Designing of New Optical Immunosensors Based on 2-Amino-4-(anthracen-9-yl)-7-hydroxy-4H-chromene-3-carbonitrile for the Detection of Aeromonas hydrophila in the Organs of Oreochromis mossambicus Fingerlings

A one-pot greener methodology has been adopted for the synthesis of a simple 4H-chromene core-based fluorescent tag of (S)-2-amino-4-(anthracen-9-yl)-7-hydroxy-4H-chromene-3-carbonitrile (AHC), and its structure has been analyzed using NMR spectroscopy. The physicochemical properties of AHC were well-studied by UV–vis and fluorescent spectroscopy techniques. As a result of excellent emitting property (ϕ ≈ 0.75), it has been coupled with anti-AH through amide linkage, and the AHC-tagged anti-AH has been used as an immunoassay for the selective detection of Aeromonas hydrophila in the presence of interfering pathogens. Under optimized conditions, immunosensors could successfully quantify A. hydrophila from 4 to 736 CFU/mL, and the LOD was 2 CFU/mL. Saliently, the immunoassay has been successfully demonstrated for the analysis of A. hydrophila in the organs of Oreochromis mossambicusfingerlings, and results have shown a very good agreement with our optimized neat AH fluorimetric titration results.

These methods are only focused on the isolation of Aeromonas species from food and water samples.
From the detection point of view, till date, only polymerase chain reaction (PCR) method has been mainly addressed for the recognition of AH. A simple PCR method has been reported for the detection of AH in raw milk within the limit of 2 log 10 CFU/g, and the detection rate was found to be 23% for this method and 14% for culture method. 23 In addition, some microarray-based method is constructed using DNA probes to study the population dynamics of microbial communities, such as marine bacteria in coastal waters in which aeromonads were found to make up a large proportion of the microbial flora. 24 Another microarray method has also been reported for the detection of AH cytotoxic enterotoxin-inducing genes in macrophages. 25−27 Some researchers have developed probes for the detection of various Aeromonas species. 28,29 The chromene moieties often appear as an important structural component in both biologically active and natural compounds. Chromene fragments occur in alkaloids, flavonoids, tocopherols, and anthocyanins. Moreover, functionally substituted chromenes have played increasing roles as promising compounds in the field of medicinal chemistry. 30 −33 On the basis of deep tunneling of all reports for AH, let us conclude that no immunosensor has been reported so far for the detection of AH in laboratory as well as in real samples. Among the already-reported PCR techniques, DNA probe methods are used only for isolation and to find the detection rate of AH and not for precise quantification. These methods are costly, more time-consuming, and a lot of steps have to be taken care of. On the basis of this, we assure that our group is reporting for the first time about a fluorescent-based immunoassay for the very selective and ultrasensitive detection of A. hydrophila. The 4H-chromenecore(S)-2-amino-4-(anthracen-9-yl)-7-hydroxy-4H-chromene-3-carbonitrile (AHC) was specially designed via a one-pot greener approach using ultrasonication method with high yield in a short time, that is, 20 min, and it was applied as a tag for the qualitative and quantitative detection of A. hydrophila from 4 to 736 CFU/mL with an LOD of 2 CFU/mL, and the immunoassay developed has been applied for the quantification of AH in the organs of Oreochromis mossambicusfingerlings.

■ RESULTS AND DISCUSSION
Conformation of Tagging of AHC with Anti-AH by U V − V i s S p e c t r a . I n g e n e r a l , 1 -e t h y l -3 -( 3dimethylaminopropyl)carbodiimide hydrochloride (EDC)/Nhydroxysuccinimide (NHS) reagent offers a very good coupling platform for the immunosensors based on the amide linkage between antibodies and sensing materials. 34−36 In this aspect, UV−vis spectral analysis assists us to confirm the tagging of anti-AH with our synthesized AHC. The normal amide formation in EDC/NHS coupling generally occurs based on the reaction between −COOH and −NH 2 group. 37,38 In our case, the amide formation occurred between the −COOH group of anti-AH and −NH 2 group of our AHC probe in MES buffer at optimized condition. 39 First, absorption maxima of AHC were recorded in phosphatebuffered saline (PBS) working buffer, and it showed two major absorption maxima at 218 and 277 nm because of the existence of π−π* and n−π* transitions, respectively (Figure 1a). We optimized the equivalents of AHC with respect to the number of −COOH group present in the anti-AH by UV−vis spectral analysis at lower and higher concentrations of AHC. Two equivalents (2 mL, 0.001 M) and four equivalents (4 mL, 0.001 M) of AHC were treated with one equivalent of anti-AH (containing four −COOH group). 40 The corresponding absorbance changes were recorded, and it was clearly shown that 4 equiv of AHC at lower concentrations (0.001 M)

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Article containing four −NH 2 groups may be allowed to form amide link with four −COOH groups of anti-AH, thereby leading to maximum absorbance (Figure 1b). Whereas at higher concentration of AHC (0.01 M), the absorbance intensity was lower which may be because of the poor solubility of AHC. Hence, the precipitate was obtained. Initially, AH has showed a maximum absorbance because of twisted international critical table (ICT) state of the AH structure. After the formation of amide linkage, the peaks at 218 and 277 nm were blue-shifted to 212 and 269 nm, respectively, which may be because of the inhibition of initial twisted intramolecular charge transfer as a result of complex formation between anti-AH (acceptor) and AHC (donor) via amide linkage ( Figure  1c), and hence decrement in absorbance was absorbed. We have also cross-checked if any unbound AHC was present in the resulting anti-AH−AHC. Briefly, the settled anti-AH− AHC was washed with phosphate-buffered solution for 5 min and then centrifuged. The resulting supernatant solution was analyzed by UV−vis spectra. No peak corresponding to free AHC appeared for supernatant solution in the UV spectra. This seems to be that all of the 4 equiv of AHC were utilized for the amide linkage. Therefore, the UV−vis spectral shift has clearly evinced and supported the completion of anti-AH tagging with our AHC probe. The corresponding UV data are shown in Figure 1d.
Confirmation of Tagging of AHC with Anti-AH by Emission Analysis. The tagging was further studied with the help of fluorescent emission analysis. Precisely, the initial emission maximum of AHC was analyzed at the absorption maxima of 277 nm in PBS buffer, and the corresponding emission was occurred at 390 and 505 nm. As a result of amide coupling, the emission maxima at 505 nm was blue-shifted to 485 nm along with a drastic decrement in the emission intensity as in the case of UV−vis absorption spectra which may once again confirm the existence of inhibited twisted ICT mechanism during the coupling of AHC with anti-AH. In general, antibody-fluorescent tagging protocol may lead to a blue shift and decrement in fluorescent intensity because of amide coupling. 41,42 First, the AHC has shown maximum fluorescence because of the twisted ICT state mechanism. 43 After the coupling of anti-AH via amide bond, the initial twisted ICT was inhibited, and the repulsive interaction between the donor−NH 2 and −COOH of anti-AH may rise the energy level of anti-AH/AHC and hence shift to the lower wavelength was observed (blue shift) The Stoke's shift of the AHC was found to be 228 nm which shows very excellent luminescent properties of AHC. 44 After the anti-AH was tagged with AHC, the Stoke's shift was calculated as 216 nm. Generally, proteins, bacterial-based tagging, will give a large Stoke's shift greater than 200 nm-based. 45−47 Likewise, our anti-AH tagged with AHC has shown excellent Stoke's shift, and the respective emission spectra are shown in Figure 2.
Development of Fluorescent Immunoassay for A. hydrophila Using Anti-AH−AHC. Generally, we have to be more aware on Aeromonas colonization as human pathogens because of its association with gastrointestinal diseases. Our developed simple fluorescent immunoassay will be a better analytical tool to quantify the colonies of A. hydrophila. Although varying the concentration of AH from 10 −1 to 10 −12 CFU/g, the emission intensity of peaks at 390 and 485 nm was increased with increasing AH concentrations without any peak shift; however, at higher concentration of AH, the above peaks

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Article were merged into a single new peak at 411 nm with increment in intensity. The Stoke's shift of 208 nm from its excitation wavelength was observed during this interaction. The respective emission data along with the linear regression plot are shown in Figure 3. The sensitivity of the developed immunoassay was analyzed using colony counting method based on the dilution of stock culture of A. hydrophila, and the corresponding linear range of detection was found from 4 to 736 CFU/mL with the LOD of 2 CFU/mL. Selectivity Study of Anti-AH−AHC Immunoassay toward A. hydrophila. The important aspect of any immunoassay is its selectivity toward the targeted pathogen among all possible interfering pathogens. The antibody-tagging technology opens a new pathway for the most selectivity toward particular pathogens. 40,47 The fluorescent labeling has been keynoted as a very good immunoplatform without using any blocking agents as in the case of electrochemical immunoassay. 48 In our present immunoassay, we have tagged the AHC with anti-AH, and hence there is no doubt that the resulting anti-AH−AHC will be attached with its pathogen more specifically. To check the selectivity of the above immunoassay, the pathogens that have an analog behavior to AH, such as Escherichia coli, Bacillus, Pseudomonas aeruginosa, and Staphylococcus aerus were analyzed. There was a slight decrease in the emission intensity of the peaks at 390 and 485 nm and was noted for all other pathogens instead of increase in intensity as in the case of AH. Therefore, the results firmly support the selectivity of anti-AH−AHC toward A. hydrophilaand shown in Figure 4.
Competitive Performance of the Immunoassay in the Presence of Other Interfering Pathogens. The selective immunosensing of anti-AH−AHC was proved via a selective recognition study and firmly confirmed the immunocomplex formation only between AH and anti-AH−AHC ( Figure 2). The selective nature was again confirmed by performing the competitive analysis using other possible interfering pathogens such as E. coli, Bacillus, P. aeruginosa, and S.aerus ( Figure 5). Interaction of all of these pathogens slightly quenches the initial fluorescent intensity of anti-AH−AHC which may be because of nonfluorescent complex formation via FRET quenching. 45 The competitive test was performed by incubating 1 equiv of 2 mL of anti-AH−AHC with 1 equiv (10 μL, 10 −1 CFU/g) of AH and 1 equiv (10 μL, 10 −1 CFU/g) of all interfering pathogens, and the corresponding fluorescent intensity changes were recorded. Remarkably, there was no quenching in the fluorescent intensity for anti-AH−AHC. Hence, the designed immunoassay has proved to be very selective only for the AH in the presence of other interfering pathogens.
Observation of Fluorescent and Naked Eye Color Changes Using UV−Vis Trans Illuminator. Generally, fluorescent sensors show excellent color changes toward various analytes such as biomolecules, 49 metal ions, 50 and pathogens, specifically. 51 In our case, we have used AHC as a fluorescent tag because of its excellent quantum yield of 0.90 which is comparable with the standard rhodamine 6G dye. First, the AHC has exhibited orange fluorescent color under UV−vis lamp in MES buffer medium (pH 5.4). After the amide coupling with anti-AH, the initial color was diminished and changed slightly into yellowish green. Finally, after the formation of an immunocomplex between the AH and anti-AH−AHC, a bright-green fluorescent color change was

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Article observed, and images are given in Figure 6a. Under naked eye condition, initially, the AHC was deep orange in color; after coupling with anti-AH, it became slightly yellow in color, and then, finally, it turned into deep-bright-green fluorescent after binding with AH ( Figure 6b). These results supported the utility of AHC as a fluorescent tag for the detection of AH.
The accumulation of AH in the organs of fish grown in contaminated water (where they were grown for different days) was analyzed through imaging the color change of anti-AH−AHC during binding with AH using a transilluminator. The fluorescent color of anti-AH−AHC increases with the increasing growing time and is shown in Figure 7. All of these observations also supported the accumulation of AH in the fish organs and selectivity of immunoassay toward AH pathogen.
Sensing Mechanism of the AH/Anti-AH−AHC Fluorescence Immunoassay. Among all fluorescent-sensing mechanisms, intra/intermolecular charge-transfer-based mechanism has been commonly existing in various fluorescent sensing assays. 52−54 After tagging, the formation of amide linkage between anti-AH and AHC may be responsible for the spectral shift in both UV−vis as well as fluorescence emission spectra with decrement in absorbance/emission intensity. On the basis of the blue shift, it is confirmed that the synthesized AHC has shown maximum absorbance/emission which may be because of the twisted ICT mechanism among the molecule itself from donor −NH 2 to acceptor anthracene moiety. 55 After tagging with anti-AH, the initial twisted intramolecular charge-transfer mechanism among AH molecule was inhibited because of the amide coupling between donor −NH 2 of AHC and −COOH of anti-AH. Therefore, the donating nature of −NH 2 to anthracene moiety was arrested. As a result, a blue shift was observed which may be because of the electronic charge repulsion between the two strong electron charge entities of the −NH 2 group in AHC and −COOH group in anti-AH via amide bond formation, and this repulsion finally might increase the energy levels of the anti-AH−AHC, which results in the blue shift. Interestingly, when the pathogen AH was incubated with the anti-AH−AHC, the fluorescent intensity was increased enormously. This phenomenon may be because of aggregated induced emission (AIE) of the resulting immunocomplex formation. After the incubation with pathogen, all of the AHC molecules in anti-AH−AHC might come closer to each other, and thereby the antharacene moiety in AHC can undergo dimerization or aggregation. In general, fluorescent intensity of anthracene-containing molecules will be enhanced in solution/solid phase because of the aggregation-induced phenomenon. 56−58 Therefore, the fluorescent intensity of the immunoassay was increased. 51,59,60 Therefore, based on these UV−vis and emission spectral changes, it is confirmed that two interesting mechanisms such as inhibition of twisted ICT and AIE are existing during the formation of anti-AH−AHC and AH/anti-AH−AHC immunocomplexes, respectively, under optimized conditions (Scheme 1).
Determination of Quantum Yield (ϕ). Using the emission data, the fluorescence quantum yield (ϕ) at various steps were estimated by integrating the area under fluorescence curves using the following equation. 46 The absorbance value of AHC, anti-AH, and AH/anti-AH−AHC was chosen from where, ϕ is the quantum yield; I is the integrated area under the corrected emission spectra; A is the absorbance at the

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Article excitation wavelength; λ ex is the excitation wavelength; η is the refractive index of the solution; and the subscripts u and s refer to the unknown and the standard, respectively. The quantum yield of rhodamine 6G dye was calculated as 0.94 in absolute ethanol medium at an excitation wavelength of 470 nm. 62 The quantum yield of AHC, anti-AH−AHC, and AH/anti-AH− AHC was calculated as 0.80, 0.328, and 0.90, respectively, and calculations were given at the end of the section. The aggregation may lead to an increment in quantum yield after binding with AH. [56][57][58]63,64 Analysis of Accumulation of A. hydrophila in Organs of O. mossambicusFingerlings. The fingerlings of O. mossambicuswere grown in A. hydrophila contaminated water for 7 days and the images of growing vessels are shown in the Supporting Information ( Figure S1). The detailed protocol was given in the above Materials and Methods section. The accumulation of AH in three different organs such as guts, liver, and tissue of O. mossambicusfingerlings and the remaining portion of AH in growing medium were analyzed. In addition, the photo images of the organs dissected from O. mossambicus fingerlings for 7 days are shown in the Supporting Information ( Figure S2).
After dissection, the organs of the fish were collected for each day, and samples were prepared as per the procedure given in Materials and Methods section and analyzed for the AH accumulation using the emission method (Figure 8a−c). Among these organs, higher accumulation of AH was found at guts than at liver and tissue. The results obtained were compared and also confirmed using the colony counting method. The colony formation of AH on a disc was also analyzed at the biologically simulated conditions of fish guts ( Figure S3). The calculated CFU values are given in Table 1 and compared with the plate count method.
For cross-checking, the amount of AH in contaminated water was also tested using emission method for each day. The concentration of AH was found higher in the medium on the first day, and it started to decrease as the day goes up to fifth day, whereas in fish organs, a reverse trend was observed. However, after the fifth day, the amount of AH found in fish organs and the medium attained saturation (Figure 8d

■ CONCLUSIONS
Hence, a very first fluorescent-based immunoassay is developed for the specific and ultrasensitive detection of A. hydrophila. We have designed a natural 4H-chromene corebased, AHC, as a fluorescent tag via the one-pot greener approach using ultrasonication method. Then, it was tagged

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Article with the anti-AH successfully and applied for the qualitative and quantitative detection of A. hydrophila from 4 to 736 CFU/mL with the LOD of 2 CFU/mL. To further enhance the application of our developed immunoassay, it was applied for the real-time quantification of A. hydrophila accumulation in organs of O. mossambicusfingerlings. Results have shown excellent agreement with the plate-counting method, and we assure that it would be a better immunoanalytical tool for the quantification of A. hydrophilain fish and some food samples.

■ MATERIALS AND METHODS
We have used analytical grade solvents and double distilled water throughout the studies. Malononitrile, resorcinol, anthracene-1-carboxaldehyde, HCl, disodium mono hydrogen phosphate, monosodium dihydrogen phosphate, glacial acetic acid, ammonium acetate, sodium acetate, absolute ethanol, high-performance liquid chromatography methanol, ammonium chloride, liquid ammonia, piperidine, NaOH, and KCl were purchased from Sigma-Aldrich Chemical Company. The acetate salts of metal ions analyzed were purchased from Merck and Avra Chemicals. NMR studies were carried out using Bruker-400 MHz spectrometer for 1 H and 13 C NMR analysis using CDCl 3 and DMSO-[d 6 ] as solvents containing a trace quantity of tetramethylsilane as the internal standard, and the chemical shifts are reported in parts per million at 25°C. UV spectra were recorded using Shimadzu single-beam UV−vis spectrophotometer. Finally, fluorescent measurements were done with the help of Cary Eclipse spectrophotometer having a 450 W xenon lamp. Bandwidths of 5 and 2.5 nm were maintained as excitation and emission slit widths, respectively, throughout the experiments.

Article
One-Pot Greener Synthesis of AHC. 4H-chromene derivative was synthesized via one-pot greener approach using ultrasonication method. In brief, antharacene-1-carboxaldehyde (1.0 mmol), malononitrile (1.0 mmol), and resorcinol (1.0 mmol) were dissolved one by one in 5 mL of absolute ethanol and stirred for 2 min. Then, catalytic amount of piperidine (300 μL) was added with constant stirring. Then, the resulting solution mixture was kept in an ultrasonication bath (1.5 Hz) and was sonicated for 20 min (Scheme S1). In between the reaction times, the crude reaction mixture was analyzed for the completion of the reaction by using TLC (by observing a single spot). After the confirmation of reaction, the reaction mixture was poured into ice-cold water, and a light yellow precipitate was obtained. This solid was filtered and washed thoroughly for three times with diethyl ether and absolute ethanol, and finally, it was dried over anhyd. CaCl 2 . It was further separated through organic separation method using diethyl ether and water mixture as a solvent. The ether layer was separated, evaporated, and then the obtained yellow color solid was recrystallized using absolute hot ethanol to obtain a pure product. The yield was calculated to be 98.5% (1.92 g). The structure of the synthesized 4H-chromene derivatives (1a−d) was thoroughly characterized by NMR technique, and the corresponding coupling constant values are given below. 1 (Figures 9 and 10).
Protocol for the Tagging of AHC with the AH Antibody. The AHC was tagged with anti-AH via standard EDC/NHS coupling protocol. 30−32 In briefly, 1 equiv of anti-AH (1 mL, 100 μg/μL) in MES buffer and 4 equiv (4 mL, 0.001 M) of AHC were mixed, and then 100 μL of each solution of EDC and NHS were added one by one to the above solution. It was gently stirred for 1−2 h and then kept for incubation at 4°C for 1 day. Then, the resultant tagged anti-AH/AHC (0.002 μg) was allowed to stand at room temperature for 5−10 min, and then it was completely centrifuged at 30 000 rpm for 20 min at 4°C. The resulting pellets were collected and washed thoroughly with PBS buffer for five times. The unbound AHC was removed by simply washing with PBS buffer and confirmed by analyzing the supernatant solution using UV−vis spectra until the disappearance of standard UV−vis peak of AHC. The final anti-AH/AHC in buffer solution was stored at 4°C for further studies (Scheme S2).
Bacterial Cultivation. Pure culture of AH bacteria was procured from MTCC1739 strain, Pune, India. It was grown initially in tryptic soya broth and maintained in Luria Bertani medium at 30°C for 16 h for further studies. The bacterial cells were then separated through centrifugation (6000 rpm, 20 min) and then rinsed thrice with PBS. The culture was serially diluted with physiological saline solution, and the viable cell number was determined via the most probable number method.
Development of Immunosensor (AH/Anti-AH−AHC). Under optimized conditions, 2 mL of AHC/anti-AH (0.002 μg) was mixed thoroughly with 10 μL of AH pathogens diluted in different CFU, and then they were incubated at 4°C for 25 min. The major absorption maxima at 277 nm were fixed as excitation wavelength, and the corresponding fluorescence spectra were recorded at 411 nm, and all of these experiments were carried out in the phosphate buffer (PBS, pH = 7.4) medium throughout the studies.
Fish Maintenance and Treatment Procedure. Fingerlings of O. mossambicus were grown in artificial condition (plastic turf in laboratory condition) for 1 month, subsequently; they were transferred in to 250 mL conical flask in the presence of sterile water (200 mL) and kept for acclimatization for 15 days inside the sterile laminar hood. After acclimatization, AH was inoculated in seven different conical flasks along with fingerlings of O. mossambicus, and 100 μg/mL concentration of filter-sterilized glucose was added to enhance the growth of AH. From first day to seventh day, fingerlings were sacrificed using sterile dissection apparatus in

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Article sterile condition, and liver, guts, and tissues samples were collected in sterile PBS (pH = 7.4), and a loop-full suspension was inoculated in the AH growth-specific media to check the increasing number of bacterial colony. O. mossambicus in sterile water without AH was used as a control, and the experiment was performed thrice in a triplicate manner.