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Automated multiplexed ecl immunoarrays for cancer biomarker proteins

By Karteek Kadimisetty, Spundana Malla, Naimish P. Sardesai, Amit A. Joshi, Ronaldo C. Faria, Norman H. Lee and James F. Rusling


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 mitropumps 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. Ultra low 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

Topics: electrogenerated chemiluminescence, ultrasensitive detection, microfluidic devices, prostate-cancer, amplification, diagnostics, arrays, proteome, valves, serum
Publisher: 'American Chemical Society (ACS)'
Year: 2015
DOI identifier: 10.1021/acs.analchem.5b00421
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