Highly sensitive and specific high-throughput detection of Aβ42 in clinical specimens using a G-quadruplex-structured fluorescent aptamer analysis platform.

The early diagnosis of Alzheimer's disease (AD) relies on highly sensitive detection of the biomarker amyloid-β 42 (Aβ42). Currently used methods for detecting Aβ42, such as enzyme-linked immunosorbent assay (ELISA) and mass spectrometry, have limitations including insufficient sensitivity, high cost, and limited throughput. This study aimed to develop a high-affinity, specific G-quadruplex aptamer for Aβ42 through rational sequence trimming and optimization. Both theoretical calculations and biolayer interferometry experimental data demonstrated that the 29-bp aptamer exhibited good binding affinity to Aβ42 protein, systematically validating the robust and specific interaction between this G-quadruplex and Aβ42. Building on this, thioflavin T was introduced as a fluorescent mediator to successfully construct a high-throughput fluorescent aptamer analysis platform based on G-quadruplex-assisted structural transformation. The analysis platform exhibited an excellent linear response within the Aβ42 concentration range of 0.01-100 pg/mL, with a limit of detection as low as 3 fg/mL, enabling ultrasensitive detection and demonstrating remarkable selectivity. Spiked experiments using plasma and serum samples showed that the method maintained a good linear relationship across the same concentration range. Furthermore, the robustness of this platform in complex biological matrices was systematically validated through parallel testing of clinical plasma samples from 13 patients with AD and 11 healthy controls. The test results showed a high correlation with the ELISA (R2 = 0.9473), demonstrating the reliability and potential clinical applicability of this method. In summary, the developed high-throughput fluorescent aptamer analysis platform enabled ultrasensitive, rapid, and scalable detection of Aβ42, offering a promising diagnostic platform and a robust sensing strategy for the early clinical screening of AD.