Simultaneous quantification of thyroid hormones using an ultrasensitive single-molecule fourplex nanoimmunosensor in an evanescent field.

The thyroid gland, which regulates the metabolism of the human body, has a sophisticated feedback system that induces the secretion of thyroid-stimulating hormone (TSH) to regulate the levels of triiodothyronine (T3) and thyroxine (T4). In this study, a single-molecule fourplex nanoimmunosensor was developed for the simultaneous quantitative analysis of TSH, T3, and T4. The three thyroid hormones were detected with a high signal-to-noise ratio in an evanescent field using laser-induced total internal reflection fluorescence. Additionally, the use of gold nanoislands for the detection of molecular interactions between thyroid hormones and antibodies labeled with quantum dots minimized the background noise from the substrate compared with the use of microislands or microwells. The nanoimmunosensor exhibited excellent detection limits of 114-193 yM (yoctomolar = 10-24 M) for thyroid hormones. The detection sensitivity was approximately 1015-fold higher than that of the conventional enzyme-linked immunosorbent assay. Paired Student's t-test of the human blood samples revealed that the difference between the two methods was insignificant at the 98% confidence level. Therefore, the proposed single-molecule fourplex nanoimmunosensor can be used for early diagnosis and prognosis monitoring at the single-molecule level because it can accurately, rapidly, and simultaneously diagnose various thyroid diseases, such as hyperthyroidism and hypothyroidism.