Microchip-based microfluidic electrochemical arrays hold great promise for fast, high-throughput multiplexed detection of cancer biomarker proteins at low cost per assay using relatively simple instrumentation. Here we describe an inexpensive high-throughput electrochemical array featuring 32 individually addressable microelectrodes that is further multiplexed with an 8-port manifold to provide 256 sensors. The gold electrode arrays were fabricated by wet-etching commercial gold compact discs (CD-R) followed by patterned insulation. A print-and-peel method was used to create sub-microliter hydrophobic wells surrounding each sensor to eliminate cross contamination during immobilization of capture antibodies. High-throughput analyses were realized using eight 32-sensor immunoarrays connected to the miniaturized 8-port manifold, allowing 256 measurements in <1 h. This system was used to determine prostate cancer biomarker proteins prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), interleukin-6 (IL-6), and platelet factor-4 (PF-4) in serum. Clinically relevant detection limits (0.05 to 2 pg mL−1) and 5-decade dynamic ranges (sub pg mL−1 to well above ng mL−1) were achieved for these proteins utilizing precapture of analyte proteins on magnetic nanoparticles decorated with enzyme labels and antibodies.

A simple method is reported to fabricate gold arrays featuring microwells surrounding 8-electrodes from gold compact discs (CDs) for less than $0.2 per chip. Integration of these disposable gold CD array chips with microfluidics provided inexpensive immunoarrays that were used to measure a cancer biomarker protein quickly at high sensitivity. The gold CD sensor arrays were fabricated using thermal transfer of laserjet toner from a computer-printed pattern followed by selective chemical etching. Sensor elements had an electrochemically addressable surface area of 0.42 mm2 with RSD <2%. For a proof-of-concept application, the arrays were integrated into a simple microfluidic device for electrochemical detection of cancer biomarker interleukin-6 (IL-6) in diluted serum. Capture antibodies of IL-6 were chemically linked onto the electrode arrays and a sandwich immunoassay protocol was developed. A biotinylated detection antibody with polymerized horseradish peroxidase labels was used for signal amplification. The detection limit of IL-6 in diluted serum was remarkably low at 10 fg mL −1 (385 aM) with a linear response with log of IL-6 concentration from 10 to 1300 fg mL −1. These easily fabricated, ultrasensitive, microfluidic immunosensors should be readily adapted for sensitive detection of multiple biomarkers for cancer diagnostics.

Redox proteins attached to surfaces designed for biocatalysis hold promise for future clean synthetic routes. It is advantageous for these biocatalysts to operate in low-toxicity fluids with a high capacity to dissolve reactants. Here we report cross-linked films of myoglobin (Mb) and poly(L-lysine) (PLL) chemically attached to oxidized carbon cloth cathodes that in microemulsions feature the protein in a water-rich film environment with reactant in an oil-rich environment. These cross-linked Mb/PLL films were the most stable in microemuslions and had the largest turnover rates for epoxidation of styrene compared to lightly cross-linked or uncross-linked Mb/poly(styrene sulfonate) films. Up to 40-fold larger turnover rates were found in bicontinuous microemulsions compared to oil-in-water microemulsions and micelles. Enhanced turnover rates are correlated with up to 10-fold faster mass transport of solutes in the oil phases of the bicontinuous fluids.

A low cost three-dimensional (3D) printed clear plastic microfluidic device was fabricated for fast, low cost automated protein detection. The unibody device features three reagent reservoirs, an efficient 3D network for passive mixing, and an optically transparent detection chamber housing a glass capture antibody array for measuring chemiluminescence output with a CCD camera. Sandwich type assays were built onto the glass arrays using a multi-labeled detection antibody-polyHRP (HRP = horseradish peroxidase). Total assay time was ~30 min in a complete automated assay employing a programmable syringe pump so that the protocol required minimal operator intervention. The device was used for multiplexed detection of prostate cancer biomarker proteins prostate specific antigen (PSA) and platelet factor 4 (PF-4). Detection limits of 0.5 pg mL−1 were achieved for these proteins in diluted serum with log dynamic ranges of four orders of magnitude. Good accuracy vs ELISA was validated by analyzing human serum samples. This prototype device holds good promise for further development as a point-of-care cancer diagnostics tool.