Rupesh K. Mishra

A novel automated flow-based biosensor for the determination of organophosphate pesticides in milk

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 15 min. 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(-6)M to 5 × 10(-12)M. The limit of detection (LOD) obtained in milk for CPO, EPOx and MOx were 5 × 10(-12)M, 5 × 10(-9)M and 5 × 10(-10)M, respectively, with a correlation coefficient R(2)=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.

Sensitive quantitation of Ochratoxin A in cocoa beans using differential pulse voltammetry based aptasensor

In this work, we propose for the first time a sensitive Ochratoxin A (OTA) detection in cocoa beans using competitive aptasensor by differential pulse voltammetry (DPV). In the proposed method, biotin labeled and free OTA competed to bind with immobilized aptamer onto the surface of a screen printed carbon electrode (SPCE), and percentage binding was calculated. The detection was performed after adding avidin-ALP to perform avidin-biotin reaction; the signal was generated through a suitable substrate 1-naphthyl phosphate (1-NP), for alkaline phosphatase (ALP). The cocoa samples were extracted and purified using molecular imprinted polymer (MIP) columns specifically designed for OTA. The developed aptasensor showed a good linearity in the range 0.15-5 ng/mL with the limit of detection (LOD) 0.07 ng/mL and 3.7% relative standard deviation (RSD). The aptasensor displayed good recovery values in the range 82.1-85% with 3.87% RSD, thus, demonstrated the efficiency of proposed aptasensor for such matrices.

A label free aptasensor for Ochratoxin A detection in cocoa beans: An application to chocolate industries

Contamination of food by mycotoxin occurs in minute/trace quantities. Nearly 92.5% of the cocoa samples present Ochratoxin A (OTA) levels at trace quantity. Hence, there is a necessity for a highly sensitive and selective device that can detect and quantify these organic toxins in various matrices such as cocoa beans. This work reports for the first time, a facile and label-free electrochemical impedimetric aptasensor for rapid detection and quantitation of OTA in cocoa beans. The developed aptasensor was constructed based on the diazonium-coupling reaction mechanism for the immobilization of anti-OTA-aptamer on screen printed carbon electrodes (SPCEs). The aptasensor exhibited a very good limit of detection (LOD) as low as 0.15 ng/mL, with added advantages of good selectivity and reproducibility. The increase in electron transfer resistance was linearly proportional to the OTA concentration in the range 0.15-2.5 ng/mL, with an acceptable recovery percentage (91-95%, RSD = 4.8%) obtained in cocoa samples. This work can facilitate a general model for the detection of OTA in cocoa beans based on the impedimetric aptasensor. The analysis can be performed onsite with pre-constructed and aptamer modified electrodes employing a portable EIS set up.

Flow injection analysis biosensor for urea analysis in adulterated milk using enzyme thermistor

Urea in adulterated milk is one of the major health concern, it is especially harmful to pregnant women, children, and the sick. A sophisticated and reliable detection system is needed to replace current diagnostic tools for the urea in the milk. In this work, we report a flow injection analysis-enzyme thermistor (FIA-ET) bio-sensing system for monitoring of urea in adulterated milk. This biosensor was made of the covalently immobilized enzyme urease (Jack bean) on controlled pore glass (CPG) and packed into a column inside thermistor, which selectively hydrolysed the urea present in the sample. The specific heat registered from the hydrolysis of urea was found proportional to the concentration of urea present in the milk sample. The biosensor showed a linear range 1-200 mM, with % R.S.D. 0.96 for urea in 100 mM phosphate buffer, pH 7.2. Good recoveries were obtained (97.56-108.7%) for urea up to 200 mM in the spiked milk samples with % R.S.D. 0.95. In the adulterated milk, a simple filtration strategy and matrix matching technique was used to analyse urea. The response time of the sensor was evaluated for urea, which was 2 min, and it gives satisfactory output. A good comparison was observed between the urea concentrations measured through FIA-ET and the colorimetric method. These results indicate that utilizing this system could be very effective to detect low and high level of urea in adulterated milk. The immobilized urease column exhibited a good operational stability up to 180 days when used continuously at room temperature.

Sensitive analytical performance of folding based biosensor using methylene blue tagged aptamers.

This work demonstrates the development of a folding based electrochemical aptasensor using methylene blue (MB) tagged anti-Ochratoxin A (OTA) aptamers. Different aptamer coupling strategies were tested using Hexamethylenediamine, polyethylene glycol, simple adsorption and diazonium coupling mechanism. The best sensitivity was recorded by oxidation of amines using hexamethylenediamine (HDMA) on screen printed carbon electrode (SPCE). To achieve the direct detection of OTA, aptamer conjugated redox probe was used and detection was demonstrated based on the conformational changes in aptamer structure upon OTA sensing. Signaling in this class of sensors arises from changes in electron transfer efficiency upon target-induced changes in the conformation/flexibility of the aptamer probe. These changes can be readily recorded electrochemically. The developed aptasensor is unique in its own mechanism as redox probe tagged aptamer coupling such as MB has never been tried to immobilize using long carbon chain spacers as, addition of spacers would provide more sensitive detection methods. A good dynamic range 0.01-5ng/ml was obtained for OTA with Limit of detection (LOD) 0.01ng/ml and Limit of quantification (LOQ) of 0.03ng/ml respectively. The good reproducibility was recorded with RSD% of 3.75. The obtained straight line equation was y=0.4035x+0.90311, r=0.9976. We believe that the sensor design guidelines outlined here represents a general strategy for developing new folding-based electrochemical aptasensors. The developed aptasensor was extended to screen cocoa samples for OTA contamination. The cocoa samples were extracted and purified using molecular imprinted polymer (MIP) columns. The aptasensor displayed good recovery values in the range 84-85% thus, exhibited the effectiveness of proposed aptasensor for such complex matrices.

Label free aptasensor for Lysozyme detection: A comparison of the analytical performance of two aptamers

This work presents a comparison of two different aptamers (Apts) (COX and TRAN) for the detection of a ubiquitous protein Lysozyme (Lys) using Apt-based biosensors. The detection is based on the specific recognition by the Apt immobilized on screen printed carbon electrodes (SPCEs) via diazonium coupling reaction. The quantitative detection of Lys protein was achieved by electrochemical impedance spectroscopy (EIS). A very good linearity and detection limits for the quantitation of Lys were obtained from 0.1 to 0.8 μM and 100 nM using Apt COX and from 0.025 to 0.8 μM and 25 nM using Apt TRAN respectively. The obtained results showed that the developed aptasensors exhibit good specificity, stability and reproducibility for Lys detection. For real application, the aptasensors were tested in wine samples and good recovery rates were recorded in the range from 94.2 to 102% for Lys detection. The obtained recovery rates confirm the reliability and suitability of the developed method in wine matrix. The developed method could be a useful and promising platform for detection of Lys in different applications.

Continuous minimally-invasive alcohol monitoring using microneedle sensor arrays

The present work describes an attractive skin-worn microneedle sensing device for the minimally invasive electrochemical monitoring of subcutaneous alcohol. The device consists of an assembly of pyramidal microneedle structures integrated with Pt and Ag wires, each with a microcavity opening. The microneedle aperture was modified by electropolymerizing o-phenylene diamine onto the Pt wire microtransducer, followed by the immobilization of alcohol oxidase (AOx) in an intermediate chitosan layer, along with an outer Nafion layer. The resulting microneedle-based enzyme electrode displays an interference-free ethanol detection in artificial interstitial fluid without compromising its sensitivity, stability and response time. The skin penetration ability and the efficaciousness of the biosensor performance towards subcutaneous alcohol monitoring was substantiated by the ex vivo mice skin model analysis. Our results reveal that the new microneedle sensor holds considerable promise for continuous non-invasive alcohol monitoring in real-life situations.

A high sensitivity micro format chemiluminescence enzyme inhibition assay for determination of Hg(II).

A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx) and horseradish peroxidase (HRP) for determination of mercury Hg(II) in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II) was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II) was found to be linear in the range 5–500 pg·mL−1 with 3% CV in inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II) was measurable as low as 1 pg·mL−1 within 15 min. About 10-fold more specificity of the developed assay for Hg(II) analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II) and lead Pb(II). Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II), Cd(II) and Pb(II), inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II) in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%.

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.

A High-Throughput Enzyme Assay for Organophosphate Residues in Milk

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.