Rapid and label-free detection of Aflatoxin-B1viamicrofluidic electrochemical biosensor based on manganese (III) oxide (Mn3O4) synthesized by co-precipitation route at room temperature.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin, naturally occurring in food items, and it causes several types of lethal diseases. Therefore, a rapid and convenient detection method for AFB1 is the first step toward overcoming the effect of AFB1. The current work presents the development of an efficient microfluidic electrochemical-based biosensor using tri-manganese tetroxide nanoparticles (Mn3O4nps) for AFB1 detection. The Mn3O4nps were synthesized at room temperature through the co-precipitation route. Its phase purity, structural and morphological studies have been characterized through x-ray diffraction, Raman spectroscopy, energy-dispersive x-ray, Fourier transform infrared spectroscopy and transmission electron microscopy. The mask-less UV-lithography was carried out to fabricate the three-electrode chip and microfluidic channel of the microfluidic electrochemical biosensing system. The designed microfluidic immunosensor (BSA/Ab-AFB1/Mn3O4/ITO) was fabricated using the three-electrode chip, microfluidic channel in poly-dimethyl siloxane. The fabricated sensor exhibited the 3.4µA ml ng-1cm-2sensitivity and had the lowest lower detection limit of 0.295 pg ml-1with the detection range of 1 pg ml-1to 300 ng ml-1. Additionally, the spiked study was also performed with this immunoelectrode and a recovery rate was obtained of 108.2%.

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.

Microfluidic biosensor for beta-amyloid(1-42) detection using cyclic voltammetry.

Numerous studies have identified beta-amyloid(1-42) protein (Abeta42) in the cerebrospinal fluid as a potential biomarker of Alzheimer's disease. It is of particular interest to establish the diagnosis before reaching the stage of clinical severity. The current methods for studying amyloid detection, however, is often time-consuming, expensive, and labor intensive, making the analytical process very slow. Thus, a critical need exists for an analytical system that would enable the rapid investigation of amyloid formation with a very small amount of amyloidogenic peptides and reagents. In our present work, we report a simple microfluidic biosensor to analyze very small quantities of Abeta42 peptide on gold surface that were modified with Au nano-particles onto the thiol groups of self-assembled 1,6-hexandithiol cross-linkers. The vital advantage of this method includes retaining the bioactivity and environment similar to nature for protein immobilization while using minimal amounts of reagents and highly sensitive detection.

The selection of highly specific and selective aptamers using modified SELEX and their use in process analytical techniques for Lucentis bioproduction.

Aptamers for Lucentis were selected using 10 rounds of a modified and highly stringent SELEX process. Affinity column chromatography was used for the binding, partitioning, and elution steps, and the regeneration of ssDNA was performed via asymmetric PCR in the SELEX process. The interaction of aptamers with Lucentis was studied by means of the HADDOCK web server docking program. In addition, the secondary structures of aptamers were interrogated using the mfold web server to check common regions responsible for better affinity towards Lucentis. The two best aptamers for Lucentis (aptamers 1 and 25) were found to have dissociation constant (K d) values between 23 and 35 nM by means of thermofluorimetric and non-faradaic impedance spectroscopy (NFIS) analysis. The low dissociation constants in the nanomolar range showed the high specificities of the aptamers for Lucentis. Selectivity tests were also performed using both aptamers with different proteins in which negligible responses were obtained from interfering proteins with respect to Lucentis. Although neither of the two aptamers showed prominent responses to the interfering proteins, slightly better selectivity was shown by aptamer 1. The same aptamers were tested for their application in the detection of Lucentis in spiked and real media broth samples. For this detection test, interdigitated (IDT) gold electrodes on a glass substrate were fabricated using standard photolithography and thermal deposition techniques. NFIS measurements were used for the label-free detection of Lucentis in samples. The linear ranges of detection for aptamers 1 and 25 were found to be 22-100 nM and 40-100 nM, respectively. The LODs for aptamers 1 and 25 were calculated to be 22 nM and 40 nM, respectively, which were significantly better than the values from a HPLC-based detection method (about 240 nM). The real sample analysis results were cross-checked via a standard HPLC method, and better correlation was found between the HPLC and aptamer 1 results than the aptamer 25 results; hence, aptamer 1 can be further analyzed and tested for use in affinity column chromatography and detection-kit/chip-based PAT for Lucentis bioproduction.

A decoupler-free simple paper microchip capillary electrophoresis device for simultaneous detection of dopamine, epinephrine and serotonin.

This paper demonstrates a new and simplified configuration for capillary electrophoresis-amperometric detection (CE-AD) using a paper microfluidic chip incorporating inexpensive wax printing and screen printing based methods and then used for electrophoretic separation and simultaneous in-channel amperometric detection of three clinically relevant neurochemicals in a single run without using any decouplers. Detection of neurochemicals e.g., dopamine, epinephrine and serotonin is crucial for early prediction of neurological disorders including Parkinson's, Alzheimer's, dementia, as well as progressive neuro-psychiatric conditions such as depression, anxiety, as well as certain cardiovascular diseases. The plasma concentrations of such neurochemicals are as important as those present in cerebrospinal fluid (CSF) and can be useful for rapid and convenient biosensing. However, simultaneous detection of such neurochemicals in a complex mixture such as human serum requires their separation prior to detection. With the developed microchip, separation and detection of the neurochemicals were exhibited within 650 seconds without pre-treatment and the procedure was validated with spiked fetal bovine serum samples. Beside this, the developed paper microfluidic chip has potential to be integrated in point-of-care diagnosis with onsite detection ability. Moreover, the use of a straight channel capillary, a screen-printed carbon electrode without decoupler, in-channel amperometric detection and low sample volume requirements (2 µL) are shown as additional advantages.

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.

Surface Functionalized Prussian Blue-coated Nanostructured Nickel Oxide as a New Biosensor Platform for Catechol Detection.

An amperometric biosensor has been developed for highly efficient and sensitive detection of catechol using Prussian blue (PB)-coated nickel oxide (NiO) nanoparticles (NPs) as a matrix for the immobilization of tyrosinase enzyme. The NiO NPs were synthesized by sol-gel method using sodium dodecyl sulphate as anionic surfactant and the surface of the synthesized NiO NPs was modified with PB to enhance electrocatalytic activity and to prevent surface aggregation. After confirmation of successful synthesis of the PB-NiO NPs from transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopic (EDS) studies, the prepared NPs were deposited onto a working electrode of a commercially available screen printed carbon electrode (SPCE) substrate. The tyrosinase enzyme was covalently immobilized onto the PB-NiO deposited SPCE for selective detection and estimation of catechol through electrochemical methods via cyclic voltammetry (CV) and chronoamperometric techniques. The functionalization of tyrosinase on the electrode surface was verified by atomic force microscopy (AFM) and scanning electron microscopic (SEM) techniques and the electrochemical response studies of the proposed biosensor showed high sensitivity of 0.954 µA/µM for catechol in a wide linear range (1 - 50 µM) with low detection limit (LOD) of 0.087 µM. The developed sensor also exhibited a fast response time of 27 s and decent selectivity for catechol detection.

Smartphone based non-invasive salivary glucose biosensor.

The present work deals with the development of a non-invasive optical glucose biosensor using saliva samples and a smartphone. The sensor was fabricated with a simple methodology by immobilization of Glucose oxidase enzyme along with a pH responsive dye on a filter paper based strip. The strip changes color upon reaction with glucose present in saliva and the color changes were detected using a smartphone camera through RGB profiling. This standalone biosensor showed good sensitivity and low interference while operating within 20 s response time. We used various means for improvements such as the use of slope method instead of differential response; use of a responsive pH indicator and made numerous tweaks in the smartphone app. Calibration with spiked saliva samples with slopes for (R + G + B) pixels revealed an exponentially increasing calibration curve with a linear detection range of 50-540 mg/dL, sensitivity of 0.0012 pixels sec-1/mg dL-1 and LOD of 24.6 mg/dL. The biosensor was clinically validated on both healthy and diabetic subjects divided into several categories based on sex, age, diabetic status etc. and correlation between blood and salivary glucose has been established for better standardization of the sensor. Correlation of 0.44 was obtained between blood and salivary glucose in healthy individuals whereas it was 0.64 and 0.94 in case of prediabetic and diabetic patients respectively. The developed biosensor has the potential to be used for mass diagnosis of diabetes especially in such areas where people remain prohibited from routine analysis due to high healthcare cost. Apart from that, a smartphone would be the only device the user needs for this measurement, along with a disposable low cost test strip.

Is the absence of Right Hepatic Vein opening into Inferior Vena Cava a contraindication for right lobe liver donation in Living Donor Liver Transplantation? Common hepatic venous trunk-A rare hepatic vein anomaly: A case report and review.

INTRODUCTION: In majority of the living liver donors, the left and the middle hepatic veins form a common trunk and the right hepatic vein drains by a separate trunk into the IVC forming two ostial openings. PRESENTATION OF CASE: This report presents a rare challenge to the operating surgeon in which the three major hepatic veins form a common trunk and drain into the IVC through a single ostial opening. It was detected preoperatively by the routine donor imaging studies. DISCUSSION: To our knowledge, this type of rare venous anatomy in the setting of living donor liver transplantation has not been described in the literature before. A few studies have described similar anatomy in the cadaveric liver specimen of some particular ethnicity. CONCLUSION: This type of a rare anomaly poses challenge to the donor operation and requires a sound expertise on the knowledge of hepatic venous anatomy to perform the donor hepatectomy with the appropriate maneuvering.

Optical microbial biosensor for detection of methyl parathion pesticide using Flavobacterium sp. whole cells adsorbed on glass fiber filters as disposable biocomponent.

An optical microbial biosensor was described for the detection of methyl parathion pesticide. Whole cells of Flavobacterium sp. were immobilized by trapping in glass fiber filter and were used as biocomponent along with optic fiber system. Flavobacterium sp. has the organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into detectable product p-nitrophenol. The immobilized microbial biocomponent was disposable, cost-effective and showed high reproducibility and uniformity. The detection of methyl parathion by the use of disposable microbial biocomponent with optical biosensor was simple, single step and direct measurement of very low quantity of the sample. The home made reaction vessel was small and needed only 75 microl of sample. A lower detection limit 0.3 microM methyl parathion was estimated from the linear range (4-80 microM) of calibration plot of organophosphorus hydrolase enzymatic assay. The applicability to synthetic methyl parathion spiked samples was demonstrated.

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.

Reusable Glucose Sensor Based on Enzyme Immobilized Egg-shell Membrane.

In this paper, an egg-shell membrane has been used for efficient immobilization and stabilization of glucose oxidase. This membrane was used for developing a simple and reusable method for estimation of glucose in biological samples. The proposed sensor was effectively used in a wide glucose concentration range (1 - 1000 mM) with fast response time of 70 s for higher concentrations and 120 s for lower concentrations. The results of response study for the fabricated sensor show limit of detection of 4.761 mM with high sensitivity over the entire concentration range (1 - 1000 mM). Most interestingly, the membrane used in the fabricated sensor could be repeatedly used for glucose analysis 150 times and it exhibited a shelf-life of more than 6 weeks. The proposed sensor was also demonstrated for estimation of glucose in human blood samples.

Preparation of polyvinyl alcohol-polyacrylamide composite polymer membrane by gamma-irradiation for entrapment of urease.

Composite polymer membrane of polyvinyl alcohol (PVA) and acrylamide was prepared on cheesecloth support by gamma-irradiation induced free radical polymerization. The enzyme urease was entrapped in the membrane during polymerization and was cross-linked within the matrix using glutaraldehyde. The membranes could be reused a number of times without significant loss of urease activity.

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 individual's 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.

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.

An integrated PCR microfluidic chip incorporating aseptic electrochemical cell lysis and capillary electrophoresis amperometric DNA detection for rapid and quantitative genetic analysis.

A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 µl min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.

DNA hybridization sensor based on pentacene thin film transistor.

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 glass substrate for the sensing of DNA hybridization. The ss-DNA (polyA/polyT) or ds-DNA (polyA/polyT hybrid) were immobilized directly on the surface of the pentacene, producing a dramatic change in the electrical properties of the devices. The electrical characteristics of devices were studied as a function of DNA immobilization, single-stranded vs. double-stranded DNA, DNA length and concentration. The TFT device was further tested for detection of λ-phage genomic DNA using probe hybridization. Based on these results, we propose that a "label-free" detection technique for DNA hybridization is possible through direct measurement of electrical properties of DNA-immobilized pentacene TFTs.

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.