Sunil K. Arya

EIS-based biosensor for ultra-sensitive detection of TNF-α from non-diluted human serum

Serum background is a critical issue for biosensor development as it interferes with the detection of target molecules and may give rise to false positive signal. We present here highly sensitive and selective TNF-α biosensor which is able to detect TNF-α from non-diluted human serum using magnetic bead coupled antibody and electrochemical impedance spectroscopy (EIS) techniques. The process is designed to detect TNF-α from human serum in three stages; (1) abundant protein backgrounds are depleted from the serum using magnetic bead coupled albumin and IgG antibodies, (2) after background depletion TNF-α is captured using magnetic bead coupled TNF-α antibody, and (3) the captured TNF-α is eluted from the magnetic beads and measured using EIS technique in which comb structured gold microelectrodes array (CSGM) is utilized to enhance the detection sensitivity. The system is able to achieve the limit of detection (LOD) at 1 pg/ml (57 fM) and a linear relationship between increasing TNF-α concentrations and charge-transfer resistance in a dynamic range of 1-1000 pg/ml.

Anti-EpCAM modified LC-SPDP monolayer on gold microelectrode based electrochemical biosensor for MCF-7 cells detection

A succinimidyl 6-(3-[2-pyridyldithio]-propionamido) hexanoate (LC-SPDP) self-assembled monolayer (SAM) prepared onto a 500 μm (diameter) gold microelectrode (Au) surface has been utilized for covalent immobilization of anti-EpCAM antibody. Amino group on anti-EpCAM antibody was covalently bound with succinimidyl group on SAM via amide bond and unreacted active groups of LC-SPDP were blocked using 1% ethanol amine (EA). These anti-EpCAM/LC-SPDP/Au electrodes were characterized using cyclic voltammetric (CV) and fluorescence techniques, respectively. The anti-EpCAM/LC-SPDP/Au electrodes were exposed to solutions with different MCF-7 cell concentrations and CV technique was used to determine the cell concentration. Further, CV studies on blank 500 and 50 μm (diameter) gold microelectrodes were used to identify cell via molecular profiling using ferrocene amidopentyl carboxylic acid based redox tagging and magnetic beads based enhancement. CV results confirm that the anti-EpCAM/LC-SPDP/Au based biosensor could detect MCF-7 cells in the range of 1×10(5)-1×10(8) with correlation coefficient of 0.999 and detection limit of 1×10(5) cells ml(-1) i.e. 100 cells in solution used for incubation (1 μl). Molecular profiling studies suggest that smaller size microelectrode (50 μm; diameter) with magnetic beads based enhancement can be employed to identify cell type. This work establishes the feasibility of using microelectrode based platform for breast cancer specific MCF-7 cell concentration estimation and their molecular profiling.

Aptamer-based array electrodes for quantitative interferon-γ detection

Present work describes the methylene blue tagged thiolated aptamer-modified gold micro-array based biosensor for specific detection of IFN-γ. The microchips with the microelectrode array were fabricated using standard silicon microfabrication technologies, and modified with methylene blue tagged aptamer using standard gold thiol chemistry. Electrodes were characterized and tested using Cyclic Voltammetric (CV) and Square Wave Voltammetry (SQW) measurements in a standard three-electrode format at room temperature. On an aptamer modified electrode, aptamer density was estimated to be about 4.4 × 10(12)molecules/cm(2). In IFN-γ studies, oxidation peak currents were found to decrease and more than 50% signal suppression was achieved at 500 ng/ml. Further, the magnitude of signal suppression was found to be logarithmically proportional to the IFN-γ in the concentration range of 1-500 ng/ml, with a detection limit of 1.3 ng/ml (i.e. 0.8 fmol in used sample volume of 10 µl). Biosensor showed negligible signal changes (5%) in a very high non-specific protein background, while still able to differentiate target protein IFN-γ at 5 ng/ml. The results indicated that our sensor binds selectively to target molecules, and the non-specific binding where adsorption of BSA protein molecules may be effectively omitted from consideration.

4-Fluoro-3-nitrophenyl grafted gold electrode based platform for label free electrochemical detection of interleukin-2 protein

A new platform based on 4-Fluoro-3-nitrophenyl (FNP) grafted gold disk electrode prepared via electrochemical reduction of 4-fluoro-3-nitrobenzene diazonium ion has been developed and utilized for biosensor fabrication. Anti-interleukin-2 (anti-IL2) antibody has been covalently immobilized onto FNP/Au surface and utilized for label free electrochemical impedance based detection of cytokine IL2. FNP acts as a bridge (cross-linker) between gold surface and anti-IL2, where fluoro group of FNP undergoes nucleophilic substitution by amino group of biomolecule and results in its covalent immobilization. The immobilization process and fabricated electrode have been characterized using contact angle (CA) measurements, cyclic voltammetry (CV) and electrochemical impedance (EIS) technique. CV studies show that FNP grafted surface provides conductive surface for anti-IL2 immobilization. The EIS response of studies as a function of IL2 concentrations exhibits a detection in linear range from 1 pg ml(-1) to 10 ng ml(-1) with minimum detectable concentration of 1 pg ml(-1). The electrode has been found to be selective against other cytokine molecules.

On-chip electrochemical immunoassay platform for specific protein biomarker estimation in undiluted serum using off-surface membrane matrix

The manuscript presents a new biosensor platform using bioreceptors modified porous 2-dimensional (2D) membrane based off-surface matrix for on-chip electrochemical immunoassay. Antibody based bioreceptors modified 2D matrix of porous polycarbonate (PC) membrane with densely packed 20µm holes as off-surface matrix was incorporated in very close proximity of the sensor surface and integrated with fluidic system for reagent flow and incubation chamber. Covalent attachment of antibodies on 2D PC membrane based off-matrix was achieved using 4-fluoro-3-nitro-azidobenzene (FNAB) cross-linker. Anti-TNF-α/FNAB/PC membrane was integrated over array of micro fingers of gold based sensor chip using double side tape spacer and StartingBlock phosphate buffer saline- Tween-20; (PBS-T20) blocking buffer was utilized to minimize nonspecific binding. Differential pulse voltammetric studies of Anti-Tnf-α/FNAB/PC-Au for protein biomarker (TNF-α) detection and estimation in undiluted serum indicated that the immunosensor system can detect TNF-α linearly in 100pg/ml to 100ng/ml range with insignificant interference from other cytokines and serum proteins. Further, immunosensor exhibited high sensitivity of 194nA/(ng/ml) and 240nA/(ng/ml), respectively for single and double membrane based system. Thus, use of 2D membrane based off surface matrix may present the new platform to sensitively measure biomarkers electrochemically to pg/ml range with insignificant nonspecific binding and false signal in undiluted serum.

Off surface matrix based on-chip electrochemical biosensor platform for protein biomarker detection in undiluted serum

The manuscript describes a concept of using off surface matrix modified with capturing biomolecule for on-chip electrochemical biosensing. 3D matrix made by laser engraving of polymethyl methacrylate (PMMA) sheet as off surface matrix was integrated in very close vicinity of the electrode surface. Laser engraving and holes in PMMA along with spacing from surface provide fluidic channel and incubation chamber. Covalent binding of capturing biomolecule (anti-TNF-α antibody) on off-surface matrix was achieved via azide group activity of 4-fluoro-3-nitro-azidobenzene (FNAB), which act as cross-linker and further covalently binds to anti-TNF-α antibody via thermal reaction. Anti-TNF-α/FNAB/PMMA matrix was then integrated over comb structured gold electrode array based sensor chip. Separate surface modification followed by integration of sensor helped to prevent the sensor chip surface from fouling during functionalization. Nonspecific binding was prevented using starting block T20 (PBS). Results for estimating protein biomarker (TNF-α) in undiluted serum using Anti-TNF-α/FNAB/PMMA/Au reveal that system can detect TNF-α in 100pg/ml to 100ng/ml range with high sensitivity of 119nA/(ng/ml), with negligible interference from serum proteins and other cytokines. Thus, use of off surface matrix may provide the opportunity to electrochemically sense biomarkers sensitively to ng/ml range with negligible nonspecific binding and false signal in undiluted serum.

CMOS based high density micro array platform for electrochemical detection and enumeration of cells

A highly sensitive label-free complementary-metal-oxide-semiconductor (CMOS) based high density micro-array for electrochemical detection and enumeration of breast tumor cell (MCF-7) is presented. The electrochemical impedance spectroscopy (EIS) based detection platform exhibited detection at single cell resolution (22 μm) and enumeration with mapping accuracy of ~80%. Maximum tumor-cell impedance increase of 28% was recorded.