Single-wall carbon nanotube forest arrays for immunoelectrochemical measurement of four protein biomarkers for prostate cancer

Abstract

Protein arrays that measure multiple protein cancer biomarkers in clinical samples hold great promise for reliable early cancer detection. Herein, we report a prototype 4-unit electrochemical immunoarray based on single-wall carbon nanotube forests for the simultaneous detection of multiple protein biomarkers for prostate cancer. Immunoarray procedures were designed to measure prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interleukin-6 (IL-6) simultaneously in a single serum sample. All of these proteins are elevated in serum of patients with prostate cancer, but they have widely different relative levels of serum concentration. Horseradish peroxidase (HRP) was used as label on detection (secondary) antibodies in a sandwich immunoassay scheme. Biotinylated secondary antibodies (Ab(2)) that bind specifically to streptavidin-HRP conjugates provided 14-16 labels per antibody and gave the necessary higher sensitivity required for PF-4 and IL-6 detection at physiological levels. Conventional singly labeled Ab(2)-HRP conjugates were sufficient for PSA and PSMA detection. Immunoarrays were used to measure four biomarkers in clinical human serum samples of prostate cancer patients and controls with excellent correlation to referee enzyme-linked immunosorbent (ELISA) assays.

Figure 1

Amperometry at −0.3 V and 2500 rpm after placing arrays in buffer containing 1 mM hydroquinone and then injecting H₂O₂ to 0.4 mM for SWNT immunoarrays incubated with antigen standards in 10 µL undiluted calf serum for 1.25 h followed by Ab₂-HRP (A & B) or Ab₂-streptavidin-HRP (C & D) in 10 µL 0.4% w/v casein and 0.05% tween 20 PBS buffer: (A) PSA, (B) PSMA, (C) PF-4, and (D) IL-6.

Figure 2

Immunoarray calibration plots with standards in calf serum for (A) PSA, (B) PSMA, (C) PF-4, (D) IL-6 (n = 3).

Figure 3

Amperometry at −0.3 V and 2500 rpm after placing array in buffer containing 1 mM hydroquinone and then injecting H₂O₂ to 0.4 mM results for SWNT immunoarrays incubated with 40 µL human serum samples for 1.25 h followed by Ab₂-HRP (A & B) or Ab₂-streptavidin-HRP (C & D) in 10 µL 0.4% w/v casein and 0.05% tween 20 PBS buffer; (A) PSA, (B) PSMA, (C) PF-4 and (D) IL-6.

Figure 4

Correlation plots of SWNT immunoarray results for human serum samples against results from ELISA determinations for the same samples (A) PSA, (B) PSMA, (C) PF-4, (D) IL-6.