随着中药现代化和全球化贸易的扩展，中药材养生的理念逐渐推广，“药食同源”类中药材的市场需求量逐年增加。“药食同源”类中药材通常富含淀粉、油脂、蛋白质等营养物质，在其种植、采收、加工、运输及贮藏的过程中，若处置不当，极易发生霉变而被高毒性的真菌毒素污染。其中，黄曲霉毒素B₁（AFB₁）具有较强的肝肾毒性和“致癌、致畸、致突变”作用，已被列为IA类致癌物。建立灵敏、准确的技术手段用于大批量富含淀粉的“药食同源”类中药材中AFB₁污染水平的现场快速检测，对保障药材质量和安全及消费者的身心健康具有重要意义。

AFB₁的传统检测方法如液相色谱及液质联用技术因对样品的前处理和操作人员的要求高，且检测设备昂贵，极大地限制了大批量中药材现场检测的可能性。生物传感器集合了生物识别元件的特异捕获及纳米材料的灵敏信号变化的性能，具有操作简单、可实现现场快速低成本检测等优点而倍受关注。核酸适配体是生物传感器中常见的识别元件之一，作为能够以高亲和力与靶标高特异性识别的寡核苷酸单链，适配体具有体外筛选、合成成本低、批间差异小、易修饰、靶标范围广等优势，已被用于不同领域的各种靶标分子的特异识别、捕获及检测中。

目前已报道了数条针对AFB₁的适配体序列，然而，这些序列在淀粉类中药材的实际检测应用中却无法表现出良好的特异性能。这可能是由于该类中药材自身基质复杂、干扰较大，导致明显的交叉反应，从而降低了适配体的亲和力和特异性，也凸显目前适配体筛选技术的局限性。因此，亟需采用更可靠的技术筛选出具有较强抗中药材基质干扰能力，适合淀粉类中药材基质AFB₁特异识别的适配体序列，并用于构建灵敏的适配体传感器，以实现淀粉类中药材中AFB₁的现场快速、低成本检测。基于此，本论文的具体研究内容如下：

一、基于“精准-SELEX”技术的抗淀粉类中药材基质干扰的AFB₁适配体筛选

采用基于基质和靶标干扰物反筛、基于特异靶标正筛的“精准-SELEX”技术筛选具有抗淀粉类中药材基质干扰的AFB₁核酸适配体。首先，构建了基于本研究文库序列的qPCR荧光定量标准曲线，拟合的回归方程为Log₁₀C = -0.2814 Cq + 5.3363，R² = 0.9973 （C为溶液中DNA序列的浓度，Cq为qPCR荧光信号起跳值）。qPCR能够对pM级的单链DNA（ssDNA）仍产生灵敏响应，可实现对筛选中掉落文库的灵敏定量监测。然后，进行“精准-SELEX”筛选，结合链霉亲和素修饰的磁珠、含随机序列的ssDNA文库及生物素修饰的文库反义引物，将文库间接固定于磁珠上。通过引入灵活的反筛机制，分别以PBS-甲醇缓冲液、莲子、薏苡仁和麦芽基质及潜在的同族（AFB₂、AFG₁、AFG₂）和非同族毒素干扰物（OTA、DON、T-2、ZEN）进行反筛，以AFB₁靶标进行正筛。经过17轮筛选程序，正筛回收率达5.423%，文库对AFB₁的亲和力远高于各反筛靶标（可达37 - 953倍），表明该文库已具有足够的亲和力和特异性。对最后一轮文库扩增后进行高通量测序，并对数量前100名的序列进行序列比对及同源性分析，发现文库中主要有六大家族，各家族的代表序列分别为A-5、A-1、A-3、A-4、A-9和A-2。

二、抗基质干扰AFB₁适配体的亲和力测定及序列截短优化

基于对各家族代表性序列分析，需选择具有最佳亲和力的序列进行截短优化，以用于后续实际样品检测。首先，利用AFB₁与适配体结合后自身荧光增强的特性，检测各候选适配体序列对AFB₁的亲和力，各序列均表现出了nM级的解离常数（Kd为5.58 - 705.1 nM）。选择亲和力最优的适配体序列A-4，结合二级和三级结构分析，发现A-4序列包含两个长茎环结构，将其分别截短为新适配体序列并命名为A-41及A-42，通过圆二色谱测定确定了两条序列与AFB₁的结合能力。基于分子对接对A-41、A-42序列分别进行截短，发现A-41的结合区域在其顶部的环结构，A-42的结合区域则位于尾部的双链结构。分别以这两个结构域为中心对序列进行优化后，以荧光增强实验对预测结果进行验证。发现A-42截短后的序列A-42-2（Kd = 3.61 nM）具有优于A-4的亲和力，表明A-42-2是A-4与AFB₁结合的主要区域。优化后的A-42-2序列在基质中仍表现出良好的AFB₁结合能力（Kd = 5.55 nM），与其在缓冲液中的亲和力差值小于2 nM。抗基质干扰的AFB₁适配体A-42-2的成功筛选，表明以实际样品检测需求为导向的“精准-SELEX”筛选流程能够获得适合复杂中药材基质的具有特定性能的适配体。

三、基于A-42-2序列的比色-荧光双读出适配体检测淀粉类中药材中AFB₁体系构建

为验证适配体A-42-2的可靠性，并实现3种淀粉类中药材基质中AFB₁的灵敏准确检测，将A-42-2序列与金纳米颗粒（AuNPs）和荧光碳点（CDs）相结合，利用AuNPs与寡核苷酸之间的吸附作用和AuNPs与CDs之间的内滤效应，构建了基于A-42-2序列的用于淀粉类中药材中AFB₁检测的比色-荧光双读出适配体传感器。首先，合成了用于产生比色信号的AuNPs和荧光信号的CDs，并对主要的理化参数如检测体系中AuNPs的加入量、适配体浓度、盐浓度、CDs原液稀释倍数及AuNPs与A-42-2的孵育时间等进行系统优化。在最佳条件下，该双读出适配体传感器可实现对3种淀粉类中药材基质中0.25 - 30 nM（1.95 – 234.4 μg/kg）范围内AFB₁准确检测。比色和荧光方法的检测限分别可达0.2205 nM（1.72 μg/kg）和0.04837 nM（0.20 μg/kg），在3种淀粉类中药材样品中3个（高、中、低）加标浓度下的回收率分别为90.38% - 102.60% （RSD <7.78%）和91.61% - 113.77% （RSD <16.33%）。采用该比色-荧光适配体传感器15批莲子、薏苡仁和麦芽实际样品进行AFB₁的检测，其中4批样品中检测到AFB₁，阳性率为26.67 %，且这些阳性样品均以UFLC-MS/MS法进行确证。基于所筛选的A-42-2序列的比色荧光双读出适配体传感器操作简单、检测成本低、不依赖大型仪器且环境友好，能够满足大批量淀粉类中药材中AFB₁污染水平的现场快速检测需求。

本研究提出的“精准-SELEX”策略可筛选出适合淀粉类中药材（莲子、薏苡仁、麦芽）中AFB₁识别和捕获的高特异性适配体，并可用于构建实现复杂中药材基质中AFB₁超灵敏快速检测的比色-荧光双读出传感器，为复杂中药材基质中AFB₁的高效检测提供了可靠的技术手段，对保证中药材的质量及用药安全具有重要意义。

With the modernization and expended global trade of traditional Chinese medicine (TCMs), the concept of health care by TCMs is widely promoted, resulting in the rapid increase in requirement of medicinal and edible TCMs. While, these medicinal and edible TCMs that are rich in starch, oil and proteins are prone to be polluted by different mycotoxins in the processes of planting, harvesting, processing, transportation and storage due to the inappropriate treatments. Among various mycotoxins, aflatoxin B₁ (AFB₁) displays the most severe hepatorenal toxicity, and carcinogenic, teratogenic, and mutagenic effects, which has been classified as Class IA carcinogen by WHO. Thus, it is of great urgency and importance to develop sensitive and accurate methods for the on-site rapid determination of AFB₁ contamination level in large amounts of medicinal and edible TCMs rich in starch, so as to ensure their quality and safety, as well asthe health of consumers.

A large number of traditional techniques, such as liquid chromatography (LC) and LC-Mass (LC-MS) spectrometry have been proposed for AFB₁ detection in many fields. However, they have been greatly limited for broaden application for on-site AFB₁ detection in large quantities of TCMs due totheir drawbacks like complex sample pretreatment, expensive and heavy equipment, high cost, and the requirement of professional operators. Biosensors that combine specific biometric recognition elements and sensitive signal nano materials into one detection system, have attracted much attention due to their significant advantages of simple operation, fast on-site and low-cost detection. Nucleic acid aptamer is one of the most commonly used recognition elements in biosensors. As the single-stranded oligonucleotide that can recognize the targets with high affinity and specificity, aptamers have performed a lot of excellent advantages, such as in-vitro screening, low-cost synthesis, low difference between batches, easy modification and a wide range of targets. Now, aptamers have been employed for the specific recognition, capture and detection of various small and large molecular targets in different fields.

Although several aptamer sequences against AFB₁ have been selected and applied, while, they are insufficient and cannot perform excellent detection abilities for the practical application in TCMs rich in starch. This might be due to their lowered affinity and specificity resulting from the unavoidable cross-reactions from the complex matrices and strong interferences of TCMs. And this also highlights the limitations of current screening technology for AFB₁ aptamer. Therefore, it is urgent to propose more reliable strategy to screen aptamer sequences against AFB₁with strong resistance to the matrix effect and suitability for the specific recognition of AFB₁ in TCMs rich in starch, and further to construct sensitive aptasensor to realize the on-site rapid and low-cost detection of AFB₁. Thus, the main contents of this research are listed as follows:

1. Screening of AFB₁ aptamers with strong anti-matrix-interference ability by a “Precision-SELEX” protocol.

The AFB₁ aptamerwith strong anti-matrix-interference ability was selected and isolated by a new “precision-SELEX” (P-SELEX) protocol based on the introduced counter screening with the matrix extract and potential interferences, as well as positive screening with target AFB₁. First, the qPCR fluorescence standard curve for quantitation was constructed with a linear regression equation of Log₁₀C = -0.2814 Cq + 5.3363, R² = 0.9973 (C stands for the concentration of ssDNA, Cq is the take-off value of qCPR fluorescence signal). Owing to its strong response to ssDNA solution even at pM level, the qPCR method could realize sensitive monitoring of the dropped sequences in the screening process. Regarding the P-SELEX procedure, the streptavidin modified magnetic beads (SA-MBs), ssDNA library with random sequences in the middle, and biotin modified antisense primer, were combined to indirectly immobilize the library on the surface of SA-MBs. Based on the thoughtfully designed counter P-SELEX protocol, three kinds of complex matrices rich in starch including Lotus seed, Coix seed and Malt were taken as the counter screening samples to effectively eliminate the unknown interferences, while, target AFB₁ was utilized for positive screening. After a 17-round of selection containing four designed screening stages (no counter target for the 1st stage, and buffer solution, matrix and common interferents, other AFs for counter screening in the subsequent 3 stages, respectively), many aptamer sequences against AFB₁ with excellent anti-interference ability, as well as high affinity and specificity, were isolated. The recovery rate of positive screening reached 5.423%. The affinity of ssDNA pool to AFB₁ was much higher (37-953 times) than that to counter targets. These all proved the sufficient affinity and specificity of the ssDNA pool in the final round. High-throughput sequencing was carried out after the final ssDNA pool was amplified. Through the sequence alignment and homology analysis of the top 100 DNA sequences, for 6 main families in the final pool were determined with the representative sequences as A-5, A-1, A-4, A-9 and A-2, respectively.

2.  Dissociation constant (Kd) measurement and aptamer truncation of candidate aptamers for AFB₁.

On the foundation of sequence analysis of the representive sequences, the DNA sequence with the best affinity was selected and optimized for the subsequent actual application in real TCM samples. The combination of aptamer and the target AFB₁ can enhance the fluorescence of AFB₁, which have been utilized for determining the binding affinity of aptamer against AFB₁. The fluorescence enhancement determination confirmed that the candidate aptamers showed Kd values at nM level (5.58-705.1 nM) by. A-4 sequence with the best affinity was selected for the subsequent truncation and application. Combined with the secondary and tertiary structure analysis, two long stem-ring structures in the A-4 were truncated and named as A-41 and A-42 sequences, respectively, and the binding ability of which were verified by circular dichroism analysis. Based on molecular docking, the binding region of A-41 sequence was found in the ring structure on the top, and the binding region of A-42 sequence was in the double chain segment at the tail. After optimization, the final truncated A-41-2 and A-42-2 aptamers were applied for Kd measurement. It was found that A-42-2 aptamer showed even better affinity than A-4 (3.61 nM), indicating that A-42-2 aptamer was the main domain of A-4 sequence binding to AFB₁. What’s more, A-42-2 aptamer still performed excellent binding ability (Kd = 5.55 nM) to AFB₁ even in the mixed solution of the three kinds of TCM samples, which was only less than 2 nM that in the buffer. This proved the successful selection of anti-matrix-interference AFB₁ aptamer, and showed that the P-SELEX procedure guided by the detection needs of actual application can finally obtain the ideal aptamers with specific properties suitable for complex TCM matrices.

3. Detection of AFB₁ in TCMs rich in starch by the optimum aptamer A-42-2 based colorimetric and fluorescent dual-readout aptasensor.

To verify the reliability of A-42-2 aptamer and realize the sensitive and accurate detection of AFB₁ in the TCMs rich in starch, a colorimetric and fluorescent dual-readout aptasensor based on the absorption between AuNPs and aptamers, as well as the internal filtration effect (IFE) between AuNPs and CDs,was constructed. Firstly, AuNPs that could generat colorimetric signals and CDs for fluorescent responses were synthesized and characterized. Then, a series of key conditions, such as the ratio of AuNPs in the reaction system, the concentrations of A-42-2 aptamer and NaCl, the dilution fold of CDs solution, and incubation time of aptamer A-42-2 with AuNPs that might influence the detection performance of the aptasensing platform, were investigated and optimized. Under the optimum conditions, the mewly-developed dual-readout aptasensor could realize reliable detection of AFB₁ in the range of 0.25 to 30 nM (1.95-234.4 μg/kg in matrix) with the limits of detection (LODs) as low as 0.22 nM (1.72 μg/kg) for colorimetric measurement and 0.048 nM (0.20 μg/kg) for fluorescent detection. The recovery rates of three (high, medium and low) spiked concentration of AFB₁ in three kinds of TCM samples were 90.38% to 102.60% (RSD < 7.78%) for colorimetric measurement and 91.61% to 113.77% (RSD < 16.33%) for fluorescent detection, respectively. Finally, 15 batches of actual medicinal and edible samples of lotus seed, coix seed and malt were tested by using the developed aptasensor, and AFB₁ was detected in a total of 4 samples with a positive occurence rate of 26.67%. All these positive samples were confirmed by using an UFLC-MS/MS method. The A-42-2 aptamer based colorimetric and fluorescence dual-readout sensor with the advantages of low cost, simple operation, no dependence on large instruments, and no pollution to the environment, can realize the on-site rapid AFB₁ detection in large quantities of TCMs rich in starch.

In conclusion, the highly-specific AFB₁ aptamer for rapid recognition and capture of trace AFB₁ in complex TCM samples was selected through the thoughtfully designed P-SELEX strategy, further for the successful construction of a colorimetric and fluorescence dual-readout aptasensor for ultra-sensitive detection of AFB₁ in real complex matrices. This research provided a reliable tool for the efficient aptamer screening and ultrasensitive monitoring of AFB₁ in TCMs to ensure the quality and safety of them.