Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College of Chongqing University, Chongqing, 400044, PR China.
Chongqing University Three Gorges Hospital, Chongqing, 404000, PR China.
Anal Chim Acta. 2024 Nov 15;1329:343255. doi: 10.1016/j.aca.2024.343255. Epub 2024 Sep 19.
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.
碱性磷酸酶（ALP）水平异常与乳腺癌、前列腺癌、骨损伤、牙龈炎和肝功能异常有关。监测 ALP 水平对于这些疾病的更好诊断和治疗非常重要。比色法检测 ALP 在信号读取方面非常便携，但仍然存在灵敏度低的问题。SERS 可以实现高灵敏度检测，但不能与大型精密仪器分离。因此，研究人员致力于优化传感器的各个方面，如灵敏度、检测时间和操作程序，以实现便携式和快速的 ALP 检测。使用简单系统组件的等温扩增满足了快速、便携检测的当前需求。我们开发了一种新颖的一锅高效 ALP 检测策略，称为 IHP-GT。IHP-GT 仅使用一个探针（IGHP）作为模板进行一步级联扩增。磷酸化引物 P 与 IGHP 结合，形成 P/IGHP 结构。此时，G-四链体关闭，没有信号产生。在存在 ALP 的情况下，引物 P 去磷酸化以去除限制，然后以 IGHP 为模板进行级联扩增，释放完整的 G-四链体结构。单链 G-四链体将弯曲形成二级结构，便于从引物 AT 开始进行二级扩增，产生 PrG 和 P'。PrG 结构将触发三重扩增，实现级联扩增。级联扩增产生的 G-四链体结构具有促进引物 AT 扩增和与 ThT 结合产生荧光信号的双重作用。IHP-GT 方法在不到 90 分钟的时间内提供了对 ALP 的高度敏感检测，并已成功用于分析人血清样本中的 ALP。此外，IHP-GT 可用于筛选 ALP 抑制剂。重要的是，我们将 IHP-GT 反应成分冻干成粉末形式，便于用户进行 poc 测试。
