Membrane-Based Microwave-Mediated Electrochemical Immunoassay for the In Vitro, Highly Sensitive Detection of Osteoporosis-Related Biomarkers

Abstract

Highly sensitive and multiplexed in vitro detection of osteoporosis-related biochemical markers were carried out based on the membrane-based microwave-mediated electrochemical immunoassay (MMeEIA), where we can dramatically reduce the sample preparation time by shortening the incubation time of conjugation to obtain sensitive detection based on three dimensional conjugation of antibodies with target antigens in nylon membrane disk. C-terminal cross-linked telopeptide of type I collagen (CTx), Osteocalcin (OC), parathyroid hormone (PTH), and N-terminal propeptide of type I collagen (P1NP), which can be utilized to monitor the progress of osteoporosis, were quantified using their corresponding antibody immobilized in membranes. Coefficient of variations in this intra- and inter-assays were within 8.0% for all markers. When compared with data obtained from clinically used standard equipment (Roche modular E170), their coefficients of determination, R² values, are mostly more than 0.9. They show that the results obtained from MMeEIA are in good agreement with that from the conventional clinical instruments.

Keywords: C-terminal cross-linked telopeptide of type I collagen (CTx); N-terminal propeptide of type I collagen (P1NP); bone turnover marker; membrane-based microwave-mediated electrochemical immunoassay (MMeEIA); osteocalcin (OC); osteoporosis; parathyroid hormone (PTH).

Figure 1

Schematic diagram showing the process of MMeEIA and surface modification of membrane disk for antibody binding.

Figure 2

(a) Differential pulse voltammograms of a single analyte CTx (Solid line: 10 ng/mL, Dotted line: negative control). Standardization curve between the peak current and the concentration of (b) CTx, (c) OC, (d) P1NP, and (e) PTH by MMeEIA, respectively. All experiments were carried out three times. The bar graph represents the mean ± SD (n = 3).

Figure 3

Results of cross-reactivity: (a) CTx antigen (0.01–10 ng/mL range) interaction with OC, P1NP, and PTH pair antibodies; (b) OC antigen (0.1–1000 ng/mL range) interaction with CTx, P1NP, and PTH pair antibodies; (c) P1NP antigen (1–1000 ng/mL range) interaction with CTx, OC, and PTH pair antibodies; and (d) PTH antigen (0.01–10 ng/mL range) interaction with CTx, OC, and P1NP pair antibodies.

Figure 4

Measurement results of clinical samples using MMeEIA method and their correlation with the standard results using E170 (Roche): (A) CTx; (B) OC; (C) P1NP; (D) PTH. The Pearson correlation factor between measured concentration and standard results were 0.94, 0.96, 0.88, and 0.98, respectively.