鸡肉营养价值高，富含水分，是理想的肉类来源。但近年来，鸡肉安全问题频现对鸡肉消费市场产生了巨大冲击，引发了政府及群众的高度关注。其中，鸡肉中的食源性致病菌，因其广泛的污染途径、难以被察觉以及强大的致病力等问题，更应受到人们的重视。志贺氏菌作为鸡肉中的四大必检菌之一，是引发细菌性痢疾最为常见的病原菌之一，极易导致人类胃肠道疾病的发生。传统的志贺氏菌检测技术普遍存在着设备昂贵、检测时间过长、灵敏度低、检测效率低下等问题。因此，开发方便、灵敏、快捷的志贺氏菌检测方法对于保障消费者的健康至关重要。近年来荧光检测技术在微生物检测领域展现出广阔的应用前景，其中上转换纳米颗粒（UCNPs）由于其独特的发光特性，可以在许多方面弥补传统荧光检测技术的不足，因此越发受到人们的关注。综上所述，本课题以鸡肉中的志贺氏菌作为研究对象，基于适配体特异性识别，磁性分离，荧光共振能量转移（FRET）及荧光内滤效应（IFE）等技术，分别构建了两种新型荧光生物传感器，并将其用于志贺氏菌的快速检测和分析，旨在克服传统检测方法的不足，为快速检测食源性致病菌提供新思路和新方法。具体研究内容如下：

1.稀土掺杂的上转换纳米颗粒制备

以镧系稀土元素（钆、钇、镱、饵）为原材料，搭建以高温热分解法为基础的UCNPs合成平台，制备粒径大小均匀，形貌统一，分散性优良，荧光强度高的油溶性的上转换材料（OA-UCNPs），并利用阿仑磷酸对OA-UCNPs进行表面氨基化修饰，使其成为水溶性的上转换材料（ADA-UCNPs）。采用透射电镜、傅里叶中红外、X-衍射光谱、荧光光谱等表征手段，对制备的UCNPs的晶型以及荧光性能进行表征，针对油溶性纳米颗粒存在的颗粒过大、形貌不一的问题，通过对实验过程中的温度条件（均质温度和结晶温度）进行优化，筛选出最佳的反应温度组合。接着通过配体交换的方式，利用阿仑膦酸对OA-UCNPs进行功能化修饰，获取能够用于构建荧光传感器的ADA-UCNPs，并对其进行透射电镜、傅里叶中红外表征，结果表明修饰过程对于UCNPs的晶型和荧光性能无影响，可以用于后续志贺氏菌的检测实验中。

2.基于UCNPs@GNPs生物荧光传感器的鸡肉中志贺氏菌检测

本研究基于荧光共振能量转移效应，构建一种上转换-金纳米生物荧光传感器，实现志贺氏菌的快速检测。由“Au-S”键形成的志贺氏菌适配体修饰的金纳米颗粒（GNPs）与互补链修饰的上转换荧光材料通过碱基互补配对相结合。当体系中不存在志贺氏菌时，由于GNPs的吸收峰和UCNPs的荧光发射峰在500-600 nm间发生重叠，且在适配体与互补链的“桥梁”下金纳米颗粒与上转换纳米颗粒距离小于10 nm，促使荧光共振能量转移效应的发生，荧光淬灭。由于志贺氏菌和适配体之间更强的亲和力，随着志贺氏菌浓度增加，GNPs与UCNPs逐渐分离，荧光强度增加，利用荧光强度变化，可在1 h内实现对志贺氏菌的快速检测。在最优条件下，志贺氏菌在1.2×102-1.2×108 CFU/mL的浓度范围内与上转换的荧光强度呈现良好的线性关系，检测限为30 CFU/mL。同时，本方法以鸡肉中的志贺氏菌为检测对象进行真实样本测试，得出该传感器在鸡肉中志贺氏菌的加标回收率为86.7%-113.4%，所得检测结果与传统细菌培养技术检测结果基本一致。研究表明该方法具有良好的实用性和准确性，可以用于鸡肉中志贺氏菌的快速检测。 

3.基于MNPs@UCNPs生物荧光传感器的鸡肉中志贺氏菌检测

本研究利用荧光内滤效应、适配体识别技术、酶促反应与磁分离技术，构建一种基于磁性上转换荧光生物传感器的志贺氏菌的检测新方法。志贺氏菌适配体功能化的辣根过氧化物酶（HRP）与互补链修饰的磁性上转换荧光材料（MNPs@UCNPs）通过碱基互补配对相结合，在没有志贺氏菌的情况下，磁分离后的体系中仍含有辣根过氧化物酶，其能将无色的3, 3',5, 5'-四甲基联苯胺（TMB）氧化成蓝色的oxTMB，由于oxTMB的吸收峰和MNPs@UCNPs的荧光发射峰重叠，导致荧光在545 nm处淬灭。由于志贺氏菌和适配体之间更强的亲和力，随着志贺氏菌浓度增加，磁分离后体系中HRP含量降低，荧光强度增加，利用荧光强度变化，可在1 h内实现对志贺氏菌的快速检测。在最优条件下，志贺氏菌在2.3×102-2.3×108 CFU/mL的浓度范围内与磁性上转换荧光强度呈现良好的线性关系，检测限为32 CFU/mL。同时，本方法以鸡肉中志贺氏菌为检测对象进行真实样本测试，得出该传感器在鸡肉中志贺氏菌的加标回收率为89.2%-108.5%，所得检测结果与传统细菌培养技术检测结果基本一致。研究表明本方法构建的MNPs@UCNPs荧光传感器可用于鸡肉中志贺氏菌的检测当中，并具有良好的实用性和准确性。

Chicken has high nutritional value and is rich in water, making it an ideal source of meat. However, in recent years, the frequent occurrence of chicken safety problems has had a huge impact on the chicken consumption market, which has aroused great concern of the government and the masses. Among them, the contamination of foodborne pathogens in chicken should be paid more attention because of its wide range of contamination routes, difficult to be detected and strong pathogenicity. As one of the four major bacteria in chicken, Shigella is one of the most common pathogens causing bacillary dysentery, which can easily lead to gastrointestinal diseases for human. The traditional detection methods of Shigella have many problems, such as expensive equipment, long detection time, low sensitivity and low detection efficiency. Therefore, it is very important to develop convenient, sensitive and fast detection methods for Shigella to protect the health of consumers. In recent years, fluorescence detection technology has shown broad application prospects in the field of microbial detection, and upconversion nanoparticles（UCNPs） have attracted more and more attention because of their unique luminescence characteristics, which can make up for the shortcomings of traditional fluorescence detection technology in many aspects. In summary, based on aptamer-specific recognition, fluorescence resonance energy transfer（FRET）, magnetic separation and fluorescence internal filtering effect, two novel fluorescent biosensors were constructed for the rapid detection and analysis of Shigella in chicken. The purpose of this topic is to overcome the shortcomings of traditional detection methods and provide new ideas and methods for rapid detection of foodborne pathogens. The specific research contents are as follows:

1. Preparation of rare earth doped upconversion nanoparticles

Using lanthanide rare earth elements（gadolinium, yttrium, ytterbium and erbium）as raw materials, a platform for synthesizing UCNPs based on high temperature thermal decomposition was built to prepare OA-UCNPs with uniform particle size, uniform morphology, excellent dispersion and high fluorescence intensity. Then, the surface of OA-UCNPs was modified by Alannic acid to form water-soluble ADA-UCNPs. The crystal structure and fluorescence properties of UCNPs were characterized by Transmission electron microscopy（TEM）, Fourier transform mid-infrared spectroscopy（FT-IR）, X-ray diffraction（XRD）and fluorescence spectroscopy. In order to solve the problem that the oil-soluble nanoparticles are too large and have different morphologies, the temperature combination of best reaction was screened out by optimizing the temperature conditions（homogenization temperature and crystallization temperature）in the experimental process. Then, the OA-UCNPs were functionalized with alendronic acid by ligand exchange to obtain ADA-UCNPs that can be used to construct fluorescent sensors. Finally, the ADA-UCNPs were characterized by TEM and FT-IR to verify that the modification process had no effect on the crystal form and fluorescence properties of UCNPs, which could be used in the subsequent detection experiments of Shigella.

2. Detection of Shigella in chicken based on UCNPs@GNPs bioluminescent sensor

Based on the fluorescence resonance energy transfer effect, an upconversion-gold nanobiofluorescence sensor was constructed for the rapid detection of Shigella. Shigella aptamer-modified gold nanoparticles（GNPs）formed by "Au-S" bonds were combined with complementary strand-modified upconversion fluorescent materials through base complementary pairing. In the absence of Shigella, the absorption peak of GNPs and the fluorescence emission peak of UCNPs overlapped between 500 nm and 600 nm, and the distance between gold nanoparticles and upconversion nanoparticles under the "bridge" between aptamer and complementary strand was less than 10 nm, which promotes the occurrence of fluorescence resonance energy transfer effect and fluorescence quenching. Due to the stronger affinity between Shigella and aptamer, GNPs and UCNPs were gradually separated with the increase of Shigella concentration, and the fluorescence intensity increased, which could be used for rapid detection of Shigella within 1 h. Under the optimal conditions, the upconversion fluorescence intensity showed a good linear relationship with the concentration of Shigella in the range of 1.2×102-1.2×108CFU/mL, and the detection limit was 30 CFU/mL. At the same time, the real samples of Shigella in chicken were tested by this method, and the recoveries of Shigella in chicken were 86.7%-113.4%, which were basically consistent with the results of traditional bacterial culture technology. The results showed that the method had good practicability and accuracy, and could be used for the rapid detection of Shigella in chicken.

3. Detection of Shigella in chicken based on MNPs@UCNPs bioluminescent sensor.

In this study, a novel method for the detection of Shigella based on magnetic upconversion fluorescence biosensor was constructed by combining fluorescence internal filtering effect, aptamer recognition technology, enzymatic reaction and magnetic separation technology. Shigella aptamer-functionalized horseradish peroxidase（HRP）was combined with complementary strand-modified magnetic up-conversion fluorescent materials（MNPs@UCNPs）through base complementary pairing. In the absence of Shigella, the system after magnetic separation still contained horseradish peroxidase, which can oxidize colorless 3, 3', 5, 5' -tetramethylbenzidine（TMB）to blue oxTMB. The fluorescence was quenched at 545 nm due to the overlap of the absorption peak of oxTMB and the fluorescence emission peak of MNPs@UCNPs. Due to the stronger affinity between Shigella and aptamer, the content of HRP in the magnetic separation system decreased and the fluorescence intensity increased with the increase of Shigella concentration, and the rapid detection of Shigella can be achieved within 1 h by using the change of fluorescence intensity. Under the optimal conditions, the upconversion fluorescence intensity showed a good linear relationship with the concentration of Shigella in the range of 2.3×102-2.3×108 CFU/mL, and the detection limit was 32 CFU/mL. At the same time, the real samples of Shigella in chicken were tested by this method, and the recovery rate of Shigella in chicken was 89.2%-108.5%, which was basically consistent with the detection results of traditional bacterial culture technology. The results showed that the MNPs@UCNPs fluorescent sensor constructed by this method could be used for the detection of Shigella in chicken, and had good practicability and accuracy.