无标记高通量光电生物传感阵列的构建及其凝血酶抑制剂筛选应用

Label-free optical biosensing techniques like surface plasmon resonance (SPR) sensors can constantly monitor interactions between biomolecules, which are essential to a wide range of research fields like life sciences, drug screening and clinic diagnosis. However, these kinds of sensors usually suffer from high cost of instrumentation and sensing chips. In this project, we plan to develop inexpensive, label-free and high-throughput photoelectrochemical (PEC) biosensing techniques based on single-channel electrochemical analyzers by combining steric effect-based sensing principles with light-addressing detection strategies. To achieve this goal, three main research stages will be performed: (1) we will construct biorecognition interfaces on Bi2S3 semiconductor films with narrow band gap, high conversion efficiency and good uniformity, on which label-free and highly sensitive PEC biosensing methods for real-time monitoring of biomolecule interactions will be established; (2) high-density biosensing arrays and microfluidic channels will be integrated into the above label-free PEC biosensing interfaces by techniques like inkjet printing and lithography; then, taking advantages of the high conversion efficiency of Bi2S3 in long wavelength regions, red laser microbeams with high power density and low damage will be used as high spatial-resolved light sources to carry out light-addressing detection of the above PEC biosensing arrays, which will establish label-free, highly sensitive and high-throughput PEC biosensing techniques; (3) the resulting PEC biosensing systems will be employed for the efficient screening of thrombin inhibitors, on the basis of the influences of potential inhibitors on thrombin-induced interfacial biodeposition during blood coagulation. In summary, the PEC biosensors developed in this project can not only achieve label-free and high-throughput biosensing resembling SPR sensors, but also accommodate inexpensive and portable detection devices like pH meters, which provide a portable, cost-effective and highly sensitive biosensing platform with high time- and spatial- resolution for biomedical researches.

SPR等无标记光学生物传感技术能实时动态监测生物分子相互作用，在生物医学领域应用广泛，但其设备昂贵、检测成本高。本项目拟将位阻效应传感原理与寻址激发检测方案相结合，采用常规单通道电化学检测仪器，发展低成本的无标记高通量光电化学传感方法。通过在窄带隙Bi2S3薄膜表面固定生物识别分子，利用生物识别产生的位阻效应及其对光电信号的抑制，构建能动态监测生物分子相互作用的无标记光电传感界面；在该传感界面上集成生物识别阵列及微流控通道，利用高功率、低损伤的长波光源进行寻址激发检测，建立无标记、高通量的光电化学传感体系；基于凝血酶的凝血催化反应导致的传感界面生物沉积，将其应用于凝血酶的活性分析及抑制剂筛选。本项目所建立的光电化学传感方法，具有类似SPR传感器的无标记高通量生物检测能力，同时兼容pH计等手持式检测设备，将为生物医学研究提供低成本、高灵敏、便携式的时空分辨分析技术。

电化学及光电化学传感器具有高灵敏、高集成和低成本等优势，然而其多目标/高通量检测仍是巨大的挑战。现有的多信号标记方案一般只能实现数个检测通量，要实现数十乃至数百通量需要复杂的多电极阵列及昂贵的多通道检测设备。本项目提出了单电极分区寻址检测的光电化学传感策略，能有效解决这一关键科学问题，主要工作包括：（1）建立了以模板过滤和激光雕刻为代表的新型电极阵列构筑技术；（2）建立了富勒烯C60及Bi2S3光电材料/薄膜的高效制备及生物标记方案；（3）搭建了一系列高均匀或高空间分辨寻址光源及自动寻址检测装置；（4）发展出基于单个电极分区寻址检测的无标记多目标及高通量光电生物传感体系，成功实现肿瘤标志物、农药残留及凝血酶抑制剂的高灵敏、高通量检测或筛查。比如，利用两个直径50mm的PVDF滤膜，通过简单的过滤方案，即可构筑出检测通量达42的纸片状单通道可寻址光电化学传感器，结合手持式检测设备，能在10分钟内实现数十通量农残样品的快速批量检测，而且其检测通量可以根据需要进一步拓展至数百样品。上述研究从材料、技术、原理和装置等寻址光电化学传感器的多个方面开展了工作，其研究成果突破了现有单通道电化学测试体系的通量限制，为多目标/高通量电化学及光电化学传感提供了新的思路，也为体外诊断及食品安全等领域提供了高灵敏、高通量、便携式的生化分析平台。相关研究成果在Analytical Chemistry、ACS Sensors、Biosensors & Bioelectronics和Carbon等国内外期刊发表标注基金资助论文8篇，同时申请4项国家发明专利（已授权3项）。