用于血清低水平游离胆红素测定的金纳米簇LB膜荧光传感器研究

Determination of free bilirubin is a routine item for blood test in clinical analysis. There are still some disadvantages in the present methods towards free bilirubin determination，those are relatively poor selectivity and sensitivity at low level concentration. Fluorescent sensors based on films of human serum albumin (HSA) directed gold nanoclusters (AuNCs) are proposed to determine free bilirubin in serum at low level concentration. The sensing mechanism is based on fluorescence quenching. When HSA binds bilirubin, the fluorescence intensity of the membrane decreases with increasing concentration of free bilirubin. Conventional methods for membrane preparation, such as casting, usually produce thick films that hinder mass transport to give prolonged response time. And the sensing elements are disorderly distributed in membrane, resulting in limited sensitivity. In order to reduce thickness to nanometer level, monolayers will be prepared by Langmuir-Blodgett technology in order to accelerate mass transport between the analytes and the sensors, resulting in quick response. In the other hand, the alignment of AuNCs can be controlled by surface pressure and deposition layers on substrate. To further improve sensitivity and selectivity of the sensors, the LB films will be functionalized by glucuronic acid and carbon quantum dots (CQDs) respectively. (1) HSA will react with glucuronic acid to introduce more binding sites for bilirubin sensing. After functionalization, one HSA molecule can bind more bilirubin molecules, offering amplified signal to enhance sensitivity. Human serum is a complicated mixture with unknown components. A ratiometric fluorescence strategy is proposed to eliminate matrix effect. (2) Carbon quantum dots (CQDs) that are silent to free bilirubin will be convalently conjugated to AuNCs LB films, acting as an inner reference fluorescence probes. The quantification of free bilirubin is based on the fluorescence intensity ratio of CQDs and AuNCs. This research makes efforts to reveal some principles for bilirubin sensor design and to elucidate a few rules for controlling the response, selectivity and sensitivity of the sensor based on AuNCs fluorescent LB films. The research can also provide a foundation and supply some proofs for the fabrication and application of bilirubin sensor.

血清游离胆红素(Bu)是临床常规检测项目,目前的检测方法存在选择性不强、对低浓度水平Bu灵敏度不高等缺点。项目拟制备人血清白蛋白(HSA)-金纳米簇（AuNCs）荧光膜，利用Bu与HSA结合使薄膜荧光猝灭，用于低水平血清Bu的测定。为克服传统成膜方法的缺点，采用LB膜技术实现膜厚降至纳米级以加快传感器响应速率，通过改变膜表面压及膜沉积层数调控HSA-AuNCs在膜空间的有序分布以改善传感器灵敏度。在此基础上，为进一步提高传感器灵敏度和选择性，对LB膜进行功能化改进：（1）膜与葡萄糖醛酸偶联，增加胆红素结合位点，使荧光响应信号有效放大；（2）在膜中引入不与Bu作用的碳量子点作为荧光参比探针，采用比率荧光方法，有效消除血清的基质干扰。通过研究，探索基于AuNCs荧光LB膜的Bu传感器设计原理，研究传感器响应时间、选择性和灵敏度的调控规律，为胆红素荧光传感器的构建和应用提供思路和基础数据。

血清胆红素是判断肝功能及诊断各类肝脏疾病的一项重要指标。同时，胆红素水平的高低也与心血管病风险呈负相关。本项目采用人血清白蛋白(HSA)-金纳米簇（AuNCs）为荧光信号单元，构建胆红素传感器实现对血清中低水平胆红素的测定，以满足肝脏类等疾病的准确诊断和风险评估需求。. (1)首先，研究了均相溶液条件下金纳米簇对胆红素的响应，发现灵敏度不高，为μmol/L水平，无法满足低浓度胆红素测定的需求。同时均相测定还存在易污染检测体系、探针只能一次使用的缺点。. (2)其次，对金纳米簇的成膜方法进行了研究。分别采用了厚膜及超薄膜技术来构建传感器。一是分别采用了聚苯胺 LB 膜层及聚多巴胺膜作为粘结层来固定金纳米簇以实现对胆红素的测定。实验发现这两种方法能获得性能稳定的荧光膜，对胆红素的响应灵敏度比均相测定提高了约10倍。但是金纳米簇在中介膜层上的结合是无序的，无法控制膜厚，因而阻碍了传感器灵敏度的提高。二是采用有序超薄膜技术，即LB膜技术及层层自组装膜技术（LBL技术）来直接制备金纳米簇膜，实验发现通过降低膜的厚度，可以有效提高膜传感器的灵敏度，能检测亚纳摩尔水平的胆红素。. (3)最后，采用比率荧光策略及薄膜功能化策略，进一步提高传感器的灵敏度及抗干扰能力。通过将能发射蓝色及红色荧光的金纳米簇或是碳量子点-金纳米簇交联物，采用LBL技术同时组装形成超薄膜，构成胆红素比率荧光传感器，实际检测限可达10nM。在薄膜中引入更多的胆红素结合位点，将葡萄糖醛酸功能化与可调控膜分子分布的LB膜技术相结合，检测限为0.27nmol/L，可实现nmol/L水平胆红素的测定。. 研究结果表明，在构建薄膜传感器时，降低膜厚至单分子层级及对成膜分子空间分布的有序性进行调控，是提高传感器灵敏度的有效途径，这为提高传感器的性能提供了一种通用可行的路径。通过多种传感策略联用，实现血清低水平胆红素的测定，研究成果在对一些与氧化应激有关的疾病评估中具有较好的应用前景。