A new detection mode for gold nanoparticle-linked immunosorbent assay (GNLISA) based on a clock reaction: instrument- and enzyme-free visual quantitative detection of prostate-specific antigen (PSA)

Abstract

The enzyme-linked immunosorbent assay (ELISA) has been serving as both the workhorse and the gold standard in immunoassays due to its high specificity, sensitivity, and accuracy, despite its known shortcomings and limitations. In this work, we report our effort to improve the current ELISA protocol significantly by achieving the following four goals all together: first of all, we achieved enzyme-free signal generation in ELISA by replacing the conventional enzyme-based color generation step with an enzyme-free pure chemical color change process from a clock reaction. Secondly, we achieved the use of time lapse characteristically associated with a clock reaction as the quantitative readout signal for analyte concentration. Thirdly, gold nanoparticles (AuNPs) were employed for dual functions, both as a secondary antibody carrier and a catalytic regulator for the clock reaction. And last but not least, the clocking time associated with the colorimetric detection in our modified ELISA can be read out either by the naked eye assisted with a stopwatch free from any benchtop instrument or more accurately on any benchtop instrument with a built-in timer and with pre-set absorbance associated with the characteristic absorption wavelength of the clock reaction. The viability of our method is demonstrated by the quantitative detection of PSA in human serum and validated by both instrument-based measurements and commercial ELISA kits. The naked-eye detection range for PSA was found to be from 1.00 to 100 ng ml⁻¹ with a limit of detection (LOD) of 0.96 ng ml⁻¹, which is lower than the typical threshold value (4 ng ml⁻¹), and a sensitivity of ca. 3.1 ng mL⁻¹ min⁻¹ for the early diagnosis of prostate cancer.