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Kanungo L.,Biosensor Laboratory | Bhand S.,Biosensor Laboratory
Journal of Analytical Methods in Chemistry | Year: 2013

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 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. © 2013 Lizy Kanungo and Sunil Bhand. Source


Mishra R.K.,University of Perpignan | Mishra R.K.,Biosensor Laboratory | Mishra R.K.,Aix - Marseille University | Alonso G.A.,Autonomous University of Guerrero | And 3 more authors.
Sensors and Actuators, B: Chemical | Year: 2015

This work presents an application of automatic flow based biosensor to detect binary (chlorpyriphos-oxon (CPO) and malaoxon (MO)) organophosphate (OP) mixtures in milk, based on artificial neural network (ANN). Genetically modified acetylcholinesterase (AChE) B394 and B4 were used as a biological recognition element for sensor development. AChE binds with OPs irreversibly, creating an anionic phosphonyl species. The enzymes were coupled on screen printed electrodes (SPEs) and inserted in a flow cell connected to the potentiostat and syringe pump. In order to model the combined response of CPO and MO, a total set of 19 mixtures were prepared using ANN. The modeling was validated with an external test of 6 milk samples spiked with CPO and MO mixtures. The spiked concentrations of CPO and MO were ranged from 5 × 10-10 to 5 × 10-12 M and 1.01 × 10-10 to 9.17 × 10-11 M, respectively. These concentrations were determined using factorial designing (FD) method and the obtained and expected recovery values in milk showed good co-relation. The average % recovery yields for CPO and MO are 109.53 and 100.66, respectively. © 2014 Elsevier B.V. All rights reserved. Source


Mishra R.K.,University of Perpignan | Mishra R.K.,Biosensor Laboratory | Dominguez R.B.,University of Perpignan | Dominguez R.B.,CINVESTAV | And 3 more authors.
Biosensors and Bioelectronics | Year: 2012

This work describes the development of an automated flow-based biosensor that employs genetically modified acetylcholinesterase (AChE) enzymes B394, B4 and wild type B131. The biosensor was based on a screen printed carbon electrode (SPE) that was integrated into a flow cell. Enzymes were immobilised on cobalt (II) phthalocyanine (CoPC) modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). The automated flow-based biosensor was successfully used to quantify three organophosphate pesticides (OPs) in milk samples. The OPs used were chlorpyriphos-oxon (CPO), ethyl paraoxon (EPOx) and malaoxon (MOx). The total analysis time for the assay was less than 15min. Initially, the biosensor performance was tested in phosphate buffer solution (PBS) using B394, B131 and B4 biosensors. The best detection limits were obtained with B394; therefore, this biosensor was used to produce calibration data in milk with three OPs in the concentration range of 5×10-6M to 5×10-12M. The limit of detection (LOD) obtained in milk for CPO, EPOx and MOx were 5×10-12M, 5×10-9M and 5×10-10M, respectively, with a correlation coefficient R2=0.9910. The automated flow-based biosensor successfully quantified the OPs in different fat-containing milk samples. There were no false positives or false negatives observed for the analytical figures of merit for the constructed biosensors. This method is inexpensive, sensitive, portable, non-invasive and provides real-time results. This analytical system can provide rapid detection of highly toxic OPs in food matrices such as milk. © 2011 Elsevier B.V. Source


Pal S.,Biosensor Laboratory | Sharma M.K.,Indian Institute of Technology Delhi | Danielsson B.,Acromed Invest AB | Willander M.,Linkoping University | And 2 more authors.
Biosensors and Bioelectronics | Year: 2014

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 0.062mM 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 0.0005mM to 2mM. 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). © 2013 Elsevier B.V. Source


Kanungo L.,Biosensor Laboratory | Pal S.,Biosensor Laboratory | Bhand S.,Biosensor Laboratory
Biosensors and Bioelectronics | Year: 2011

An ultra-sensitive sandwich ELISA was developed for detection of AFM1 in milk. The assay involved the immobilization of rat monoclonal antibody of AFM1 in 384 microtiter plate to capture AFM1 antigen. This was detected by tracer secondary rabbit poly-clonal antibody labelled with horseradish peroxidase upon addition of a luminol-based substrate. Milk samples with different fat percentage were analyzed after pre-treatment. Linear range of AFM1 detection 250-6.25. pg/mL was achieved in 3% fat milk. The miniaturised assay (10μL) enabled ultra trace analysis of AFM1 in milk with much improved lower limit of detection at 0.005. pg/mL. A sensitive magnetic nanoparticles (MNPs) based ELISA was also developed and coupled with micro plate ELISA for analysis in milk. The hybrid-assay, by coupling the 1°Ab immobilized MNPs column with microwell plate assay enabled simultaneous measurement of low (0.5. pg/mL) and high AFM1 contamination (200. pg/mL). The most promising feature of this MNPs-ELISA is the small column size, high capture efficiency and lower cost over other reported materials. The proposed assay can be deployed for simultaneous analysis and monitoring of AFM1 in milk. © 2010 Elsevier B.V. Source

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