基于BLI技术的Lipocalin 1适配体传感器在糖尿病视网膜病变早期诊断 中的研究

Diabetic retinopathy (DR) is a worldwide cause of blindness, with high rate of incidence and blindness. Early diagnosis and intervention can effectively reduce the risk of blindness. However, commonly used methods of examination are often used for DR diagnosis and disease assessment, and thus cannot meet the needs of clinical early diagnosis. Lipocalin 1 (LCN 1), which is derived from human tears, is a specific biomarker of DR. As a target for early diagnosis of DR, it is simple, convenient and non-invasive with several other advantages.. Aptamer, as a novel biological recognition molecule, is widely used in analytical diagnostics, medical research and other fields. Previously, our group has successfully established the platform of aptamer screening and optimization, and the developed biosensor through the couplet of aptamer and biolayer interferometry (BLI) technology has a detection limit of up to the ppt level. Therefore, our group plans to obtain aptamers against LCN 1 with high affinity and specificity through this platform and improve their performance by enzyme digestion, site mutation, lock nucleic acid substitution and other optimization strategies. Furthermore, on the basis of the combination of aptamers and BLI technology, we plan to introduce enzyme-linked reaction to significantly amplify the detection signal. In this way, a BLI-based aptasensor is to be developed for the detection of LCN 1, which provides an effective tool for the early diagnosis, intervention and treatment of DR.

糖尿病视网膜病变（DR）是一种世界性致盲眼病，发病率和致盲率均较高。早期的诊断和干预能有效降低其致盲风险，而常用的检查方法往往用于病情的确诊和评估，难以满足临床早期诊断的需求。人泪液中的lipocalin 1（LCN 1）蛋白是DR特异的生物标志物，以其为DR早期诊断的靶点具有简单、方便、无创等优势。. 适配体作为一种新型的生物识别分子，在分析诊断、医药研究等领域应用广泛。本课题组前期已成功建立适配体筛选和优化平台，并将其与生物膜干涉（BLI）技术联用，构建的传感器检测限达到ppt级别。因此，本研究拟通过该平台筛选获得与LCN 1特异结合的高亲和力适配体；通过酶切、定点突变、锁核酸替代等优化策略改善其性能；通过在适配体与BLI技术联用的基础上，引入酶联反应，进一步放大检测信号，构建一种基于BLI技术的适配体传感器用于LCN 1的检测，为DR的早期诊断、干预和治疗提供有效工具。

糖尿病视网膜病变（DR）是糖尿病最严重的微血管并发症之一, 现已成为全球工作年龄人群第一位的致盲性眼病。随着糖尿病患者的增多，病程的延长，DR的患病率和致盲率仍在逐年升高。由于DR的发病机制十分复杂，迄今为止尚无治愈的方法，主要通过早期诊断和及时干预才能有效降低其致盲风险。然而，目前临床上常用的检查方法往往更加适合于DR中晚期患者的诊断和评估，难以满足临床早期诊断的需求。. 人泪液中的lipocalin 1 (LCN 1)是DR特异性的生物标志物，通过对其灵敏和特异的检测将为DR的早期诊断提供可能。鉴于此，在本项目研究中：（1）本团队通过MB-SELEX技术的筛选，成功获得了与LCN 1特异性结合的高亲和力核酸适配体APT12；（2）通过截短、突变等多种优化策略的组合应用，得到了核酸适配体APT12TM，其结合亲和力、靶向特异性和结构稳定性均得到显著改善；（3）通过分子模拟与对接等实验研究，阐明了核酸适配体APT12TM与LCN 1的分子识别机制，为进一步核酸适配体传感器的设计、优化与验证提供了重要的实验指导；（4）通过将APT12TM作为分子识别工具，成功构建了一种基于G-四聚体辅助结构转换的荧光适配体传感器。该传感器对LCN 1不仅具有较高的灵敏度和特异性，而且对人工泪液中加标的样品具有较高的回收率、较低的变异系数和较好的准确性。因此，本项目所构建的传感器，通过对泪液中的生物标志物LCN 1进行灵敏、特异和快速的检测，从而为DR的早期快速诊断提供了有效工具。