癌症是世界上对人类生命安全健康威胁最大的因素之一，目前全球癌症患者的人数每年都在增加，根据世界卫生组织-国际癌症研究机构发布的数据，仅在2021年，全球就有多达1929万新的癌症病例，包括996万死亡病例。目前，癌症的检测大多依靠医院的各种检查，不仅费用高，耗时长，而且给癌症患者的治疗增加了许多困难。因此，迫切需要一种价格低廉、响应度高的肿瘤标志物检测装置。

有机电化学晶体管(Organic Electrochemical Transistors，OECT)具有电解质门控(与生物环境紧密接触)和在水溶液中具有良好的稳定性等特点，因此其特别适合在活体内操作。OECT在生物疾病监测等方面得到了广泛应用，包括电生理记录、神经元刺激、肿瘤疾病检测等。

本课题通过静电纺丝技术和原位热还原法制备了负载金纳米颗粒的碳纳米纤维(Au@CNFs)。通过Au-S键对其进行生物特异性修饰，在Au@CNFs表面形成一层生物自组装单层，将其用作OECT的栅极，对癌症患者所分泌的程序性死亡配体1(Programmed cell death Ligand 1, PD-L1)外泌体进行特异性检测，以此作为本课题的主要创新点。主要工作内容如下：

(1) 本文基于有机电化学晶体管技术，以聚(3,4-乙烯二氧噻吩):聚苯乙烯磺酸(PEDOT:PSS)为导电沟道，首先采用蒸镀工艺制备不同沟道宽长比器件，讨论沟道宽长比对器件响应度、跨导、开启时间的影响。后续探究了栅极材料对OECT性能的影响，栅极分别为：传统的铂电极和通过静电纺丝和原位热还原技术制备得到的负载金纳米颗粒的碳纳米纤维(Au@CNFs)。以选出更为优良的栅极材料为后续的肿瘤标志物传感器的搭建创造基础。

(2) 本文对负载金纳米颗粒的碳纳米纤维(Au@CNFs)进行表面形貌、化学结构和电化学性能表征。首先采用扫描电子显微镜(SEM)、透射电子显微镜(TEM)对Au@CNFs表面形貌进行表征；通过激光共聚焦显微拉曼光谱仪、傅里叶变换红外光谱仪(FTIR)，X射线衍射光谱仪(XRD)对Au@CNFs的化学结构进行了表征。采用四探针及将Au@CNFs制备成类叉指电极，对其电化学性能进行表征。实验结果表明Au@CNFs具有较大的比表面积、良好的导电性、优异的电化学性能，其表面的金纳米颗粒具有良好的生物活性，为后续肿瘤标志物检测奠定良好的基础。

(3) 本文通过PD-L1功能化OECT实现对肿瘤标志物—PD-L1外泌体的特异性检测。采用蒸镀工艺制备OECT的源漏电极，以静电纺丝和原位热还原法合成的Au@CNFs作为OECT的栅极。通过生物自组装法(SAM)对Au@CNFs进行生物特异性修饰，实现对人体肿瘤细胞分泌的PD-L1外泌体特异性检测。首先，通过TEM、能谱仪(EDS)对修饰后的Au@CNFs进行微观表征；采用循环伏安曲线(CV)对修饰前后的Au@CNFs的导电性能进行了表征。通过实验证明经过功能化修饰的OECT其对PD-L1外泌体的检测限度低至10 pg/mL，对不同浓度的PD-L1外泌体有良好的区分度。

(4) 本文通过蒸镀金电极和电化学沉积金电极作为OECT的栅极，来对比三种材料对PD-L1外泌体检测响应度的影响。采用相同方式对三种电极进行生物修饰，通过实验证明采用静电纺丝和原位热还原法合成的Au@CNFs在水溶液中的稳定性和响应度均优于另外两种电极。本文对PD-L1功能化OECT的抗干扰性能进行了测试。测试了体液中常见的葡萄糖、尿酸、抗坏血酸等干扰物质。最后对人体细胞分泌出的PD-L1阳性样本(A549)和PD-L1阴性样本(L929)临床样本进行测试，研究结果表明该生物传感器对外泌体阳性/阴性样本具有优异的区分度。表明本研究制备的PD-L1功能化OECT生物传感器具有优异的选择性及抗干扰能力，具有一定的临床应用前景。

Cancer is one of the greatest threats to the safety and health of human life around the world and the number of cancer patients worldwide is increasing every year, according to data released by the World Health Organisation-International Agency for Research on Cancer, in 2021 In 2021 only, there were as many as 19.29 million new cancer cases worldwide, including 9.96 million deaths. At present, most cancer detection is based on various examinations in hospitals, which is not only expensive and time-consuming, but also adds many difficulties to the treatment of cancer patients. Therefore, there is an urgent need for an inexpensive and highly sensitive cancer marker detection device.

 

Organic electrochemical transistors (OECT) are particularly suitable for in vivo operation due to their electrolytic gating (close contact with the biological environment) and good stability in aqueous solutions. OECT has been widely used in the monitoring of biological diseases, including electrophysiological recording, neuronal stimulation, detection of tumour diseases and more.

 

In this study, carbon nanofibres (Au@CNFs) loaded with gold nanoparticles were prepared by electrospinning and in situ thermal reduction technology. It is biologically modified by Au-S bonds, forming a bio-assembled monolayer on the surface of the Au@CNFs, which is used as a carrier of OECT to specifically detect exosomes of programmed cell death ligand 1 (PD-L1) secreted by cancer patients, as the main innovation of this project. The main work contents are as follows:

 

(1) Based on organic electrochemical transistor technology, Poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) was used as the conductive channel. Source-drain electrodes with different channel width-to-length ratios were first prepared by vapor deposition to discuss the influence of channel width-to-length ratio on device response sensitivity, transconductance, and turn-on time. The influence of the gate electrode material on the OECT performance was then explored. The gate electrodes were platinum electrodes and Au@CNFs prepared by electrostatic spinning and in-situ thermal reduction technology. The selection of improved gate materials is used to create the basis for the subsequent construction of tumour marker sensors.

 

(2) The surface morphology, chemical structure, and electrical properties of Au@CNFs loaded with gold nanoparticles were characterized. The surface morphology of Au@CNFs was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The structure of Au@CNFs was characterized by laser confocal Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction spectroscopy (XRD). The electrochemical properties of Au@CNFs were further characterized by ResMap Four Point Probe and the prepared similar interdigital electrode. The results show that Au@CNFs have a large specific surface area, good conductivity, and

excellent electrochemical performance, the gold nanoparticles on the surface with good bioactivity, providing a good foundation for subsequent biological specificity detection.

 

(3) In this thesis, PD-L1 functionalised OECT was used to achieve specific detection of PD-L1 exosomes, a tumour marker. The source-drain electrode of OECT was prepared by vapour deposition process, and Au@CNFs synthesized by electrostatic spinning and in situ thermal reduction were used as the gate of OECT. The Au@CNFs were biospecifically modified by biological self-assembly to enable specific detection of PD-L1 exosomes secreted by human tumour cells. Firstly, the modified Au@CNFs were microscopically characterised by TEM, EDS; the conductivity of the Au@CNFs before and after modification was characterised using CV. It was demonstrated that the functionally modified OECT had a low detection limit of 10 pg/mL for PD-L1 exosomes and good discrimination between different concentrations of PD-L1 exosomes.

 

(4) Subsequently, the influence of the three materials on the sensitivity of PD-L1 exosome detection was compared by vapor-deposited gold electrodes and electrochemically deposited gold electrodes as the gate of OECT. The same method was used for bio-modification of the three electrodes, and the stability and sensitivity of Au@CNFs synthesized by electrostatic spinning and in situ thermal reduction were experimentally demonstrated to be superior to the other two electrodes in aqueous solution. In this study, the anti-interference performance of PD-L1 functionalised OECT was tested. Interfering substances such as glucose, uric acid and ascorbic acid, which are commonly found in body fluids, were tested. Finally, clinical sample testing was carried out on PD-L1-positive samples (A549) and PD-L1-negative samples (L929) secreted by human tumour cells, the results showed that the biosensor has excellent discrimination between exosome-positive/negative samples. The experimental results show that the biosensor prepared in this experiment has good selectivity and anti-interference ability, laying the foundation for future applications in tumour disease detection.

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