Vigneshwaran Mani

Ultrasensitive immunosensor for cancer biomarker proteins using gold nanoparticle film electrodes and multienzyme-particle amplification.

A densely packed gold nanoparticle platform combined with a multiple-enzyme labeled detection antibody-magnetic bead bioconjugate was used as the basis for an ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum. Sensitivity was greatly amplified by synthesizing magnetic bioconjugates particles containing 7500 horseradish peroxidase (HRP) labels along with detection antibodies (Ab2) attached to activated carboxyl groups on 1 microm diameter magnetic beads. These sensors had sensitivity of 31.5 microA mL ng(-1) and detection limit (DL) of 0.5 pg mL(-1) for prostate specific antigen (PSA) in 10 microL of undiluted serum. This represents an ultralow mass DL of 5 fg PSA, 8-fold better than a previously reported carbon nanotube (CNT) forest immunosensor featuring multiple labels on carbon nanotubes, and near or below the normal serum levels of most cancer biomarkers. Measurements of PSA in cell lysates and human serum of cancer patients gave excellent correlations with standard ELISA assays. These easily fabricated AuNP immunosensors show excellent promise for future fabrication of bioelectronic arrays.

Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL⁻¹ levels. MPs were conjugated with ∼90,000 antibodies and ∼200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL⁻¹ for PSA and 0.30 pg mL⁻¹ for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.

Attomolar Detection of a Cancer Biomarker Protein in Serum by Surface Plasmon Resonance Using Superparamagnetic Particle Labels

Methods to measure protein biomarkers with ultralow detection limit (DL) and high sensitivity promise to provide valuable tools for early diagnosis of diseases such as cancer, and for monitoring therapy and post-surgical recurrence.[i, ii] Surface plasmon resonance (SPR) utilizing nanoparticle-antibody labels for signal amplification in immunoassays is an emerging approach for detecting proteins in biomedical samples.[iii–x] Herein, we show for the first time that clustering of superparamagnetic labels on SPR surfaces leads to unprecedented sensitivity and ultralow DL for protein biomarkers in serum. Specifically, antibody bioconjugates on 1 µm diameter superparamagnetic particles (MP) used for off-line antigen capture enabled SPR detection of cancer biomarker prostate specific antigen (PSA) in serum at an ultralow DL of 10 fg mL−1 (~300 aM). This approach opens doors for accurate diagnostics based on new protein biomarkers with inherently low concentrations.

Resistive-pulse measurements with nanopipettes: detection of Au nanoparticles and nanoparticle-bound anti-peanut IgY

Solid-state nanopores have been widely employed in sensing applications from Coulter counters to DNA sequencing devices. The analytical signal in such experiments is the change in ionic current flowing through the orifice caused by the large molecule or nanoparticle translocation through the pore. Conceptually similar nanopipette-based sensors can offer several advantages including the ease of fabrication and small physical size essential for local measurements and experiments in small spaces. This paper describes the first evaluation of nanopipettes with well characterized geometry for resistive-pulse sensing of Au nanoparticles (AuNP), nanoparticles coated with an allergen epitope peptide layer, and AuNP-peptide particles with bound antipeanut antibodies (IgY) on the peptide layer. The label-free signal produced by IgY-conjugated particles was strikingly different from those obtained with other analytes, thus suggesting the possibility of selective and sensitive resistive-pulse sensing of antibodies.

Paper-Based Electrochemiluminescent Screening for Genotoxic Activity in the Environment

A low cost, microfluidic paper electrochemical device (μPED) was fabricated using screen printing of electrodes and heat transfer of patterned wax paper onto filter paper. The μPED features films of a light-emitting ruthenium metallopolymer, microsomal metabolic enzymes, and DNA to detect potential genotoxic pollutant activity in environmental samples. Unlike conventional analytical methods that detect specific pollutant compounds, the μPED was designed to rapidly measure the presence of genotoxic equivalents in environmental samples with the signal related to benzo[a]pyrene (B[a]P) as a reference standard. The analytical end point is the detection of DNA damage from metabolites produced in the device using an electrochemiluminescence output measured with a charge-coupled device (CCD) camera. Proof-of-concept of this measurement was established for smoke, water, and food samples. The μPED provides a rapid screening tool for on-site environmental monitoring that specifically monitors the genotoxic reactivity of metabolites of toxic compounds present in the samples.

Magnetic particles in ultrasensitive biomarker protein measurements for cancer detection and monitoring

IMPORTANCE OF THE FIELD: Devices for the reliable detection of panels of biomarker proteins facilitated by magnetic bead-based technologies have the potential to greatly improve future cancer diagnostics. The reason for this review is to highlight promising research on emerging procedures for protein capture, transport and detection featuring magnetic particles. AREAS COVERED IN THIS REVIEW: The review covers applications of magnetic particles in protein immunoassays in emerging research and commercial methods, and stresses multiplexed protein assays for reliable future cancer diagnostics. Research literature over the past dozen years has been surveyed and specific examples are presented in detail. EXPERT OPINION: Magnetic particles are important components of emerging protein detection systems. They need to be integrated into simple inexpensive systems for accurate, sensitive detection of fully validated panels of biomarker proteins to be widely useful in clinical cancer diagnostics.

Highly efficient binding of paramagnetic beads bioconjugated with 100,000 or more antibodies to protein-coated surfaces.

We report here the first kinetic characterization of 1 μm diameter superparamagnetic particles (MP) decorated with over 100,000 antibodies binding to protein antigens attached to flat surfaces. Surface plasmon resonance (SPR) was used to show that these antibody-derivatized MPs (MP-Ab(2)) exhibit irreversible binding with 100-fold increased association rates compared to free antibodies. The estimated upper limit for the dissociation constant of MP-Ab(2) from the SPR sensor surface is 5 fM, compared to 3-8 nM for the free antibodies. These results are explained by up to 2000 interactions of MP-Ab(2) with protein-decorated surfaces. Findings are consistent with highly efficient capture of protein antigens in solution by the MP-Ab(2) and explain in part the utility of these beads for ultrasensitive protein detection into the fM and aM range. Aggregation of these particles on the SPR chip, probably due to residual magnetic microdomains in the particles, also contributes to ultrasensitive detection and may also help drive the irreversible binding.

A membrane-less enzymatic fuel cell with layer-by-layer assembly of redox polymer and enzyme over graphite electrodes †

Layer-by-layer (LBL) assembly of alternate osmium redox polymers and glucose oxidase, at anode, and laccase, at cathode, using graphite electrodes form a membrane-less glucose/O(2) enzymatic fuel cell providing a power density of 103 μW cm(-2) at pH 5.5.

Microfluidic Electrochemical Array for Detection of Reactive Metabolites Formed by Cytochrome P450 Enzymes

A novel, simple, rapid microfluidic array using bioelectronically driven cytochrome P450 enzyme catalysis for reactive metabolite screening is reported for the first time. The device incorporates an eight-electrode screen-printed carbon array coated with thin films of DNA, [Ru(bpy)(2)(PVP)(10)](ClO(4)) {RuPVP}, and rat liver microsomes (RLM) as enzyme sources. Catalysis features electron donation to cyt P450 reductase in the RLMs and subsequent cyt P450 reduction while flowing an oxygenated substrate solution past sensor electrodes. Metabolites react with DNA in the film if they are able, and damaged DNA is detected by catalytic square wave voltammetry (SWV) utilizing the RuPVP polymer. The microfluidic device was tested for a set of common pollutants known to form DNA-reactive metabolites. Logarithmic turnover rates based on SWV responses gave excellent correlation with the rodent liver TD(50) toxicity metric, supporting the utility of the device for toxicity screening. The microfluidic array gave much better S/N and reproducibility than single-electrode sensors based on similar principles.