median half-life of 2 h in both strains, which corresponds with the cellular doubling time. Nucleolar and ribosomal proteins showed short half-lives, whereas mitochondrial proteins and other energy production enzymes presented longer half-lives. Except for some proteins involved in lysine biosynthesis, we observed a high correlation in protein half-lives between prototroph and auxotroph strains. Overall, our results demonstrate the feasibility of using prototrophs for proteomic turnover studies and provide a reliable data set of protein half-lives in exponentially growing yeast.

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein–ligand and protein–protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched (¹⁵N-labeled) p51 and unlabeled p66. To enable detection of ¹⁵N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% ¹⁵N model. Our results demonstrated that ¹⁵N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but ¹⁵N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure.

For targeted protein quantification by liquid chromatography mass spectrometry (LC/MS), an optimal approach for efficient, robust and hi-throughput sample preparation is critical, but often remains elusive. Here we describe a straightforward surfactant-aided-precipitation/on-pellet-digestion (SOD) strategy that provides effective sample cleanup and enables high and constant peptide yields in various matrices, allowing reproducible, accurate and sensitive protein quantification. This strategy was developed using quantification of monocolnocal antibody in tissues and plasma as the model system. Surfactant treatment before precipitation substantially increased peptide recovery and reproducibility from plasma/tissue, likely because surfactant permits extensive denaturation/reduction/alkylation of proteins and inactivation of endogenous protease inhibitors, and facilitates removal of matrix components. The subsequent precipitation procedure effectively eliminates the surfactant and nonprotein matrix components, and the thorough denaturation by both surfactant and precipitation enabled very rapid on-pellet-digestion (45 min at 37 °C) with high peptide recovery. The performance of SOD was systematically compared against in-solution-digestion, in-gel-digestion and filter-aided-sample-preparation (FASP) in plasma/tissues, and then examined in a full pharmacokinetic study in rats. SOD achieved the best peptide recovery (∼21.0–700% higher than the other three methods across various matrices), reproducibility (3.75–10.9%) and sensitivity (28–30 ng/g across plasma and tissue matrices), and its performance was independent of matrix types. Finally, in validation and pharmacokinetic studies in rats, SOD outperformed other methods and provided highly accurate and precise quantification in all plasma samples without using stable isotope labeled (SIL)-protein internal standard (I.S.). In summary, the SOD method has proven to be highly robust, efficient and rapid, making it readily adaptable to large-scale clinical and pharmaceutical quantification of biomarkers or biotherapeutics.

As an important biomarker for early cancer diagnostics and a valuable therapeutic target, telomerase has attracted extensive attention concerning its detection and monitoring. Herein, a homogeneous electrochemical strategy based on T7 exonuclease-aided target recycling amplification is proposed for a simple, rapid, and highly sensitive assay of human telomerase activity from crude cancer cell extracts. In this strategy, a 5′ methylene blue (MB)-labeled hairpin (HP) probe is designed, which can hybridize with the telomerase reaction products to initiate the subsequent digestion by T7 exonuclease, and a large amount of MB-labeled mononucleotides are released to result in the significantly amplified electrochemical signal. By taking advantage of the high amplification efficiency of T7-aided target recycling, the present assay enables the detection of telomerase activity at the single-cell level, which is superior or comparable to that of the reported literature. Furthermore, the assay was carried out in a homogeneous solution without complex modification or immobilization procedures, which has the merits of simplicity, rapid response, and improved recognition efficiency compared with heterogeneous biosensors. With the ability of fast detection, outstanding sensitivity, and excellent selectivity, this strategy offers a convenient and specific method for telomerase activity detection, which exhibits great potential in the practical application in telomerase-based early stage cancer diagnosis.

Almost all of the important functions of DNA are realized by proteins which interact with specific DNA, which actually happens in a limited space. However, most of the studies about the protein–DNA binding are in an unconfined space. Here, we propose a new method, nanopore-based DNA-probe sequence-evolution (NDPSE), which includes up to 6 different DNA-probe systems successively designed in a nanoscale confined space which unveil the more realistic characteristics of protein–DNA binding phenomena. There are several features; for example, first, the edge-hindrance and core-hindrance contribute differently for the binding events, and second, there is an equilibrium between protein–DNA binding and DNA–DNA hybridization.

Understanding the biology of bacteria is critical for exploiting their beneficial properties and for preventing and treating bacterial diseases. Nanobioscience is an area that has recently seen major scientific progress. Here, we demonstrate that a raspberry-shaped nanostructure with a high density of gold nanoparticles acts like an excellent antenna due to its optical properties, which permit sensitive detection and analysis of bacterial cells. By using antibodies, these nanoantennas can be engineered to recognize only specific bacterial species. This system provides a new technique that will allow for more sensitive detection of specific bacteria.

In our study, various bacteria, including Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Acinetobacter calcoaceticus, Serratia marcescens, Shewanella oneidensis) and Gram-positive (Bacillus subtilis) bacteria, were straightforwardly immobilized into the conducting polymers (CPs) by electrochemical deposition. The doping state of bacteria in the polymer films (polypyrrole and poly(3,4-ethylenedioxythiophene)) varied according to the polymerization conditions. The viability of bacteria in the polymers and of those adsorbed on various substrates was evaluated. The activity of bacteria doped on the polymer film was evaluated by cyclic voltammetry in a thin-layer cell.