Dataset Information

High-Throughput Electrochemical Microfluidic Immunoarray for Multiplexed Detection of Cancer Biomarker Proteins.

ABSTRACT: 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.

SUBMITTER: Tang CK

PROVIDER: S-EPMC5058420 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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High-Throughput Electrochemical Microfluidic Immunoarray for Multiplexed Detection of Cancer Biomarker Proteins.

Tang Chi K CK Vaze Abhay A Shen Min M Rusling James F JF

ACS sensors 20160721 8

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 (C ...[more]

PMID: 27747294

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Project description:Parathyroid hormone-related peptide (PTHrP) is recognized as the major causative agent of humoral hypercalcemia of malignancy (HHM). The paraneoplastic PTHrP has also been implicated in tumor progression and metastasis of many human cancers. Conventional PTHrP detection methods like immunoradiometric assay (IRMA) lack the sensitivity required to measure target peptide levels prior to the development of hypercalcemia. In general, sensitive, multiplexed peptide measurement by immunoassay represents challenges that we address in this paper. We describe here the first ultrasensitive multiplexed peptide assay to measure intact PTHrP 1-173 as well as circulating N-terminal and C-terminal peptide fragments. This versatile approach should apply to almost any collection of peptides that are long enough to present binding sites for two antibodies. To target PTHrP, we employed a microfluidic immunoarray featuring a chamber for online capture of the peptides from serum onto magnetic beads decorated with massive numbers of peptide-specific antibodies and enzyme labels. Magnetic bead-peptide conjugates were then washed and sent to a detection chamber housing an antibody-modified 8-electrode array fabricated by inkjet printing of gold nanoparticles. Limits of detection (LODs) of 150 aM (∼1000-fold lower than IRMA) in 5 μL of serum were achieved for simultaneous detection of PTHrP isoforms and peptide fragments in 30 min. Good correlation for patient samples was found with IRMA (n = 57); r(2) = 0.99 assaying PTHrP 1-86 equiv fragments. Analysis by a receiver operating characteristic (ROC) plot gave an area under the curve of 0.96, 80-83% clinical sensitivity, and 96-100% clinical specificity. Results suggest that PTHrP1-173 isoform and its short C-terminal fragments are the predominant circulating forms of PTHrP. This new ultrasensitive, multiplexed assay for PTHrP and fragments is promising for clinical diagnosis, prognosis, and therapeutic monitoring from early to advanced stage cancer patients and to examine underlying mechanisms of PTHrP overproduction.

Dataset Information

High-Throughput Electrochemical Microfluidic Immunoarray for Multiplexed Detection of Cancer Biomarker Proteins.

Publications

High-Throughput Electrochemical Microfluidic Immunoarray for Multiplexed Detection of Cancer Biomarker Proteins.

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