Manish Bothara

Biogenic nanoporous silica-based sensor for enhanced electrochemical detection of cardiovascular biomarkers proteins

The goal of our research is to demonstrate the feasibility of employing biogenic nanoporous silica as a key component in developing a biosensor platform for rapid label-free electrochemical detection of cardiovascular biomarkers from pure and commercial human serum samples with high sensitivity and selectivity. The biosensor platform consists of a silicon chip with an array of gold electrodes forming multiple sensor sites and works on the principle of electrochemical impedance spectroscopy. Each sensor site is overlaid with a biogenic nanoporous silica membrane that forms a high density of nanowells on top of each electrode. When specific protein biomarkers: C-reactive protein (CRP) and myeloperoxidase (MPO) from a test sample bind to antibodies conjugated to the surface of the gold surface at the base of each nanowell, a perturbation of electrical double layer occurs resulting in a change in the impedance. The performance of the biogenic silica membrane biosensor was tested in comparison with nanoporous alumina membrane-based biosensor and plain metallic thin film biosensor. Significant enhancement in the sensitivity and selectivity was achieved with the biogenic silica biosensor, in comparison to the other two, for detecting the two protein biomarkers from both pure and commercial human serum samples. The sensitivity of the biogenic silica biosensor is approximately 1 pg/ml and the linear dose response is observed over a large dynamic range from 1 pg/ml to 1 microg/ml. Based on its performance metrics, the biogenic silica biosensor has excellent potential for development as a point of care handheld electronic biosensor device for detection of protein biomarkers from clinical samples.

Nanomonitors: Protein Biosensors for Rapid Analyte Analysis

A technology for electrical detection of proteins has been developed using electrical conductance measurements. It is based on developing high density, low-volume multiwell plate devices. The scientific core of this technology lies in integrating nanoporous membranes with microfabricated chip platforms. This results in the conversion of individual pores into wells of picoliter volume. Specific antibodies are localized and isolated into individual wells. The formation of the antibody-antigen binding complex occurs in individual wells. The membrane allows for robust separation among individual wells. This technology has the capability to achieve near real-time detection with improved sensitivity and selectivity.

Nanomonitor Technology for Glycosylation Analysis

Changes in protein glycosylation have great potential as markers for the early diagnosis of cancer and other diseases. The current analytical tools for the analysis of glycan structures need expensive instrumentation, advanced expertise, is time consuming and therefore not practical for routine screening of glycan biomarkers from human samples in a clinical setting. We are developing a novel ultrasensitive diagnostic platform called ‘NanoMonitor’ to enable rapid label-free glycosylation analysis. The technology is based on electrochemical impedance spectroscopy where capacitance changes are measured at the electrical double layer interface as a result of interaction of two molecules. The NanoMonitor platform consists of a printed circuit board with array of electrodes forming multiple sensor spots. Each sensor spot is overlaid with a nanoporous alumina membrane that forms a high density of nanowells. Lectins, proteins that bind to and recognize specific glycan structures, are conjugated to the surface of nanowells. When specific glycoproteins from a test sample bind to lectins in the nanowells, it produces a perturbation to the electrical double layer at the solid/liquid interface at the base of each nanowell. This perturbation results in a change in the impedance of the double layer which is dominated by the capacitance changes within the electrical double layer. The nanoscale confinement or crowding of biological macromolecules within the nanowells is likely to enhance signals from the interaction of glycoproteins with the lectins leading to a high sensitivity of detection with the NanoMonitor as compared to other electrochemical techniques. Using a panel of lectins, we were able to detect subtle changes in the glycosylation of fetuin protein as well as differentiate glycoproteins from normal versus cancerous cells. Our results indicate that NanoMonitor can be used as a cost-effective miniature electronic biosensor for the detection of glycan biomarkers.

Nanomonitors: Nanomaterial Based Devices Towards Clinical Immunoassays

The immobilization of biomolecules on a solid substrate and their localization in “small” regions are major requirements for a variety of biomedical diagnostic applications, where rapid and accurate identification of multiple biomolecules is essential. In this specific application we have fabricated nanomitors for identifying specific protein biomarkers based on the electrical detection of antibody-antigen binding events. The nanomonitor, lab-on-a-chip device technology is based on electrical detection of protein biomarkers. It is based on developing high density, low volume multi-well plate devices. The scientific core of this technology lies in integrating nanomaterial with micro fabricated chip platforms and exploiting the improve surface area to volume to improve the detection. The devices that have been developed utilize electrical detection mechanisms where capacitance and conductance changes due to protein binding are used as “signatures” for biomarker profiling. In comparison to optical methods, the electrical detection technique is non-invasive as well as a label free. The signal acquisition is simple and it uses the existing data acquisition and signal analysis methods