Hairpin probe-based one-pot multiplex isothermal amplification combined with bifunctional G-quadruplex (IHP-GT) for the detection of alkaline phosphatase.

Abnormal alkaline phosphatase (ALP) levels have been linked to breast cancer, prostate cancer, bone damage, gingivitis and abnormal liver function. Monitoring ALP levels is important for better diagnosis and treatment of these diseases. Detection of ALP by colorimetric methods is very portable in terms of signal reading, but still suffers from low sensitivity. SERS can achieve high sensitivity detection, but cannot be separated from large precision instruments. Therefore, researchers have worked to optimize various aspects of the sensor, such as sensitivity, detection time, and operating procedures, to enable portable and rapid ALP detection. Isothermal amplification using simple system components meets the current demand for rapid, portable assays. We have developed a novel one-pot high-efficiency ALP assay strategy called IHP-GT. IHP-GT performs a one-step cascade amplification using only one probe (IGHP) as a template. The phosphorylated primer P binds to IGHP, forming a P/IGHP structure. At this point, the G-quadruplex closes and no signal is generated. In the presence of ALP, primer P is dephosphorylated to remove the restriction and then amplified in a cascade using IGHP as a template to release the full G-quadruplex structure. The single-stranded G-quadruplex will bend to form a secondary structure, facilitating secondary amplification starting with primer AT to produce PrG and P'. The PrG structure will trigger triple amplification, enabling cascade amplification. The G-quadruplex structure produced by cascade amplification has the dual role of promoting amplification of primer AT and binding to ThT to produce a fluorescent signal. The IHP-GT method provides a highly sensitive detection of ALP in less than 90 min and has been successfully used to analyze ALP in human serum samples. In addition, IHP-GT can be used to screen for ALP inhibitors. Importantly, we lyophilized the IHP-GT reaction components into powder form for user-friendly poc testing.