Rohit Chand

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.

Homogeneous Electrochemical Assay for Protein Kinase Activity

Herein, we report a homogeneous assay for protein kinase activity using an electrochemistry-based probe. The approach involves a peptide substrate conjugated with a redox tag and the phosphate-specific receptor immobilized on an electrode surface. The peptide substrate phosphorylated by a protein kinase binds to the receptor site of the probe, which results in a redox current under voltammetric measurement. Our method was successfully applied even in the presence of citrated human blood and modified to enable a single-use, chip-based electrochemical assay for kinase activity.

Microfluidic platform integrated with graphene-gold nano-composite aptasensor for one-step detection of norovirus

Noroviruses are a foremost cause of gastroenteritis outbreaks throughout the world. On-site sample processing and detection of the viral clinical samples has always been a problem. This study reports an all-polydimethylsiloxane microfluidic chip integrated with screen-printed carbon electrode for the electrochemical detection of norovirus. The microfluidic chip contained packed silica microbeads zones to filter and enrich the norovirus infected clinical sample. Selective detection of norovirus was accomplished by functionalizing the graphene-gold nanoparticles composite modified carbon electrode with the viral capsid-specific aptamer. Norovirus specific aptamer was tagged with a ferrocene molecule, which acts a redox probe. The interaction of aptamer and norovirus resulted in a decrease in the electrochemical signal from ferrocene. The microfluidic chip and functionalized electrodes were characterized using several microscopic and electrochemical techniques. The optimized microfluidic aptasensor was employed to detect a range of norovirus concentration. Using differential pulse voltammetric analysis, a detection limit of 100 pM with a detection range from 100 pM to 3.5nM for norovirus was obtained. The application of aptasensor was also assessed by detecting norovirus in spiked blood samples. The aptasensor could easily discriminate between the target norovirus and other interfering molecules. The developed microfluidic aptasensor has the potential to be used for point-of-care one-step detection of norovirus in clinical samples.

Microchip Capillary Electrophoresis Based Separation and Detection of Cysteine and Homocysteine

Cysteine and homocysteine are the biological thiols which have an important function in various biochemical processes in our body. Alterations in their level lead to various abnormalities. Therefore, we fabricated a miniaturized platform for capillary electrophoresis that could separate and detect these amino thiols electrochemically. The device was fabricated using conventional photolithography technique on the glass substrate. The microchannel was molded in polydimethylsiloxane with gold electrodes deposited on glass for separation and detection. Based on the amperometric detection, we could detect cysteine in 93 sec while homocysteine was detected in 111 sec.

Microscale loop-mediated isothermal amplification of viral DNA with real-time monitoring on solution-gated graphene FET microchip.

Rapid and reliable molecular analysis of DNA for disease diagnosis is highly sought-after. FET-based sensors fulfill the demands of future point-of-care devices due to its sensitive charge sensing and possibility of integration with electronic instruments. However, most of the FETs are unstable in aqueous conditions, less sensitive and requires conventional Ag/AgCl electrode for gating. In this work, we propose a solution-gated graphene FET (SG-FET) for real-time monitoring of microscale loop-mediated isothermal amplification of DNA. The SG-FET was fabricated effortlessly with graphene as an active layer, on-chip co-planar electrodes, and polydimethylsiloxane-based microfluidic reservoir. A linear response of about 0.23V/pH was seen when the buffers from pH 5-9 were analyzed on the SG-FET. To evaluate the performance of SG-FET, we monitored the amplification of Lambda phage gene as a proof-of-concept. During amplification, protons are released, which gradually alters the Dirac point voltage (VDirac) of SG-FET. The resulting device was highly sensitive with a femto-level limit of detection. The SG-FET could easily produce a positive signal within 16.5min of amplification. An amplification of 10ng/μl DNA for 1h produced a ∆VDirac of 0.27V. The sensor was tested within a range of 2×102 copies/μl (10 fg/μl) to 2×108 copies/μl (10ng/μl) of target DNA. Development of this sensing technology could significantly lower the time, cost, and complications of DNA detection.

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.

Optoelectronic fowl adenovirus detection based on local electric field enhancement on graphene quantum dots and gold nanobundle hybrid

An optoelectronic sensor is a rapid diagnostic tool that allows for an accurate, reliable, field-portable, low-cost device for practical applications. In this study, template-free In situ gold nanobundles (Au NBs) were fabricated on an electrode for optoelectronic sensing of fowl adenoviruses (FAdVs). Au NB film was fabricated on carbon electrodes working area using L(+) ascorbic acid, gold chroloauric acid and poly-l-lysine (PLL) through modified layer-by-layer (LbL) method. A scanning electron microscopic (SEM) image of the Au NBs revealed a NB-shaped Au structure with many kinks on its surface, which allow local electric field enhancement through light-matter interaction with graphene quantum dots (GQDs). Here, GQDs were synthesized through an autoclave-assisted method. Characterization experiments revealed blue-emissive, well-dispersed GQDs that were 2-3nm in size with the fluorescence emission peak of GQDs located at 405nm. Both Au NBs and GQDs were conjugated with target FAdVs specific antibodies that bring them close to each other with the addition of target FAdVs through antibody-antigen interaction. At close proximity, light-matter interaction between Au NBs and QDs produces a local electric signal enhancement under Ultraviolet-visible (UV-visible) light irradiation that allows the detection of very low concentrations of target virus even in complex biological media. A proposed optoelectronic sensor showed a linear relationship between the target FAdVs and the electric signal up to 10 Plaque forming unit (PFU)/mL with a limit of detection (LOD) of 8.75 PFU/mL. The proposed sensing strategy was 100 times more sensitive than conventional ELISA method.

Recent Advances in Biosensor Development for Foodborne Virus Detection

Outbreaks of foodborne diseases related to fresh produce have been increasing in North America and Europe. Viral foodborne pathogens are poorly understood, suffering from insufficient awareness and surveillance due to the limits on knowledge, availability, and costs of related technologies and devices. Current foodborne viruses are emphasized and newly emerging foodborne viruses are beginning to attract interest. To face current challenges regarding foodborne pathogens, a point-of-care (POC) concept has been introduced to food testing technology and device. POC device development involves technologies such as microfluidics, nanomaterials, biosensors and other advanced techniques. These advanced technologies, together with the challenges in developing foodborne virus detection assays and devices, are described and analysed in this critical review. Advanced technologies provide a path forward for foodborne virus detection, but more research and development will be needed to provide the level of manufacturing capacity required.

Highly selective organic transistor biosensor with inkjet printed graphene oxide support system

Most of the reported field effect transistors (FETs) fall short of a general method to uniquely specify and detect a target analyte. For this reason, we propose a pentacene-based FET with a graphene oxide support system (GOSS), composed of functionalized graphene oxide (GO) ink. The GOSS with a specific moiety group to capture the biomaterial of interest was inkjet printed on the pentacene FET. It provided modular receptor sites on the surface of pentacene, without alteration of the device. To evaluate the performance of a GOSS-pentacene FET biosensor, we detected the artificial DNA and circulating tumor cells as a proof-of-concept. The mobility of the FET dramatically changed upon capturing the target biomolecule on the GOSS. The FET exhibited high selectivity with 0.1 pmoles of the target DNA and a few cancer cells per detection volume. This study suggests a valuable sensor for medical diagnosis that can be mass produced effortlessly at low-cost.