Amit A. Joshi

Automated Multiplexed ECL Immunoarrays for Cancer Biomarker Proteins

Point-of-care diagnostics based on multiplexed protein measurements face challenges of simple, automated, low-cost, and high-throughput operation with high sensitivity. Herein, we describe an automated, microprocessor-controlled microfluidic immunoarray for simultaneous multiplexed detection of small protein panels in complex samples. A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell detection array to achieve sensitive detection of four prostate cancer biomarker proteins in serum. The proteins are prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interlukin-6 (IL-6). The six channel system is driven by integrated micropumps controlled by an inexpensive programmable microprocessor. The reagent delivery cassette and detection array feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall carbon nanotube forests were grown in printed microwells on a pyrolytic graphite detection chip and decorated with capture antibodies. The detection chip is housed in a machined microfluidic chamber with a steel metal shim counter electrode and Ag/AgCl reference electrode for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent cassette automatically delivers samples, wash buffers, and ECL RuBPY-silica-antibody detection nanoparticles sequentially. An onboard microcontroller controls micropumps and reagent flow to the detection chamber according to a preset program. Detection employs tripropylamine, a sacrificial reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was measured by a CCD camera. Ultralow detection limits of 10-100 fg mL(-1) were achieved in simultaneous detection of the four protein in 36 min assays. Results for the four proteins in prostate cancer patient serum gave excellent correlation with those from single-protein ELISA.

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.

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.

Resistive-Pulse Measurements with Nanopipettes: Detection of Vascular Endothelial Growth Factor C (VEGF-C) Using Antibody-Decorated Nanoparticles

Quartz nanopipettes have recently been employed for resistive-pulse sensing of Au nanoparticles (AuNP) and nanoparticles with bound antibodies. The analytical signal in such experiments is the change in ionic current caused by the nanoparticle translocation through the pipette orifice. This paper describes resistive-pulse detection of cancer biomarker (Vascular Endothelial Growth Factor-C, VEGF-C) through the use of antibody-modified AuNPs and nanopipettes. The main challenge was to differentiate between AuNPs with attached antibodies for VEGF-C and antigen-conjugated particles. The zeta-potentials of these types of particles are not very different, and, therefore, carefully chosen pipettes with well-characterized geometry were necessary for selective detection of VEGF-C.

Screening reactive metabolites bioactivated by multiple enzyme pathways using a multiplexed microfluidic system

A multiplexed, microfluidic platform to detect reactive metabolites is described, and its performance is illustrated for compounds metabolized by oxidative and bioconjugation enzymes in multi-enzyme pathways to mimic natural human drug metabolism. The device features four 8-electrode screen printed carbon arrays coated with thin films of DNA, a ruthenium-polyvinylpyridine (RuPVP) catalyst, and multiple enzyme sources including human liver microsomes (HLM), cytochrome P450 (cyt P450) 1B1 supersomes, microsomal epoxide hydrolase (EH), human S9 liver fractions (Hs9) and N-acetyltransferase (NAT). Arrays are arranged in parallel to facilitate multiple compound screening, enabling up to 32 enzyme reactions and measurements in 20-30 min. In the first step of the assay, metabolic reactions are achieved under constant flow of oxygenated reactant solutions by electrode driven natural catalytic cycles of cyt P450s and cofactor-supported bioconjugation enzymes. Reactive metabolites formed in the enzyme reactions can react with DNA. Relative DNA damage is measured in the second assay step using square wave voltammetry (SWV) with RuPVP as catalyst. Studies were done on chemicals known to require metabolic activation to induce genotoxicity, and results reproduced known features of metabolite DNA-reactivity for the test compounds. Metabolism of benzo[a]pyrene (B[a]P) by cyt P450s and epoxide hydrolase showed an enhanced relative DNA damage rate for DNA compared to cyt P450s alone. DNA damage rates for arylamines by pathways featuring both oxidative and conjugative enzymes at pH 7.4 gave better correlation with rodent genotoxicity metric TD(50). Results illustrate the broad utility of the reactive metabolite screening device.

Epitope-Resolved Detection of Peanut-Specific IgE Antibodies by Surface Plasmon Resonance Imaging

Peanut allergy can be life‐threatening and is mediated by allergen‐specific immunoglobulin E (IgE) antibodies. Investigation of IgE antibody binding to allergenic epitopes can identify specific interactions underlying the allergic response. Here, we report a surface plasmon resonance imaging (SPRi) immunoassay for differentiating IgE antibodies by epitope‐resolved detection. IgE antibodies were first captured by magnetic beads bearing IgE ϵ‐chain‐specific antibodies and then introduced into an SPRi array immobilized with epitopes from the major peanut allergen glycoprotein Arachis hypogaea h2 (Ara h2). Differential epitope responses were achieved by establishing a binding environment that minimized cross‐reactivity while maximizing analytical sensitivity. IgE antibody binding to each Ara h2 epitope was distinguished and quantified from patient serum samples (10 μL each) in a 45 min assay. Excellent correlation of Ara h2‐specific IgE values was found between ImmunoCAP assays and the new SPRi method.