Zimple Matharu

Polyaniline Langmuir–Blodgett film based aptasensor for ochratoxin A detection

Ochratoxin A (OTA) produced by Aspergillus Ochraceus and Penicillium verrucosum is a very dangerous toxin due to its toxic effects in human beings and its presence in a wide range of food products and cereals. A Langmuir-Blodgett (polyaniline (PANI)-stearic acid (SA)) film based highly sensitive and robust impedimetric aptasensor has been developed for ochratoxin A (OTA) detection. DNA Aptamer (Apt-DNA) specific to OTA has been covalently immobilized onto mixed Langmuir-Blodgett (LB) monolayer comprising of PANI-SA deposited onto indium tin-oxide (ITO) coated glass plates. This Apt-DNA/PANI-SA/ITO aptaelectrode has been characterized using scanning electron microscopy, Fourier transform-infrared spectroscopy, contact angle measurements, cyclic voltammetry and electrochemical impedance spectroscopy, respectively. The Apt-DNA/PANI-SA/ITO aptasensor shows detection of OTA by electrochemical impedance spectroscopy in the linear range of 0.0001 μg/ml (0.1 ng/ml) to 0.01 μg/ml (10 ng/ml) and 1 μg/ml-25 μg/ml with detection limit of 0.1 ng/ml in 15 min. The Apt-DNA/PANI-SA/ITO aptasensor can be reused ∼13 times. The binding or affinity constant (K(a)) of aptamer with OTA, calculated using Langmuir adsorption isotherm, is found be 1.21×10(7) M(-1).

Affinity and enzyme-based biosensors: recent advances and emerging applications in cell analysis and point-of-care testing

AbstractThe applications of biosensors range from environmental testing and biowarfare agent detection to clinical testing and cell analysis. In recent years, biosensors have become increasingly prevalent in clinical testing and point-of-care testing. This is driven in part by the desire to decrease the cost of health care, to shift some of the analytical tests from centralized facilities to “frontline” physicians and nurses, and to obtain more precise information more quickly about the health status of a patient. This article gives an overview of recent advances in the field of biosensors, focusing on biosensors based on enzymes, aptamers, antibodies, and phages. In addition, this article attempts to describe efforts to apply these biosensors to clinical testing and cell analysis. FigureBiosensor for Point of care

Detecting transforming growth factor-β release from liver cells using an aptasensor integrated with microfluidics.

We developed a cell-culture/biosensor platform consisting of aptamer-modified Au electrodes integrated with reconfigurable microfluidics for monitoring of transforming growth factor-beta 1 (TGF-β1), an important inflammatory and pro-fibrotic cytokine. Aptamers were thiolated, labeled with redox reporters, and self-assembled on gold surfaces. The biosensor was determined to be specific for TGF-β1 with an experimental detection limit of 1 ng/mL and linear range extending to 250 ng/mL. Upon determining figures of merit, aptasensor was miniaturized and integrated with human hepatic stellate cells inside microfluidic devices. Reconfigurable microfluidics were developed to ensure that seeding of “sticky” stromal cells did not foul the electrode and compromise sensor performance. This microsystem with integrated aptasensors was used to monitor TGF-β1 release from activated stellate cells over the course of 20 h. The electrochemical response went down upon infusing anti-TGF-β1 antibodies into the microfluidic devices containing activated stellate cells. To further validate aptasensor responses, stellate cells were stained for markers of activation (e.g., alpha smooth muscle actin) and were also tested for presence of TGF-β1 using enzyme linked immunosorbent assay (ELISA). Given the importance of TGF-β1 as a fibrogenic signal, a microsystem with integrated biosensors for local and continuous detection of TGF-β1 may prove to be an important tool to study fibrosis of the liver and other organs.

Effect of Nanoporous Gold Thin Film Morphology on Electrochemical DNA Sensing

Advances in materials science and chemistry have led to the development of a wide range of nanostructured materials for building novel electrochemical biosensors. A systematic understanding of the challenges related to electrode morphology involved in designing such sensors is essential for developing effective biosensing tools. In this study, we use nanoporous gold (np-Au) thin film electrode coatings with submicrometer thicknesses, as a model system to investigate the influence of nanostructuring on DNA-methylene blue (MB) interactions and their application to DNA biosensors. The interaction of single- and double-stranded DNA immobilized onto morphologically different np-Au films with MB was electrochemically interrogated via square wave voltammetry (SWV). The electrochemical signal from these electrodes in response to MB decayed progressively with each SWV scan. The decay rate was governed by accessibility of the electrochemically active np-Au surface by the analyte. The optimum frequency for extracting the maximum signal via SWV was influenced by the film morphology, where the optimum frequency was lower for the nanoporous morphology with lower density of molecular access points into the porous coating. Overall, the np-Au electrodes exhibited a 10-fold enhancement in probe grafting density and approximately 10-fold higher electrochemical current upon probe-target hybridization as compared to the planar Au electrodes. The np-Au electrodes enabled sensitive detection with a dynamic range of 10 to 100 nM that shifts by 1 order of magnitude for coarsened np-Au morphology due to increased target penetration into the porous network and hence enhanced hybridization efficiency. These findings provide insight into the influence of nanostructuring on the transport mechanisms of small molecules and nucleic acids, and yield an understanding of diverse sensor performance parameters such as DNA grafting density, hybridization efficiency, sensitivity and dynamic range.

Miniature Enzyme-Based Electrodes for Detection of Hydrogen Peroxide Release from Alcohol-Injured Hepatocytes

Alcohol insult to the liver sets off a complex sequence of inflammatory and fibrogenic responses. There is increasing evidence that hepatocytes play a key role in triggering these responses by producing inflammatory signals such as cytokines and reactive oxygen species (ROS). In the present study, we employed a cell culture/biosensor platform consisting of electrode arrays integrated with microfluidics to monitor extracellular H(2)O(2), one of the major ROS types, produced by primary rat hepatocytes during alcohol injury. The biosensor consisted of hydrogel microstructures with entrapped horseradish peroxidase (HRP) immobilized on an array of miniature gold electrodes. These arrays of sensing electrodes were integrated into microfluidic devices and modified with collagen (I) to promote hepatocyte adhesion. Once seeded into the microfluidic devices, hepatocytes were exposed to 100 mM ethanol and the signal at the working electrode was monitored by cyclic voltammetry (CV) over the course of 4 h. The CV experiments revealed that hepatocytes secreted up to 1.16 μM H(2)O(2) after 3 h of stimulation. Importantly, when hepatocytes were incubated with antioxidants or alcohol dehydrogenase inhibitor prior to alcohol exposure, the H(2)O(2) signal was decreased by ~5-fold. These experiments further confirmed that the biosensor was indeed monitoring oxidative stress generated by the hepatocytes and also pointed to one future use of this technology for screening hepatoprotective effects of antioxidants.

Low density lipoprotein detection based on antibody immobilized self-assembled monolayer: investigations of kinetic and thermodynamic properties.

Human plasma low density lipoprotein (LDL) immunosensor based on surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) was fabricated by immobilizing antiapolipoprotein B (AAB) onto self-assembled monolayer (SAM) of 4-aminothiophenol (ATP). The AAB/ATP/Au immunosensor can detect LDL up to 0.252 microM (84 mg/dL) and 0.360 microM (120 mg/dL) with QCM and SPR, respectively. The SPR and QCM measurements were further utilized to study the reaction kinetics of the AAB-LDL interaction. The adsorption process involved was explored using Langmuir adsorption isotherm and Freundlich adsorption models. The thermodynamic parameters such as change in Gibbs free energy (DeltaG(ads)), change in enthalpy (DeltaH(ads)), and change in entropy (DeltaS(ads)) determined at 283, 298, and 308 K revealed that the AAB-LDL interaction is endothermic in nature and is governed by entropy. Kinetic, thermodynamic, and sticking probability studies disclosed that desorption of the water molecules from the active sites of AAB and LDL plays a key role in the interaction process and increase in temperature favors binding of LDL with the AAB/ATP/Au immunosensor. Thus, the studies were utilized to unravel the most important subprocess involved in the adsorption of LDL onto AAB-modified ATP/Au surface that may help in the fabrication of LDL immunosensors with better efficiency.

Detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode

Inflammatory cytokines are secreted by immune cells in response to infection or injury. Quantification of multiple cytokines in parallel may help with disease diagnosis by illuminating inflammatory pathways related to disease onset and progression. This paper describes development of an electrochemical aptasensor for simultaneous detection of two important inflammatory cytokines, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To enable multiplexing, IFN-γ and TNF-α aptamers were labeled with anthraquinone (AQ) and methylene blue (MB) redox reporters respectively. Random immobilization of two aptamer on gold exhibited redox peaks at -0.37 V (AQ) and -0.15 V (MB) vs. Ag/AgCl reference. When challenged with either IFN-γ or TNF-α, redox signal of the appropriate reporter changed in concentration dependent manner. To demonstrate one possible application of this sensing approach, electrodes were integrated into microfluidic devices and used to dynamically monitor cytokine release from immune cells. Two cell types, primary human CD4 T-cells and U937 monocytic cells, were used to compare differences in cytokine secretions upon stimulation. These cells were infused into the microfluidic devices and stimulated to commence cytokine production. Release of IFN-γ and TNF-α was monitored concurrently from the same small group of cells over the course of 2h. The strategy of encoding specific aptamer types with unique redox reporters allows sensitive and specific detection of multiple protein biomarkers from the same electrode.

Micropatterned surfaces functionalized with electroactive peptides for detecting protease release from cells

Matrix metalloproteinases (MMPs) regulate composition of the extracellular matrix and play a critical role in cancer, fibrosis, and wound healing. This article describes a novel peptide-based electrochemical biosensor for detecting activity of cell-secreted protease MMP9. In this sensing strategy, a peptide specific to MMP9 was modified with a redox label (methylene blue (MB)) and immobilized on microfabricated 300 μm diameter Au electrodes. Challenging the electrodes with known concentrations of MMP9 resulted in the cleavage of the MB containing peptide fragment and caused a decrease in electrical signal measured by square wave voltammetry (SWV). The limit of detection for MMP9 was determined to be 60 pM with a linear range extending to 50 nM. In preparation to detect cell-secreted MMP9, glass surfaces with Au electrode arrays were further micropatterned with poly(ethylene glycol) (PEG) gel to define annular cell adhesive regions next to electrodes and render the remainder of the surface nonfouling. The surfaces were further modified with CD14 antibody to promote attachment of monocytes. The peptide-modified electrode arrays were integrated into PDMS microfluidic devices and incubated with U-937 cells, transformed monocytes known to produce MMPs. These studies revealed a 3-fold higher electrochemical signal from ∼400 activated monocytes after 10 min activation compared to nonactivated monocytes. Whereas this article focuses on MMP9 detection, the general strategy of employing redox-labeled peptides on electrodes should be broadly applicable for detection of other proteases and should have clinical as well as basic science applications.

Nanopatterned Cadmium Selenide Langmuir–Blodgett Platform for Leukemia Detection

We present results of the studies relating to preparation of Langmuir-Blodgett (LB) monolayers of tri-n-octylphosphine oxide-capped cadmium selenide quantum dots (QCdSe) onto indium-tin oxide (ITO) coated glass substrate. The monolayer behavior has been studied at the air-water interface under various subphase conditions. This nanopatterned platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by covalently immobilizing the thiol-terminated oligonucleotide probe sequence via a displacement reaction. The results of electrochemical response studies reveal that this biosensor can detect target DNA in the range of 10(-6) to 10(-14) M within 120 s, has a shelf life of 2 months, and can be used about 8 times. Further, this nucleic acid sensor has been found to distinguish the CML-positive and the control negative clinical patient samples.

Biofouling-Resilient Nanoporous Gold Electrodes for DNA Sensing

Electrochemical nucleic acid sensors are promising tools for point-of-care diagnostic platforms with their facile integration with electronics and scalability. However, nucleic acid detection in complex biological fluids is challenging as biomolecules nonspecifically adsorb on the electrode surface and adversely affect the sensor performance by obscuring the transport of analytes and redox species to the electrode. We report that nanoporous gold (np-Au) electrodes, prepared by a microfabrication-compatible self-assembly process and functionalized with DNA probes, enabled detection of target DNA molecules (10-200 nM) in physiologically relevant complex media (bovine serum albumin and fetal bovine serum). In contrast, the sensor performance was compromised for planar gold electrodes in the same conditions. Hybridization efficiency decreased by 10% for np-Au with coarser pores revealing a pore-size dependence of sensor performance in biofouling conditions. This nanostructure-dependent functionality in complex media suggests that the pores with the optimal size and geometry act as sieves for blocking the biomolecules from inhibiting the surfaces within the porous volume while allowing the transport of nucleic acid analytes and redox molecules.