在分析检测中，采用单一的检测手段对某一分析物进行定性或者定量检测，检测结果出现假阳性或者假阴性的概率较高。在当前已开发的分析测试手段中，电化学分析是一种灵敏的、快速的检测方法，近年来发展迅速。而荧光分析也是一种具有高灵敏度、强选择性、需求样品量少、方法简便等优点的分析检测技术，在生化分析中的应用十分广泛。基于不同的被测物质（抗原、抗体、脱氧核糖核酸（DNA）、核糖核酸（RNA）），根据分子特异性识别、碱基互补配对、特异性识别剪切等手段设计多模式读出的传感分析平台是目前的一种趋势。电化学传感与荧光分析都具有较高的灵敏度，通过合理的设计与构建将电化学、荧光技术整合在一起，并结合逻辑分析、统计分析等手段来对检测物质进行共定性、共定量来实现精准检测，可有效提高检测的准确度。本论文基于电化学（光电化学与电化学）与荧光方法的结合，并整合特异性识别、反应机制和触发机制来构建多模式信号读出的传感分析平台，主要研究内容如下：

1．近红外光诱导荧光可视化与光电化学传感结合的双模式传感测试平台

本部分研究工作中，我们提出了一种基于光电化学（photoelectrochemical，PEC）分析和近红外光（near infrared，NIR）激发诱导荧光可视化（fluorescence visualization，FV）结合的双模式分析检测平台。与传统的可见光驱动的荧光可视化相比，NIR诱导的FV是一种有潜力的方法，而PEC免疫测定传感具有灵敏度高、成本低和仪器配置简单的优点。基于识别单元-分析物（即抗原，DNA和RNA）反应和三明治夹心结构，以CuInS₂微米花作为基底材料提供了原始的背景光电流；二氢卟酚e6（Ce6）与抗体修饰的上转换纳米颗粒整合形成了用于PEC和荧光可视化读出的信号标签。与传统的PEC传感不同，在近红外激光照射下，双模传感平台可以实现快速的定性（＞4ng/mL）和定量分析（0.0001 ~ 50 ng/mL）。构建的生物传感器应用在以前列腺特异性抗原（prostate specific antigen，PSA）为模板的分析中，展现出了良好的性能，表明其在临床研究以及生物学研究中具有一定的应用的潜力。将裸眼读出与PEC传感结合在一起可以帮助我们快速确定是否存在目标分析物，尤其有利于便携式定性检测。此外，PEC和NIR-FV方法可以相互作为参考方法，以提高分析的准确性。随着PEC和NIR-FV技术的进一步发展，我们所提出的设计理念有望成为一种简单、经济且迅速的传感策略。

2．三定性、双定量的多信号高精度读出传感器

我们以逐层电化学沉积修饰的ITO（indium tin oxide，ITO）/Au（gold，Au）/BiOI（bismuth oxyiodide，BiOI）作为基底材料，NaYF₄:Yb³⁺, Tm³⁺@NaGdF₄:Yb³⁺核/壳上转换纳米粒（upconversion nanoparticles，UCNPs）作为信号标签，一个具有三重定性（基于智能手机的裸眼读出、光电化学（PEC）和荧光（FL））能力，和双重定量（PEC和FL）能力的多信号读出传感器被设计用于高精度前列腺特异性抗原（前列腺癌诊断的生物标志物）的检测。基于抗原-抗体生物特异性识别反应，可实现PEC模式的传感（线性范围：0.001 ~ 50 ng/mL）；而PSA对6-羧基荧光素（6-carboxyfluorescein，FAM）染料标记螺旋肽（CGHSSKLQK-FAM）的特异性酶切可实现荧光分析（线性范围：0.01 ~ 50 ng/mL）。此外，在980 nm近红外激光的激发下，信号抗体（signal antibody，Ab₂）@UCNPs生物偶联物作为信号标签可以发出紫色荧光，从而应用于基于智能手机的定性读出。此外，对于非常低浓度的样本，我们可以选择性的借助更加灵敏的分析检测技术来进行检测，以满足不同分析指标的灵敏度要求。当PSA剪切下FAM标记的多肽后（-SKLQK-FAM）我们将其收集并稀释到合适的浓度，通过尼康超分辨荧光显微镜进行单分子荧光检测。利用单分子荧光一步漂白的特性，我们计数光漂白点的个数确定其与PSA浓度的对应关系，对PSA进行高灵敏检测，检测限可达0.005 ag/mL（S/N = 3）。本项工作结合了不同技术的优点，最大限度地提高了血清中PSA的检测准确度，为多信号读出传感器的设计提供了新思路。

3．紫外光裂解致荧光与电化学结合的双模式传感分析平台

本工作中，我们构建了一种通用的，具有电化学和荧光检测功能的双模式传感平台，并且在0.1 ~ 50 ng/mL范围内可对癌胚抗原（carcinoembryonic antigen，CEA）进行精确定量。首先将DNA适配体捕获链通过金-硫键修饰在金电极上，紧接着将FAM标记的DNA修饰在电极上，然后添加被测靶标CEA和CEA抗体-上转换纳米颗粒生物偶联物（Ab@UCNPs），并完成传感平台构建。实验原理是：一条5’端含有氧化还原标签亚甲基蓝（methylene blue，MB）的茎环结构CEA适配体被用作捕获链，另一条含有FAM与可被紫外光光裂解的接头（PCLinker）标记的DNA链用作光裂解链释放FAM。当CEA存在时，Ab@UCNPs作为信号标签与CEA结合，使原本接近电极表面的MB远离Au电极表面，产生降低的氧化还原电流；另一方面，在980 nm红外激光的照射下UCNPs发射出紫外光使PCLinker被光裂解而释放FAM产生荧光，从而实现电化学与荧光结合的双模式信号读出。对比单信号输出传感，电化学与荧光结合的双模式传感平台拥有两种信号可以相互支持的优势，具有更高的检测准确度。该分析测试平台作为一个通用型的双信号读出平台，可通过选择合适的适配体来实现多种物质的双模式检测。

In analytical detection, a single detection method is used to qualitatively or quantitatively detect an analyte, and the probability of false positive or false negative in the detection result is relatively high. Among the currently developed analytical testing methods, electrochemical analysis is a sensitive and rapid detection method, which has developed rapidly in recent years. Fluorescence analysis has the advantages of high sensitivity, strong selectivity, less sample required, and simple method, and is widely used in biochemical analysis. The development of instruments and equipment has made the detection of single fluorescent molecules possible, and the detection of single molecules has been achieved at present. For different samples (antigens, antibodies, deoxyribonucleic acid (DNA), ribonucleic acid (RNA)), the multi-modal readout sensing platform were designed according to molecular specific recognition, based on complementary pairing, specific recognition cleavage, etc. By using electrochemical, fluorescence and other methods to co-qualify and co-quantify the substances, the detection accuracy can be effectively improved. This paper is mainly based on the combination of electrochemistry (photoelectrochemistry and electrochemistry) and fluorescence methods, and integrated specific reaction mechanisms and triggering mechanisms to build a multi-mode signal readout sensing platform. The main research contents are as follows:

1．Integrating near-infrared visual fluorescence with a photoelectrochemical sensing system for dual readout detection of biomolecules

In this study, we developed a dual-mode sensing platform based on the combination of photoelectrochemical (PEC) analysis and near infrared (NIR) excitation-induced fluorescence visualization (FV) readout. Compared with traditional visible light-driven fluorescence visualization, NIR-induced FV is a promising method, and PEC immunoassay sensing has the advantages of high sensitivity, low cost, and simple instrument configuration. In the work, based on the antibody-analyte (i.e. antigen, DNA and RNA) reaction and sandwich structure, CuInS₂ microflower was used as the substrate material to provide the original background photocurrent; chlorin e6 (Ce6) and antibody-modified upconversion nanoparticles was integrated to form signal tags for PEC and fluorescence visualization readout. Different from traditional PEC immunosensors, the dual-mode sensing platform enabled both the rapid qualitative (＞ 4ng/mL) and quantitative (0.0001 ~ 50 ng/mL) analysis under near-infrared laser irradiation. The constructed biosensor showed good performance in the analysis of prostate specific antigen (PSA, as the template), indicating its great potential in clinical and biological research. Combining naked-eye readout with PEC sensing together can help us quickly determine the presence of target analytes, especially for portable qualitative detection. In addition, the PEC and NIR-FV methods can serve as reference methods for each other to improve the accuracy of the analysis. With the further development of PEC and NIR-FV technology, our proposed design idea is expected to become a simple, economical and potential sensing design strategy.

2．A three-qualitative and two-quantitative high-precision multi-signal readout sensing platform

By using the layer-by-layer electrodeposited ITO (indium tin oxide, ITO)/Au (gold, Au)/BiOI (bismuth oxyiodide, BiOI) electrode as the substrate, NaYF₄:Yb³⁺, Tm³⁺@NaGdF₄:Yb³⁺ core/shell upconversion nanoparticles (UCNPs) as the signal tags, a multi-functional sensing platform with triple qualitative (smartphone-based naked eye readout, PEC and fluorescence (FL)) capability and dual quantitative (PEC and FL) capability was developed for the high-accuracy detection of prostate-specific antigen (a biomarker for prostate cancer diagnosis). Based on the antigen-antibody biospecific recognition reaction, we realized the PEC mode sensing (linear range：0.001 ~ 50 ng/mL) ; while the specific enzyme cleavage ability of PSA to 6-carboxyfluorescein (FAM) labeled helical peptide (CGHSSKLQK-FAM) can realize fluorescence analysis (linear range：0.01 ~ 50 ng/mL). In addition, under the excitation of 980 nm near-infrared laser, the signal antibody (Ab₂)@UCNPs bioconjugate can emit purple fluorescence as a signal tag, which can be applied to smartphone-based qualitative readout. In addition, for very low-concentration samples, we can selectively use more sensitive analytical detection techniques for detection to meet the sensitivity requirements of different analytical indicators. When the FAM-labeled polypeptide (-SKLQK-FAM) was cleaved by PSA, we collected it and diluted to an appropriate concentration for single-molecule fluorescence detection by Nikon super-resolution fluorescence microscopy. Taking the advantage of the one-step photobleaching feature of single-molecule fluorescence, we counted the number of photobleaching spots to determine the corresponding relationship with the concentration of PSA, and performed sensitive detection of PSA with a detection limit of 0.005 ag/mL (S/N=3). This work combined the advantages of different technologies and maximize the serodiagnosis accuracy of PSA, also provides a new idea for the design of multi-signal readout sensors.

3．Electrochemical and ultraviolet photolysis-induced fluorescence combined dual-mode sensing platform

In this work, we constructed a general-purpose dual-mode sensing detection platform with electrochemical and fluorescence functions，and can achieve accurate quantification of carcinoembryonic antigen (CEA) in the range of 0.1 to 50 ng/mL. First, the DNA aptamer capture chain is modified on the gold electrode through gold-sulfur bond, then the short-chain DNA modified with FAM was fixed on the electrode, and then the target (CEA) and CEA antibody modified upconverting nanoparticle bioconjugates (Ab@UCNPs) was added to finish the sensing platform. The experimental principle: a CEA aptamer DNA containing a redox-labeled methylene blue (MB) stem-loop structure at the 5' end was used as the capture chain; and a short DNA strands contains a fluorescent group (FAM) and a linker that can be cleaved by ultraviolet light (PCLinker) was used as the photocleavable strands to release FAM. With the presence of CEA, Ab@UCNPs as a signal label can combined with CEA, so that the MB originally close to the electrode surface was away from the Au electrode surface, resulting in a reduced redox current; on the other hand, under the irradiation of 980 nm infrared laser, the UCNPs can emitte ultraviolet light to photocleavage PCLinker and release FAM to generate fluorescence, thereby realizing dual-mode electrochemical and fluorescence readout. Compared with single-signal output sensing, this dual-mode sensing platform has higher detection accuracy due to the advantage that the two signals can support for each other. As a universally designed dual-signal readout sensing platform, this platform can realize dual-mode detection of various substances by selecting appropriate aptamers, and have a great potential for future applications.