随着人口老龄化、亚健康及慢性病发展趋势的加快，“养”和“防”已成为人们日常保健的重心。其中，“药食同源养生观”备受关注和认可，这也导致“药食两用”中药材的需求量逐渐增加。作为一味大宗常用的“药食两用”中药材，薏苡仁具有重要的食用价值和利水渗湿、健脾止泻等药用功能。但是，受各种内在因素（高淀粉含量）及外界环境条件（高温高湿）的影响，薏苡仁在其生长、采收、加工和储藏过程中极易污染真菌毒素，其中，黄曲霉毒素B1（AFB1）和玉米赤霉烯酮（ZEN）的污染率和检出率最高。AFB1和ZEN是食品、中药材及“药食两用”中药材中常见的真菌毒素，虽含量低却毒性很大，可能会对人体产生不可逆转的急慢性毒性。因此，开发灵敏准确的方法快速检测大批量薏苡仁中AFB1和ZEN的污染水平，对保障其质量和安全及消费者的身心健康具有重要意义。

中药材中真菌毒素的现行检测方法（如高效液相色谱法及液质联用技术）通常依赖于大型、昂贵的仪器和专业的操作人员，多局限于实验室使用，较难实现中药材集散地的大批量薏苡仁样品中AFB1和ZEN的低成本快速检测。因此，迫切需要开发实用性强且灵敏准确的现场快速检测方法。电致化学发光（ECL）传感器是一种基于电化学和化学发光反应的设备便携、信号稳定、低成本且灵敏的快速检测方法，能较好的满足实际检测需求。

为此，本研究以高特异性的适配体为识别元件、环境友好且低成本合成的碳点为发光体、DNA四面体为载体、ZIF-8为信号放大单元，构建了操作简单、成本低、灵敏准确、可满足薏苡仁中痕量AFB1和ZEN检测的电致化学发光（ECL）适配体传感器，用于大批量薏苡仁实际样品的分析，以保障其质量和安全。主要从以下两个方面开展工作：

一、构建基于氮掺杂碳点/DNA四面体的电致化学发光适配体传感器灵敏检测薏苡仁中AFB1

本研究开发了一种以适配体为识别元件、氮掺杂碳点（N-CDs）为发光体、DNA四面体纳米结构（DTN）为信号载体的ECL适配体传感器，用于AFB1的灵敏检测。首先，在EDC和NHS的活化作用下，将DTN与N-CDs偶联为N-CDs/DTN复合材料。然后，将互补DNA（cDNA）链固定在Au电极（AuE）表面，并与AFB1适配体杂交。在靶标AFB1存在的情况下，它会与cDNA竞争结合适配体，导致cDNA暴露并与滴加的N-CDs/DTN结合。通过共反应物（K2S2O8）与N-CDs之间的电子转移产生ECL信号，记录由于靶标AFB1存在时产生的ECL信号强度，实现对AFB1的定量检测。通过对关键检测参数的优化，该ECL适配体传感器对AFB1的线性回归方程为IECL = 1012.442×lgCAFB1+4677.019，相关系数为0.9959，线性浓度范围为1.0×10−1 ~ 1.0×104 ng/mL，检测限（LOD）为77 pg/mL，3个浓度水平的加标回收率为89.54% ~ 111.0%。方法学实验证实了该ECL传感器的高特异性、高稳定性和高准确性，可实现大批量薏苡仁中AFB1的灵敏检测。

二、构建基于金字塔型“氮掺杂碳点/ZIF-8和DNA四面体”的电致化学发光适配体传感器同时检测薏苡仁中AFB1和ZEN

本研究以具有优良ECL性能且环境友好的两种氮掺杂碳点（阴极发光N-CDs即N-CDs1和阳极发光N-CDs即N-CDs2）为信号源，DTN为载体，ZIF-8作为信号增强单元。基于适配体与靶标结合反应的高特异性，开发了一种操作简单、灵敏度高、能实现薏苡仁中AFB1和ZEN同时快速检测的金字塔型“N-CDs/ZIF-8和DNA四面体”ECL适配体传感器。在传感器的构建过程中，每一步组装均采用电化学阻抗谱法（EIS）进行表征。N-CDs1和N-CDs2的ECL信号强度分别用于定量检测AFB1和ZEN。以AFB1为例的检测原理：首先，以合成的ZIF-8纳米材料为载体，与N-CDs1通过静电吸附形成N-CDs1/ZIF-8复合发光材料。在EDC和NHS的活化作用下，将其标记到AFB1适配体（以下简称Apt1）上得到Apt1/N-CDs1/ZIF-8信号探针。然后，将底面3个顶点均修饰了巯基（-SH）的DTN通过Au-S键组装到Au电极表面，由于DTN顶端具有与AFB1适配体互补的碱基序列，信号探针Apt1/N-CDs1/ZIF-8可通过碱基互补配对与DTN结合从而固定到Au电极表面，通过共反应物（K2S2O8）与N-CDs1之间的电子转移产生ECL信号。当AFB1存在时，它会被适配体识别并竞争结合Apt1/N-CDs1/ZIF-8，导致信号探针从电极表面脱落。随着AFB1的浓度增高，N-CDs1/ZIF-8的ECL信号呈线性下降，由此实现对AFB1的定量检测。同理实现对ZEN的同时检测。通过对检测条件的优化，最终获得强且稳定的ECL信号，以保证传感器的高灵敏度。经系统的方法学验证，该传感器对AFB1和ZEN的线性检测范围为0.01 ~ 1000 ng/mL，LOD低至3.3 pg/mL，薏苡仁样品中AFB1和ZEN的加标回收率分别为96.03% ~ 118.63%（RSD＜12.26%）和87.63% ~ 107.67%（RSD＜13.46%），从而实现对薏苡仁等复杂基质样品中AFB1和ZEN的同时灵敏检测。鉴于薏苡仁等“药食两用”中药材中多存在2种及以上真菌毒素混合污染的问题，本研究开发的双靶标ECL适配体传感器更具实用性，为中药材安全监管提供更高效的检测手段。

本研究基于适配体的高特异性识别特性、N-CDs的优异ECL性能、DNA 四面体的高负载性、ZIF-8的信号增强能力及ECL传感器的快速灵敏检测优势，构建了基于“N-CDs-DNA四面体-ZIF-8”的ECL适配体传感器，为薏苡仁等大批量“药食两用”中药材中AFB1和ZEN等真菌毒素的现场灵敏检测提供了可靠的技术手段，对保证其质量和安全及临床合理使用具有重要意义。

With the acceleration in the development trend of population aging, sub-health and chronic diseases, the recuperation and prevention of diseases have attracted more and more attention in the people's daily health care. Of them, the health concept of medicine and food homology has achieved special interests, resulting in a gradual increase in the demand for “medicinal and edible” Chinese medicinal materials (CMMs). As a kind of commonly used medicinal and edible CMMs, Coix seed has significant edible values and medicinal properties. However, under the effects of its internal factor (rich starch) and the external environmental conditions (high temperature and humidity), Coix seeds is highly susceptible to mycotoxin contamination in the growth, harvesting, processing and storage processes. Therein, aflatoxin B1 (AFB1) and zearalenone (ZEN) exhibit the highest contamination probability. AFB1 and ZEN are two commonly-monitored mycotoxins in foods, CMMs including medicinal and edible products. Although expressing low content but with high toxicity, AFB1 and ZEN can induce irreversible acute and chronic toxic effects on the human body. Therefore, it is of great significance to develop sensitive and accurate analytical methods to quickly detect trace AFB1 and ZEN in a large quantity of Coix seed samples for ensuring their quality and safety, as well as the consumers’ health.

The current methods in CMMs, such as high performance liquid chromatography and liquid chromatography tandem mass spectrometry are commonly used for mycotoxins detection. But, they often rely on large and expensive instruments and specialized operators, and are mostly limited in the laboratory, which are difficult to achieve low-cost and rapid detection of mycotoxins in large batches of CMM samples in the terminal markets. Thus, it is urgent to develop practicable, sensitive and accurate techniques for the on-site rapid detection of mycotoxins. Based on electrochemical and chemiluminescence reactions, portable instrument-assisted electrochemiluminescence (ECL) sensor can realize rapid responses, stable signals and low cost to better meet actual requirements.

Therefore, in this research, with the highly specific aptamers as the recognition elements, the environmentally-friendly and low-cost carbon dots (CDs) as the ECL probes, DNA tetrahedra as the carrier, and ZIF-8 as the signal amplification material, the ECL aptasensors were developed. They with the advantages of simple operation, low cost, high sensitivity, and fascinating accuracy could achieve the rapid detection of trace AFB1 and ZEN in a large quantity of Coix seed samples to ensure their quality and safety. The main contents were listed as follows:

1. Construction of a nitrogen-doped CDs/DNA tetrahedra based ECL aptasensor for AFB1 detection in Coix seeds

In this study, an ECL aptasensor with aptamers as the recognition elements, nitrogen-doped carbon dots (N-CDs) as the ECL probes, and DNA tetrahedral nanostructures (DTNs) as the signal carriers was constructed for the sensitive detection of AFB1. Under the activation of EDC and NHS, DTN and N-CDs were combined into N-CDs/DTN nanocomposites. Then, complementary DNA (cDNA) was immobilized on the surface of the Au electrode (AuE) and hybridized to AFB1 aptamers. In the presence of AFB1, it competed with cDNA for binding with aptamers, causing the exposure of cDNA and binding to N-CDs/DTN. Then, ECL signals were generated through the electron transfer between the co-reactants (K2S2O8) and N-CDs, which were recorded to achieve the quantitation of AFB1. By optimizing the key parameters, the linear regression equation of the ECL aptasensor for AFB1 was obtained as IECL = 1012.442×lgCAFB1 + 4677.019 with a correlation coefficient of 0.9959 in the linear concentration range of 1.0×10−1 - 1.0×104 ng/mL. A low limit of detection (LOD) of 77 pg/mL, and the satisfactory recovery rate of 89.54% - 111.0% in the spiked Coix seed samples with three concentrations of AFB1 were realized. Methodological evaluation confirmed the high specificity, excellent stability and great accuracy of the developed N-CDs/DTN-based ECL aptasensor for the sensitive detection of AFB1 in a large number of Coix seed samples.

2.  Development of a pyramid-type N-CDs/ZIF-8 and DTN based ECL aptasensor for the simultaneous detection of AFB1 and ZEN in Coix seeds

In this study, with two kinds of environmental-friendly N-CDs showing excellent ECL performance (N-CDs1 with cathodic ECL signals, and N-CDs2 with anodic ECL responses) as the signal probes, DTN as the carrier, and ZIF-8 as signal enhancement element, a pyramid-type N-CDs/ZIF-8 and DTN based ECL aptasensor that was easy to operate with high specificity and sensitivity was fabricated for the simultaneous determination of AFB1 and ZEN in Coix seeds. During the construction of the ECL aptasensor, each assembly step was characterized by using the electrochemical impedance spectroscopy (EIS) method. The ECL signal intensities of N-CDs1 and N-CDs2 were used to quantitatively detect AFB1 and ZEN, respectively. The detection principle for AFB1 was described as: firstly, ZIF-8 as the carrier was combined with N-CDs1 through the electrostatic interaction to form N-CDs1/ZIF-8 composite nanomaterials, which, under the activation of EDC and NHS, were labeled on the AFB1 aptamers (listed as Apt1) to prepare the Apt1/N-CDs1/ZIF-8 signal probes. Then, the DTN modified with sulfhydryl (-SH) groups at the three vertices on the bottom surface was adsorbed onto the surface of the Au electrode through gold-sulfur (Au-S) bonds. Since the base sequences at the top vertice of DTN was complementary to the AFB1 aptamer, the Apt1/N-CDs1/ZIF-8 signal probes could be fixed on the electrode surface, thus generating ECL signals through electron transfer between the co-reactants (K2S2O8) and N-CDs1. In the presence of AFB1, it could be recognized by its aptamer and competitively bound to Apt1/N-CDs1/ZIF-8, resulting in the release of these signal probes from the electrode surface and the following decrease of ECL signals. The decreased ECL signals of N-CDs1/ZIF-8 were linearly related with the increased concentration of AFB1, and the quantitative detection of AFB1 was realized. Based on the same detection principle, the simultaneous detection of ZEN was implemented. Through the optimization of the analytical conditions, strong and stable ECL signals were finally obtained to ensure the high sensitivity of the developed aptasensor. Through systematical validation, a wide concentration range of 0.01-1000 ng/mL and a low LOD of 3.3 pg/mL were maintained. The recovery rates of AFB1 and ZEN in the spiked Coix seed samples were 96.03%-118.63% (RSD <12.26%) and 87.63%-107.67% (RSD <13.46%), respectively, which were acceptable for the simultaneous sensitive detection of AFB1 and ZEN in Coix seed matrices. In view of the two or more mycotoxins contamination in medicinal and edible Coix seeds, the developed pyramid-type N-CDs/ZIF-8 and DTN based dual-signal ECL aptasensor with high practicability provides more efficient means for the safety supervision of CMMs.

In summary, owing to the high specificity of aptamers, the excellent ECL property of N-CDs, the outstanding compatibility of DTN, the signal amplification ability of ZIF-8, and the rapid and sensitive detection performance of ECL sensors, the developed N-CDs-DNA tetrahedra-ZIF-8 based ECL aptasensor could achieve the on-site sensitive detection of AFB1 and ZEN in a large quantity of medicinal and edible CMMs, such as Coix seed, which is of great significance to ensure the quality and safety and clinical rational use of them.