Sandeep Kumar Jha

Entrapment of live microbial cells in electropolymerized polyaniline and their use as urea biosensor.

The lyophilized biomass of bacterium Brevibacterium ammoniagenes was immobilized in polystyrene sulphonate-polyaniline (PSS-PANI) conducting polymer on a Pt twin wire electrode by potentiostatic electropolymerization. The bacterial cells retained their viability as well as urease activity under entrapped state, as confirmed with bacterial live-dead fluorescent assay and enzymatic assays. The entrapped cells were visualized using scanning electron microscope. The immobilized cells were used as a source of unpurified urease to develop a conductometric urea biosensor. The catalytic action of urease in the sensor released ammonia, thereby causing an increase in the pH of the microenvironment. The pH dependant change in the resistivity of the polymer was used as the basis of sensing mechanism. The sensor response was linear over a range of 0-75 mM urea with a sensitivity of 0.125 mM(-1). The sensor could be reused for 12-15 independent measurements and was quite stable in dry as well as buffered storage condition at 4 degrees C for at least 7 days.

Dopamine biosensor based on surface functionalized nanostructured nickel oxide platform

A dopamine biosensor has been developed using nickel oxide nanoparticles (NPs) and tyrosinase enzyme conjugate. Nickel oxide (NiO) NPs were synthesized by sol-gel method using anionic surfactant, sodium dodecyl sulphate (SDS), as template to control the size of synthesized nanoparticles. The structural and morphological studies of the prepared NPs were carried out using X-ray diffraction (XRD), transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. Afterwards, tyrosinase enzyme molecules were adsorbed on NiO NPs surface and enzyme coated NPs were deposited on indium tin oxide (ITO) coated flexible polyethylene terephthalate (PET) substrate by solution casting method. The formation of enzyme-NPs conjugate was investigated by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) techniques and used in selective detection and estimation of neurochemical dopamine by electrochemical method. The fabricated Tyrosinase/NiO/ITO electrode exhibits high sensitivity of 60.2nA/µM in linear detection range (2-100μM) with a detection limit of 1.038μM. The proposed sensor had a response time of 45s, long shelf life (45 days) with good reproducibility and selectivity in presence of interfering substances and was validated with real samples. The tyrosinase enzyme functionalized NiO platform has good bio-sensing efficacy and can be used in detection of other catecholamines and phenolic neurochemicals.

A paper strip based non-invasive glucose biosensor for salivary analysis

In our present study, we developed an optical biosensor for direct determination of salivary glucose by using immobilized glucose oxidase enzyme on filter paper strip (specific activity 1.4 U/strip) and then reacting it with synthetic glucose samples in presence of co-immobilized color pH indicator. The filter paper changed color based on concentration of glucose in reaction media and hence, by scanning this color change (using RGB profiling) through an office scanner and open source image processing software (GIMP) the concentration of glucose in the reaction medium could be deduced. Once the biosensor was standardized, the synthetic glucose sample was replaced with human saliva from donors. The individuals blood glucose level at the time of obtaining saliva was also measured using an Accuchek(™) active glucometer (Roche Inc.). In this preliminary study, a correlation of nearly 0.64 was found between glucose levels in saliva and blood of healthy individuals and in diabetic patients it was nearly in the order of 0.95, thereby validating the importance of salivary analysis. The RGB profiling method obtained a detection range of 9-1350 mg/dL glucose at a response time of 45 s and LOD of 22.2 mg/dL.

Development of potentiometric urea biosensor based on urease immobilized in PVA–PAA composite matrix for estimation of blood urea nitrogen (BUN)

A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1-1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinitytrade mark BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, beta-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity.

Capillary electrophoresis microchip for direct amperometric detection of DNA fragments.

Detection and quantitation of nucleic acids have gained much importance in the last couple of decades, especially in the post‐human genome project era. Such processes are tedious, time consuming and require expensive reagents and equipment. Therefore, in the present study, we demonstrated a simple process for the separation and analysis of small DNA fragments using capillary electrophoretic amperometric detection on an inexpensive disposable glass microchip. The device used polydimethylsiloxane engraved microchannel and Au/Ti in‐channel microelectrodes for sample detection. The DNA fragments were separated under low electric field (20 V/cm) for improved detection sensitivity and to retain the biomolecules in their native conformation. With a low sample requirement (as low as 1 μL) and high reproducibility, the proposed microchip device was successful in resolution and detection of DNA fragments of various lengths.

Analytical detection of biological thiols in a microchip capillary channel.

Sulfur-containing amino acids, such as cysteine and homocysteine play crucial roles in biological systems for the diagnosis of medical states. In this regard, this paper deals with separation, aliquot and detection of amino thiols on a microchip capillary electrophoresis with electrochemical detection in an inverted double Y-shaped microchannel. Unlike the conventional capillary electrophoresis, the modified microchannel design helps in storing the separated thiols in different reservoirs for further analysis, if required; and also eliminates the need of electrodes regeneration. The device was fabricated using conventional photolithographic technique which consisted of gold microelectrodes on a soda lime glass wafer and microchannels in PDMS mold. Multiple detections were performed using in-house fabricated dual potentiostat. Based on amperometric detection, cysteine and homocysteine were analyzed in 105 s and 120 s, respectively after diverting in branched channels. Repeated experiments proved the good reproducibility of the device. The device produced a linear response for both cysteine and homocysteine in electrochemical analysis. To prove the practicality of device, we also analyzed cysteine and homocysteine in real blood samples without any pre-treatment. Upon calculation, the device showed a very low limit of detection of 0.05 μM. The modified microchip design shall find a broad range of analytical applications involving assays of thiols and other biological compounds.

Immobilization of the urease on eggshell membrane and its application in biosensor.

Eggshell membrane is a natural material, essentially made up of protein fibers having flexibility in the aqueous solution and possessing gas and water permeability. It is used as a biomembrane for immobilization of urease for the development of a potentiometric urea biosensor. Eggshell membrane was treated with polyethyleneimine (PEI) to impart polycation characteristics. Urease was immobilized on the PEI treated eggshell membrane through adsorption. SEM study was carried out to observe the changes in surface morphology after immobilization. FTIR study of membrane was carried out to observe the changes in IR spectra after immobilization of enzyme. Immobilized membrane was associated with ammonium ion selective electrode. Biosensor exhibited sigmoidal responses for the urea concentration range from 0.5 to 10mM. The response time of the biosensor was 120 s. A single membrane was reused for 270 reactions without loss of activity. The urease-eggshell membranes were stable for 2 months when stored in buffer even at room temperature.

Development of PCR Microchip for Early Cancer Risk Prediction

An integrated continuous-flow microfluidic chip was fabricated on glass substrate with polydimethylsiloxane (PDMS)-based microchannels, cell lysis and Polymerase chain reaction (PCR) modules on the same chip. While gold-microelectrode was used for electrochemical cell lysis, indium-tin-oxide (ITO) microheater was used for thermal cycling during PCR reaction. The fabricated device was used for PCR amplification of pancreatic cancer DNA marker (SMAD4) from non-tumorigenic MCF10a human cell lines. The PCR product (193 bp) was verified for MCF10a cells by agarose gel electrophoresis after 20 cycles of reaction on the microchip, whereas no product was detected in case of tumorigenic MCF7 cells. The total time required for the entire reaction was less than 45 min. Therefore, the proposed microchip can be helpful in predicting the risk of metastatic cancer by analysis of genetic tumor markers from human samples and can also be used for other genetic analysis involving PCR reaction.

Flexible pentacene thin film transistors as DNA hybridization sensor

A DNA hybridization sensor using pentacene thin film transistors (TFTs) is an excellent candidate for disposable sensor applications due to their low-cost fabrication process and fast detection. We fabricated pentacene TFTs on flexible substrate for the sensing of DNA hybridization. The 100 mer ss-DNA (poly A/poly T) or 100 bp ds-DNA (poly A/poly T hybrid) are deposited from a solution on pentacene layer. The electrical characteristics of devices were studied as a function of DNA immobilization, single- and double-strand DNA and DNA concentrations. The DNA molecules were immobilized directly on the surface of the pentacene, thereby producing a dramatic change in the electrical properties of the devices. Based on these results, we propose that a “label-free” detection technique for DNA hybridization is possible through direct measurement of electrical properties by the immobilization of DNA on pentacene TFTs.

Miniaturisation of a capillary electrophoresis microchip for the sensing of endocrine disruptors

Although capillary electrophoresis amperometric detector (CE-AD) involving double-T microchannel configuration is a powerful analytical tool in terms of sensitivity and selectivity, its long microchannel configuration hinders further miniaturisation. Therefore a twisted CE microchannel configuration was used in the present study to fabricate CE-AD devices for detection of endocrine disruptors. The analyte separation time varied slightly for the twisted microchannel structure, whereas the detector sensitivities were similar for the two configurations. The conventional indium tin oxide amperometric detector in the device with twisted microchannel configuration was later modified with Prussian blue to enhance the sensitivity of detection.