分子诊断在疾病检测中起着重要作用。预防,和治疗,在即时测试中至关重要。在这次调查中,使用抗体控制的Cas12a生物传感器(ACCBOR)开发了一种新型的基于CRISPR/Cas12a的小分子检测平台,其中抗体将控制CRISPR/Cas12a的反式切割活性。在这个系统中,小分子标记在无靶序列(NTS)的PAM位点周围,抗体会结合在标记分子上,以防止CRISPR/Cas12a的组合,导致反式切割活性降低。生物素-,地高辛-,25-羟基维生素D3(25-OH-VD3)标记的NTS和相应的结合蛋白分别用于验证其性能,表现出极大的普遍性。最后,开发了25-OH-VD3的一锅法检测,表现出高灵敏度和优异的特异性。在30min内,血清中的检出限为259.86pg/mL。该检测平台还具有成本低的优点,操作简单(一锅法),和快速检测(~30分钟),将为其他小分子靶标的高灵敏度检测提供新的可能性。
Molecular diagnostics play an important role in illness detection, prevention, and treatment, and are vital in point-of-care test. In this investigation, a novel CRISPR/Cas12a based small-molecule detection platform was developed using Antibody-Controlled Cas12a Biosensor (ACCBOR), in which antibody would control the trans-cleavage activity of CRISPR/Cas12a. In this system, small-molecule was labeled around the PAM sites of no target sequence(NTS), and antibody would bind on the labeled molecule to prevent the combination of CRISPR/Cas12a, resulting the decrease of trans-cleavage activity. Biotin-, digoxin-, 25-hydroxyvitamin D3 (25-OH-VD3)-labeled NTS and corresponding binding protein were separately used to verify its preformance, showing great universality. Finally, one-pot detection of 25-OH-VD3 was developed, exhibiting high sensitivity and excellent specificity. The limit of detection could be 259.86 pg/mL in serum within 30 min. This assay platform also has the advantages of low cost, easy operation (one-pot method), and fast detection (∼30 min), would be a new possibilities for the highly sensitive detection of other small-molecule targets.