Micropump integrated white blood cell separation platform for detection of chronic granulomatous disease.

White blood cells (WBCs) are robust defenders during antigenic challenges and prime immune cell functioning indicators. High-purity WBC separation is vital for various clinical assays and disease diagnosis. Red blood cells (RBCs) are a major hindrance in WBC separation, constituting 1000 times the WBC population. The study showcases a low-cost micropump integrated microfluidic platform to provide highly purified WBCs for point-of-care testing. An integrated user-friendly microfluidic platform was designed to separate WBCs from finger-prick blood (⁓5 µL), employing an inertial focusing technique. We achieved an efficient WBC separation with 86% WBC purity and 99.99% RBC removal rate in less than 1 min. In addition, the microdevice allows lab-on-chip colorimetric evaluation of chronic granulomatous disease (CGD), a rare genetic disorder affecting globally. The assay duration, straight from separation to disease detection, requires only 20 min. Hence, the proposed microfluidic platform can further be implemented to streamline various clinical procedures involving WBCs in healthcare industries.

Microfluidic paper device with on-site heating to produce reactive peroxide species for enhanced smartphone enabled chemiluminescence signal.

Chemiluminescence signal amplification (CLSA) is of huge interest because of its sensitive detection in various applications such as food analysis, biomedical diagnosis and environmental monitoring. Due to this, there is a manifold attention to develop rapidly prototyped and miniaturized devices for CLSA. In this context, herein, a novel CLSA approach is demonstrated on a 3D printed microfluidic paper-based analytical device (µPADs), fabricated using Fused deposition modeling (FDM) printing technology. Influence of working temperature, ranging 30 °C-110 °C, on CL signal generation from well-established Luminol/Co+2 - H2O2 reaction was analyzed using a screen-printed flexible heater onto the 3D printed reaction platform. A smartphone-based capturing/detection system provided the amenability for a point-of-care testing system. For the first time, strong and stable CLSA was found with about 255% ± 5% increase in its signal intensity without using any additional external enhancers. The on-site working temperature was directly in proportional to the intensity of CL signal generated from Luminol/Co+2 - H2O2 reaction under optimum conditions, wherein the device had a wide linear range from 50 nM to 1 µM with a detection limit of 35 nM for H2O2 detection. The reliability of the developed amplification method was tested for practicability to detect the concentration of H2O2 in milk as real sample analysis. Overall, such CLSA mechanism in miniaturized µPADs will have strong potential for multiple CL based detection and monitoring application.

BCIG-SMAC medium and PMA-qPCR for differential detection of viable Escherichia coli in potable water.

BACKGROUND AND OBJECTIVES: Public health protection requires timely evaluation of pathogens in potable water to minimize outbreaks caused by microbial contaminations. The present study was aimed at assessing the microbiological quality of water obtained from Shantinagar (a rural area in the South Goa region of Goa, India) using 5-Bromo-4-Chloro-3-Indoxyl ß-D-glucuronide-Sorbitol MacConkey agar (BCIG-SMAC) medium and, propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assay for differential detection and quantification of viable Escherichia coli cells in water samples. MATERIALS AND METHODS: Membrane filtration method was used for both BCIG-SMAC medium and PMA-qPCR methods. To determine the efficiency of detection of viable cells, we first evaluated the PMA treatment protocol and established the standard calibration curves using previously reported primers. RESULTS: PMA-qPCR detected as low as 7 femtograms of DNA of E. coli per qPCR reaction whereas the limit of detection (LOD) of BCIG-SMAC medium was 1.8 CFU/100mL. A total of 71 water samples spanning 2017-2018 have been analyzed using BCIG-SMAC medium and PMA-qPCR, of which 95.77% (68/71) and 7.04% (5/71) were found to be total E. coli and E. coli O157:H7, respectively. PMA-qPCR study showed the viable counts of total viable E. coli cells ranging from 3 CFU/100mL to 8.2×102 CFU/100mL. The total E. coli CFU/100mL quantified by PMA-qPCR significantly exceeded (paired t-test; P<0.05) the number on BCIG-SMAC medium. CONCLUSION: The present study indicates that the microbiological quality of environmental water samples analyzed do not comply with the regulatory standard. Therefore, special attention is warranted to improve the overall portable quality of water in the perspective of public health.

A colorimetric paper-based ATONP-ALP nanobiosensor for selective detection of Cd2+ ions in clams and mussels.

A colorimetric paper-based enzyme-coupled antimony tin oxide nanoparticle (ATONP) nanobiosensor for selective detection of Cd2+ ions in clams and mussels is presented. Alkaline phosphatase (ALP) was immobilized on ATONPs via 16-phosphonohexadecanoic acid (16-PHA) to develop ATONP-ALP nanobiosensor. The biosensor was characterized using XPS, Raman spectroscopy, SEM, and EDX. ATONP-ALP nanobiosensor exhibited high selectivity towards detection of Cd2+ ion with a LOD 0.006 µg L-1 and linear range of detection 0.005-1 µg L-1. The developed biosensor was further integrated into a low-cost paper-based format. A visual color change was obtained for Cd2+ ion in the range 0.1-10 µg L-1. The developed biosensor was successfully demonstrated for the analysis of Cd2+ ions in clams with recoveries 101-104%. The ATONP-ALP nanobiosensor was validated using mussel tissue (BCR-668) and the conventional ICP-OES and ICP-MS techniques.

Supercritical Carbon Dioxide Impregnation of Gold Nanoparticles Demonstrates a New Route for the Fabrication of Hybrid Silk Materials.

How many nanoparticles can we load in a fiber? How much will leak? Underlying is the relatively new question of the "space available" in fibers for nanoparticle loading. Here, using supercritical carbon dioxide (scCO2) as a carrier fluid, we explored the impregnation in four Indian silks (Mulberry, Eri, Muga, and Tasar) with five standard sizes of gold nanoparticles (5, 20, 50, 100 and 150 nm in diameter). All silks could be permanently impregnated with nanoparticles up to 150 nm in size under scCO2 impregnation. Accompanying structural changes indicated that the amorphous silk domains reorganized to accommodate the gold NPs. The mechanism was studied in detail in degummed Mulberry silk fibers (i.e., without the sericin coating) with the 5 nm nanoparticle. The combined effects of concentration, time of impregnation, scCO2 pressure, and temperature showed that only a narrow set of conditions allowed for permanent impregnation without deterioration of the properties of the silk fibers.

A portable microfluidic device-based Fe3O4-urease nanoprobe-enhanced colorimetric sensor for the detection of heavy metals in fish tissue.

A portable microfluidic device with highly sensitive enzyme nanoprobe (Fe3O4 MNPs-urease, average size 34.6 nm) was demonstrated for the analysis of heavy metals ions (Hg2+, Cd2+ and Pb2+) in fish gill and muscle tissue. The immobilized urease nanoprobe (Km = 0.05 mM) exhibited twofold sensitivity over the free enzyme assay (apparent Km = 0.1 mM). The nanoprobe was characterized using SEM, EDAX, PSA and FT-IR. The inhibition measurements were carried out for individual as well as the mixture of metal ions (CRM standards of 9 elements (CRMmix-9)). The lower limit of quantification (LOQ) (0.5, 0.1, and 0.1 ng L-1 for Hg2+, Cd2+, and Pb2+) and lower limit of detection (LOD) was achieved at 0.1 ng L-1 with sensitivity 8-14% per decade for Hg2+, Cd2+, and Pb2+ ions. A visual result can be observed by the naked eye through the microfluidic device as well as with 96 transparent microwell plates. The order of relative inhibition was found to be CRMmix-9 > (Hg2+ + Cd2+ + Pb2+) > (Cd2+ + Pb2+) > (Pb2+ + Hg2+) > (Hg2+ + Cd2+) > Pb2+ > Cd2+ > Hg2+, respectively. The recovery % in fish tissues were found to be 88-98% for Hg2+, Cd2+ and Pb2+ ions.

A capacitive DNA sensor for sensitive detection of Escherichia coli O157:H7 in potable water based on the z3276 genetic marker: fabrication and analytical performance.

We report a label-free biosensor for the detection of Escherichia coli O157:H7 ATCC 43895 in potable water using a newly designed DNA sensing probe targeting the z3276 genetic marker. The surface of indium tin oxide (ITO) was functionalized with the novel sensing probe by covalent coupling using APTES as a crosslinker to fabricate the DNA sensor (dubbed ZEC [z[combining low line]3276 gene of E[combining low line]. c[combining low line]oli O157:H7 ATCC 43895]). The electrochemical characterization of the fabricated ZEC sensor was performed using cyclic voltammetry and electrochemical impedance spectroscopy. Atomic force microscopy and scanning electron microscopy revealed significant changes in the surface topographies of the fabricated ZEC sensor chip. Equivalent circuit analyses suggested the capacitive nature of the ZEC sensor chip, which demonstrated a declining trend of the capacitance value from 1.568 µF (Bare ITO) to 1.221 µF (after DNA hybridization). Non-faradaic sensing measurements revealed systematically declining capacitance values upon DNA hybridization, with a 10 min response time at 10 Hz frequency and 10 mV applied potential. The ZEC sensor chip exhibited linearity in the range of 0.5 to 25 pg per 10 mL for E. coli O157:H7, with ubiquitous cross-validation of each DNA concentration using quantitative PCR prior to the analyses of real water samples. The limit of detection (LOD) at 95% confidence estimated by logistic regression was 0.1 pg DNA per 10 mL of E. coli O157:H7 (equivalent to 13.67 CFU per 10 mL) with a p-value of 0.0237. Consequently, the obtained results demonstrate the possible application of the developed ZEC sensor chip for E. coli O157:H7 detection in real water samples.

A novel method for rapid and sensitive detection of viable Escherichia coli cells using UV-induced PMA-coupled quantitative PCR.

We report a specific and sensitive method to improve the coupling of propidium monoazide (PMA) with DNA derived from killed cells of Escherichia coli using UV light of 365 nm. UV light of three different intensities mainly 2.4 × 103, 4.8 × 103, and 7.2 × 103 µJ/cm2 was applied to E. coli cells each for 1, 3, and 5 min. PMA was found to be successfully cross-linked with the DNA from killed cells of E. coli at 4.8 × 103 µJ/cm2 in 3 min leading to the complete inhibition of PCR amplification of DNA derived from PMA-treated heat-killed cells. In spiked phosphate-buffered saline and potable water samples, the difference of the Cq values between PMA-treated viable cells and PMA-untreated viable cells ranged from -0.17 to 0.2, demonstrating that UV-induced PMA activation had a negligible effect on viable cells. In contrast, the difference of the Cq values between PMA-treated heat-killed cells and PMA-untreated heat-killed cells ranged from 8.9 to 9.99, indicating the ability of PMA to inhibit PCR amplification of DNA derived from killed cells to an equivalent as low as 100 CFU. In conclusion, this UV-coupled PMA-qPCR assay provided a rapid and sensitive methodology to selectively detect viable E. coli cells in spiked water samples within 4 h.

UIISScan 1.1: A Field portable high-throughput platform tool for biomedical and agricultural applications.

The colorimetric sensing technology has evolved into an essential tool for high-throughput analysis including portability and cost-effectiveness among available biomedical and agricultural screening approach. In this endeavor, the objective of work is to focus on the development of a field-portable instrument based on an Uniform Illumination Imaging System (UIIS), which will facilitate the colorimetric biochemical sensing. The developed field-portable, wavelength independent UIIS has been exploited for (a) rotavirus detection using commercial enzymatic immunoassay based microplate kit; (b) pesticide residue detection and quantification; The proposed system exhibited a good correlation in comparison to another two conventional techniques, i.e., multi-plate reader (r = 0.9991938) and LC-MS/MS (r = 0.998877399) with a short analysis time of 5 min for 95 test samples. Moreover, the feasibility of UIIS system has also been explored as field-portable enzyme-linked immunosorbent assay (ELISA) plate reader. By incorporating the Mahalanobis distance calculation, the advanced algorithm has been investigated and developed to analyze the data. The overall dataset was transformed into a matrix format to give a good correlation with a conventional plate reader, i.e., r = 0.915389612. Internet of things (IoT) enabled decision support system can be exploited by using big data analytics. Finally, test results can be shared with concerned stakeholders and the remote users. Thus, the developed UIIS will help to identify potential public health threats expeditiosly compared to conventional time consuming process of sample submission to the laboratory for analysis.

Nanoimmunosensor based on ZnO nanorods for ultrasensitive detection of 17ß-Estradiol.

Advances in nanostructured materials have facilitated the development of novel sensitive techniques for detection of environmental and clinical analytes. There is immense need for development of devices that can detect analytes at concentrations as low as few pg mL-1. The comparable size of nanostructured materials and biomolecules enabled the integration of biological systems with nanometer sized structures. Herein, we demonstrate a Zinc Oxide nanorods (ZnONRs) integrated ultrasensitive label-free biosensor with femtomolar (0.01 pg mL-1) sensitivity for the endocrine disruptor 17ß-Estradiol (E2). The ZnONRs, average width 50 nm and length 325 nm, were grown on the silver electrode surface (Ag-ZnONRs). Monoclonal antibodies of E2 (mAb-E2) were covalently immobilized on ZnONRs surface and measured using electrochemical impedance spectroscopy (EIS). A linear detection range of 0.1-200 pg mL-1 for E2 with R2 = 0.99 and % RSD = 4.35 (n = 3, assay volume 90 µL) was achieved for the developed nano-sensing system. A significant enhancement in the sensitivity was achieved in the presence of ZnONRs, enabling the limit of quantification down to 0.1 pg mL-1 with 2.7 % capacitance change per decade. In addition, a further increase in sensitivity due to assay volume reduction (20 µL) was observed enabling further scope of miniaturization.

Designed Strategies for Fluorescence-Based Biosensors for the Detection of Mycotoxins.

Small molecule toxins such as mycotoxins with low molecular weight are the most widely studied biological toxins. These biological toxins are responsible for food poisoning and have the potential to be used as biological warfare agents at the toxic dose. Due to the poisonous nature of mycotoxins, effective analysis techniques for quantifying their toxicity are indispensable. In this context, biosensors have been emerged as a powerful tool to monitors toxins at extremely low level. Recently, biosensors based on fluorescence detection have attained special interest with the incorporation of nanomaterials. This review paper will focus on the development of fluorescence-based biosensors for mycotoxin detection, with particular emphasis on their design as well as properties such as sensitivity and specificity. A number of these fluorescent biosensors have shown promising results in food samples for the detection of mycotoxins, suggesting their future potential for food applications.

Electrochemical Quartz Crystal Nanobalance (EQCN) Based Biosensor for Sensitive Detection of Antibiotic Residues in Milk.

An electrochemical quartz crystal nanobalance (EQCN), which provides real-time analysis of dynamic surface events, is a valuable tool for analyzing biomolecular interactions. EQCN biosensors are based on mass-sensitive measurements that can detect small mass changes caused by chemical binding to small piezoelectric crystals. Among the various biosensors, the piezoelectric biosensor is considered one of the most sensitive analytical techniques, capable of detecting antigens at picogram levels. EQCN is an effective monitoring technique for regulation of the antibiotics below the maximum residual limit (MRL). The analysis of antibiotic residues requires high sensitivity, rapidity, reliability and cost effectiveness. For analytical purposes the general approach is to take advantage of the piezoelectric effect by immobilizing a biosensing layer on top of the piezoelectric crystal. The sensing layer usually comprises a biological material such as an antibody, enzymes, or aptamers having high specificity and selectivity for the target molecule to be detected. The biosensing layer is usually functionalized using surface chemistry modifications. When these bio-functionalized quartz crystals are exposed to a particular substance of interest (e.g., a substrate, inhibitor, antigen or protein), binding interaction occurs. This causes a frequency or mass change that can be used to determine the amount of material interacted or bound. EQCN biosensors can easily be automated by using a flow injection analysis (FIA) setup coupled through automated pumps and injection valves. Such FIA-EQCN biosensors have great potential for the detection of different analytes such as antibiotic residues in various matrices such as water, waste water, and milk.

Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview.

Waterborne diseases have emerged as global health problems and their rapid and sensitive detection in environmental water samples is of great importance. Bacterial identification and enumeration in water samples is significant as it helps to maintain safe drinking water for public consumption. Culture-based methods are laborious, time-consuming, and yield false-positive results, whereas viable but nonculturable (VBNCs) microorganisms cannot be recovered. Hence, numerous methods have been developed for rapid detection and quantification of waterborne pathogenic bacteria in water. These rapid methods can be classified into nucleic acid-based, immunology-based, and biosensor-based detection methods. This review summarizes the principle and current state of rapid methods for the monitoring and detection of waterborne bacterial pathogens. Rapid methods outlined are polymerase chain reaction (PCR), digital droplet PCR, real-time PCR, multiplex PCR, DNA microarray, Next-generation sequencing (pyrosequencing, Illumina technology and genomics), and fluorescence in situ hybridization that are categorized as nucleic acid-based methods. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence are classified into immunology-based methods. Optical, electrochemical, and mass-based biosensors are grouped into biosensor-based methods. Overall, these methods are sensitive, specific, time-effective, and important in prevention and diagnosis of waterborne bacterial diseases.

Development of structure switching aptamer assay for detection of aflatoxin M1 in milk sample.

The discovery of in-vitro systematic evolution of ligands by exponential enrichment (SELEX) process has considerably broaden the utility of aptamer as bio-recognition element, providing the high binding affinity and specificity against the target analytes. Recent research has focused on the development of structure switching signaling aptamer assay, transducing the aptamer- target recognition event into an easily detectable signal. In this paper, we demonstrate the development of structure switching aptamer assay for determination of aflatoxin M1 (AFM1) employing the quenching-dequenching mechanism. Hybridization of fluorescein labelled anti-AFM1 aptamer (F-aptamer) with TAMRA labelled complementary sequences (Q-aptamer) brings the fluorophore and the quencher into close proximity, which results in maximum fluorescence quenching. On addition of AFM1, the target induced conformational formation of antiparallel G-quadruplex aptamer-AFM1 complex results in fluorescence recovery. Under optimized experimental conditions, the developed method showed the good linearity with limit of detection (LOD) at 5.0ngkg(-1) for AFM1. The specificity of the sensing platform was carefully investigated against aflatoxin B1 (AFB1) and ochratoxin A (OTA). The developed assay platform showed the high specificity towards AFM1. The practical application of the developed aptamer assay was verified for detection of AFM1 in spiked milk samples. Good recoveries were obtained in the range from 94.40% to 95.28% (n=3) from AFM1 spiked milk sample.

Titanium Dioxide Nanoparticles (TiO2) Quenching Based Aptasensing Platform: Application to Ochratoxin A Detection.

We demonstrate for the first time, the development of titanium dioxide nanoparticles (TiO2) quenching based aptasensing platform for detection of target molecules. TiO2 quench the fluorescence of FAM-labeled aptamer (fluorescein labeled aptamer) upon the non-covalent adsorption of fluorescent labeled aptamer on TiO2 surface. When OTA interacts with the aptamer, it induced aptamer G-quadruplex complex formation, weakens the interaction between FAM-labeled aptamer and TiO2, resulting in fluorescence recovery. As a proof of concept, an assay was employed for detection of Ochratoxin A (OTA). At optimized experimental condition, the obtained limit of detection (LOD) was 1.5 nM with a good linearity in the range 1.5 nM to 1.0 µM for OTA. The obtained results showed the high selectivity of assay towards OTA without interference to structurally similar analogue Ochratoxin B (OTB). The developed aptamer assay was evaluated for detection of OTA in beer sample and recoveries were recorded in the range from 94.30%-99.20%. Analytical figures of the merits of the developed aptasensing platform confirmed its applicability to real samples analysis. However, this is a generic aptasensing platform and can be extended for detection of other toxins or target analyte.

Ultrasensitive detection of streptomycin using flow injection analysis-electrochemical quartz crystal nanobalance (FIA-EQCN) biosensor.

This work presents the development of an ultrasensitive biosensor for detection of streptomycin residues in milk samples using flow injection analysis-electrochemical quartz crystal nanobalance (FIA-EQCN) technique. Monoclonal antibody specific to streptomycin was immobilized on to the thiol modified gold quartz crystal surface. A broad dynamic range (0.3-300 ng/mL) was obtained for streptomycin with a good linearity in the range 0.3-10 ng/mL for PBS and 0.3-50 ng/mL for milk. The correlation coefficient (R(2)) of the biosensor was found to be 0.994 and 0.997 for PBS and milk respectively. Excellent recoveries were obtained from the streptomycin spiked milk samples in the range 98-99.33%, which shows the applicability of the developed biosensor in milk. The reproducibility of the developed biosensor was found satisfactory with % RSD (n=5) 0.351. A good co-relation was observed between the streptomycin recoveries measured through the developed biosensor and the commercial ELISA kit. The analytical figures of merit of the developed biosensor confirm that the developed FIA-EQCN biosensor could be very effective for low-level detection of streptomycin in milk samples.

Flow injection analysis biosensor for urea analysis in urine using enzyme thermistor.

There is a need for analytical methods capable of monitoring urea levels in urine for patients under clinical monitoring to appraise renal function. Herein, we present a practical method to quantify levels of urea in human urine samples using flow injection analysis-enzyme thermistor (FIA-ET) biosensor. The biosensor comprises a covalently immobilized enzyme urease (Jack bean) on aminated silica support, which selectively hydrolyzes the urea present in the sample. Under optimized conditions, the developed biosensor showed a linear response in the range of 10-1,000 mM, R (2) = 0.99, and response time of 90 s in 100 mM phosphate buffer (PB) (flow rate of 0.5 mL/min, sample volume of 0.1 mL, and pH 7.2). The urea-spiked human urine samples showed minimal matrix interference in the range of 10-1,000 mM. Recoveries were obtained (92.26-99.80 %) in the spiked urine samples. The reliability and reproducibility of the developed biosensor were found satisfactory with percent relative standard deviation (% RSD) = 0.741. The developed biosensor showed excellent operational stability up to 30 weeks with 20 % loss in original response when used continuously at room temperature. These results indicate that the developed biosensor could be very effective to detect low and high levels of urea in urine samples.

Flow-based impedimetric immunosensor for aflatoxin analysis in milk products.

Label-free detection technique based on impedance was investigated for aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) analysis in milk products. The impedance change resulting from antigen-antibody interaction was studied using a two-electrode setup made up of silver (Ag) wire. Processed milk such as drinking yogurt and flavored milk samples were analyzed in a flow-based setup. Two microflow pumps were used to construct the flow system where analytes (AFM1 and AFM2) were injected and impedance was measured using functionalized Ag wire electrodes. The flow system was optimized by adjusting both inlet and outlet flows to maintain the reaction volume optimum for impedance measurements. Using Bode plot, the matrix effect was investigated for detection of AFM1 and AFM2 in various matrices. Good recoveries were obtained even at low-AFM1 concentrations in the range of 1-100 pg/mL. The influence of AFM2 on the detection of AFM1 was also investigated. The proposed method provides good scope for online monitoring of such hazardous toxins in milk products.

A miniaturized nanobiosensor for choline analysis.

A novel reusable chemiluminescence choline nanobiosensor has been developed using aligned zinc oxide nanorod-films (ZnONR). The chemically fashioned ZnONR were synthesized by hybrid wet chemical route onto glass substrates and used to fabricate a stable chemiluminescent choline biosensor. The biosensor was constructed by co-immobilization of the enzymes choline oxidase and peroxidase. The covalent immobilization of the enzymes on the ZnONR was achieved using 16-phosphonohexadecanoic acid as a cross-linker. The phosphonation of the ZnONR imparted significant stability to the immobilized enzyme as against physisorbed enzyme. A lower value of Michaelis-Menten constant (Km), of 0.062 mM for the covalently coupled enzyme over the physisorbed enzymes facilitated enhanced stability of ZnONR nanobiosensor. The ZnONR-choline biosensor has been investigated over a wide range of choline from 0.0005 mM to 2 mM. Importantly, the recovery of choline in milk samples was close to 99%. Using the developed biosensor, choline was measurable even after 30 days with 60 repeated measurements proving the stability of the sensor (Intraday RSD%=2.83 and Interday RSD%=3.51).

Fluorimetric immunoassay for multianalysis of aflatoxins.

A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25-50 pg/mL. AFM1 as low as 1 pg/mL was detected by this method with assay volume 40 µ L. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling.