Automated Multiplexed ECL Immunoarrays for Cancer Biomarker Proteins

Abstract

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 antigen proteins, 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.

Theory and Simulation for Optimising Electrogenerated Chemiluminescence from Tris(2,2′‐bipyridine)‐ruthenium(II)‐Doped Silica Nanoparticles and Tripropylamine

“…Outside the nanoparticle, TPrA, TPrA * + and TPrA * concentrations were determined according to the ECEC mechanism in Scheme 1 for a fully buffered pH of 7.4 (i. e. Eq. (3) being irreversible with k dep ¼ 3:5 Â 10 3 s À1 ) while taking into account diffusion in the solution and the diffusional perturbation created by the nanoparticle without any approximation made, i. e. none of the equations (12)(13)(14)(15)(16)(17)(18)(19) were used. The bulk TPrA concentration was 30 mM and its protonation equilibrium was set at 10 3 .…”

“…[10] This is particularly important to avoid undesired background emissions from biological analytes. [11][12][13] Secondly, the excitation system (electrode potential) is decoupled from the detection signal (light) and any potentially transferred electrical noise is filtered by diffusion of the organic reactant intermediate. This leads to very low levels of electrical perturbation of the ECL light intensity that is used as the analytical signal.…”

Imaging Analysis Based on Electrogenerated Chemiluminescence

“…And the array determinations of PSA and IL-6 in patient serum were performed and wellcorrelated with single-protein ELISA [99]. In addition, this ECL immunearray was incorporated into a microfluidic device for sensitive measurements of cancer biomarker proteins in serum [100,101]. By using the 3D printing technique, the supercapacitor-powered ECL protein immunoarray has also been fabricated [102].…”

Recent Advances and Applications of Biosensors in Novel Technology

“…Shi et al developed a bipolar electrode sensing platform, which coupled an electrochemiluminescence sensor with anodic dissolution for the sensitive detection of cancer cells [143]. In another study, the microfluidic immunoarray presented exceptional performance (within 36 min) in the instantaneous detection of the four types of prostate cancer protein biomarkers [144].…”