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Pal S.,Biosensor Laboratory | Sharma M.K.,Indian Institute of Technology Delhi | Danielsson B.,Acromed Invest AB | Willander M.,Linköping 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.


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.


Deshpande K.,Biosensor Laboratory | Mishra R.K.,Biosensor Laboratory | Bhand S.,Biosensor Laboratory
Applied Biochemistry and Biotechnology | Year: 2011

A simple, miniaturized microplate chemiluminescence assay for determination of methyl parathion (MP) was developed in 384-microwell plates. Zirconia (ZrO2) was added in microwell for adsorption of acetylcholinesterase (AChE). The developed assay is based on inhibition of AChE by MP. A good dynamic range 0.008-1,000 ng/mL was obtained for MP with limit of detection 0.008 ng/mL. Intrabatch and interbatch reproducibility for miniaturized assay was obtained with % RSD up to 3.07 and 5.66, respectively. In 384 well plate formats, 70 samples were simultaneously analyzed within 20 min with assay volume of 41.5 μL. The application of developed assay was extended for MP remediation. Column containing ZrO2 was utilized for remediation where MP was selectively adsorbed. Under optimized condition, adsorption of MP on ZrO2 was found to be 98-99% with 2-h contact time in real water samples. Adsorption of MP on ZrO2 column followed by quantification using developed bioassay provides a novel approach to monitor remediation. The applicability of assay was successfully extended for determination of MP in water samples after removal through ZrO2. © 2011 Springer Science+Business Media, LLC.


Mishra G.K.,Biosensor Laboratory | Bhand S.,Biosensor Laboratory
Proceedings of the International Conference on Sensing Technology, ICST | Year: 2012

The widespread use of antibiotics within the farming industry is one of the factors that have been linked to the appearance of these residues in milk. A flow injection analysis-electrochemical quartz crystal nano-balance (FIA-EQCN) biosensor is presented for label free analysis of sulphadiazine residues in milk. The biosensor constituted a polyclonal antibody of sulphadiazine covalently immobilized to a gold coated quartz crystal mounted on a flow cell attached with FIA-EQCN setup. The biosensor showed linearity in the range 50 to 200 μg kg -1, with minimum detection limit of 10 μg kg -1, R2 0.97 and % R.S.D. 0.83. Good recoveries were obtained (98.6-100.5%) for sulphadiazine in the spiked milk samples. A good correlation was observed between the sulphadiazine concentrations measured through FIA-EQCN and the commercial ELISA kit. These results indicate that utilizing this system could be very effective to detect low level of sulphadiazine residues in milk. © 2012 IEEE.


Bacher G.,Biosensor Laboratory | Pal S.,Biosensor Laboratory | Kanungo L.,Biosensor Laboratory | Bhand S.,Biosensor Laboratory
Sensors and Actuators, B: Chemical | Year: 2012

A highly sensitive and selective label-free impedimetric immunosensor based on silver (Ag) wire electrode for the detection aflatoxin M1 (AFM1) in milk is presented. The sensor was constructed by functionalizing Ag wire coupled with selective monoclonal antibodies of AFM1 through self assembled monolayers. The antigen-antibody interaction was quantified by measuring impedance in the frequency range (1-100 Hz) at 10 mV applied ac potential. A linear working range (6.25-100 pg mL -1) for AFM1 was obtained with 20 min analysis time. The sensor sensitivity was found to be about 2.1% impedance change per decade with limit of quantitation 6.25 pg mL -1 and limit of detection 1 pg mL -1. Good recoveries were obtained with SD = 1.13 and R 2 = 0.982 in spiked milk samples. The proposed method is useful for sensitive analysis of AFM1 in milk. © 2012 Elsevier B.V.


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.


Kanungo L.,Biosensor Laboratory | Bacher G.,Biosensor Laboratory | Bhand S.,Biosensor Laboratory
Applied Biochemistry and Biotechnology | Year: 2014

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. © 2014, Springer Science+Business Media New York.


Mishra G.K.,Biosensor Laboratory | Sharma A.,Biosensor Laboratory | Bhand S.,Biosensor Laboratory
Biosensors and Bioelectronics | Year: 2015

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 (0.3-300ng/mL) was obtained for streptomycin with a good linearity in the range 0.3-10ng/mL for PBS and 0.3-50ng/mL for milk. The correlation coefficient (R2) 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. © 2014 Elsevier B.V.


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.


Mishra R.K.,Biosensor Laboratory | Deshpande K.,Biosensor Laboratory | Bhand S.,Biosensor Laboratory
Sensors | Year: 2010

A rapid, high-sensitivity, chemiluminescence (CL) enzyme assay for the determination of organophosphate (OP) residues in milk is presented. The assay for quantification of OP residues in milk is based on the inhibition of enzyme butyrylcholinesterase (BuChE). BuChE was stabilized and preloaded in 384 well plates at 30 °C. The assay permits rapid determination of OPs in milk within 12 min including an incubation step. The enzyme assay was tested for individual and mixtures of OPs such as methyl paraoxon (MPOx), methyl parathion (MP) and malathion (MT) in milk to evaluate their synergistic effect on BuChE inhibition. Good linearity was obtained in the range 0.005-50 μg·L-1 for MPOx and 0.5-1,000 μg·L-1 for MP as well as MT in milk. Mean recovery of 93.2%-98.6% was obtained for MPOx spiked milk samples with 0.99%-1.67% reproducibility (RSD). The proposed method facilitated rapid screening of milk samples in 384 well plate formats with further miniaturization presented in 1,536 well plates.© 2010 by the authors.

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