基于时间分辨荧光纳米探针的鼠伤寒沙门氏菌和金黄色葡萄球菌检测方法研究

发布时间：2018-10-26 16:26

【摘要】：食源性致病菌易引起食源性疾病,是影响食品安全的主要因素之一。但传统的食源性致病菌的鉴定和检测方法在一定程度上存在检测步骤繁琐、耗时费力、稳定性差等不足,难以满足食品安全快速、准确的检测要求。时间分辨荧光技术可根据荧光物质寿命的长短,消除背景荧光,进而提高检测信噪比和灵敏度。目前在临床医学领域已得到广泛的应用,然而在食品安全检测领域的应用还比较少,本研究采用溶剂热法制备了两种具有时间分辨荧光特性的纳米材料,结合适配体技术、磁分离技术和荧光共振能量转移技术,构建了一系列快速、准确、灵敏的食源性致病菌检测新方法。首先,采用溶剂热法合成了具有时间分辨荧光特性的Na YF_4:Ce/Tb纳米材料(TRNPs),结合鼠伤寒沙门氏菌适配体识别元件和时间分辨荧光共振能量转移(TR-FRET)技术,构建了基于时间分辨荧光纳米材料和荧光染料共振能量转移检测鼠伤寒沙门氏菌的方法。在最优实验条件下,检测线性范围为10~2~10~6 cfu/m L(y=0.796-0.096x,R~2=0.9932),检测限为25 cfu/m L。将本方法应用于实际样品中,通过特异性研究和与平板计数方法的比较,表明该方法可用于实际样品的检测。其次,合成了表面氨基化的Fe_3O_4磁性纳米材料(MNPs)和另一种具有时间分辨荧光特性的Na Gd F_4:Eu纳米材料(TRNPs)。Na Gd F_4:Eu纳米材料与Na YF_4:Ce/Tb纳米材料可用同一波长的激发光进行激发,发射光谱不重叠。通过将鼠伤寒沙门氏菌和金黄色葡萄球菌适配体分别修饰两种TRNPs和MNPs构成荧光探针和捕获探针,构建了基于双色时间分辨荧光纳米材料荧光标记和磁分离技术同时检测鼠伤寒沙门氏菌和金黄色葡萄球菌的方法。在最佳实验条件下,鼠伤寒沙门氏菌检测线性范围为10~2~10~5cfu/m L(y=10213x-12208.92,R~2=0.9922),检测限为15 cfu/m L,金黄色葡萄球菌检测线性范围为10~2~10~5 cfu/m L(y=4803.20x-1933.87,R~2=0.9982),检测限为20 cfu/m L。将本方法应用于实际样品中,结果与平板计数法一致性良好,验证了本方法的实际应用性。最后,利用合成的Na YF_4:Ce/Tb和Na Gd F_4:Eu纳米材料,结合二硫化钨(WS_2)的高效荧光淬灭性能,建立了基于双色时间分辨荧光纳米材料和WS_2荧光共振能量转移同时检测鼠伤寒沙门氏菌和金黄色葡萄球菌的方法。在最优实验条件下,鼠伤寒沙门氏菌检测线性范围为10~2~10~6 cfu/m L(y=8670.59x-11372.41,R~2=0.9905),检测限为20cfu/m L;金黄色葡萄球菌检测线性范围为10~2~10~(6 )cfu/m L(y=4613.55x-5332.39,R~2=0.9962),检测限为33 cfu/m L。将本方法应用于实际样品的检测,结果与平板计数法一致性良好,验证了本方法的实际应用性。
[Abstract]:Food-borne pathogens are easy to cause food-borne diseases, which is one of the main factors affecting food safety. However, the traditional methods of identification and detection of foodborne pathogenic bacteria have some shortcomings, such as tedious detection steps, time-consuming and laborious, poor stability and so on, so it is difficult to meet the requirements of food safety, rapid and accurate detection. Time-resolved fluorescence technique can eliminate background fluorescence and improve detection SNR and sensitivity according to the lifetime of fluorescence material. At present, it has been widely used in the field of clinical medicine, but it is less used in the field of food safety detection. In this study, two kinds of nanomaterials with time-resolved fluorescence characteristics were prepared by solvothermal method, combined with aptamer technology. A series of rapid, accurate and sensitive methods for the detection of foodborne pathogenic bacteria were constructed by magnetic separation and fluorescence resonance energy transfer. Firstly, the Na YF_4:Ce/Tb nanomaterials (TRNPs), with time-resolved fluorescence properties were synthesized by solvothermal method, combined with an aptamer recognition element of Salmonella typhimurium and a time-resolved fluorescence resonance energy transfer (TR-FRET) technique. A method for detecting Salmonella typhimurium based on time resolved fluorescent nanomaterials and fluorescence dye resonance energy transfer was developed. Under the optimum experimental conditions, the linear range of detection was 10 ~ 2 ~ 10 ~ (10) cfu/m / L (~ (0.796-0.096) x ~ (+) ~ (2) ~ (0.9932) and the detection limit was 25 cfu/m 路L ~ (-1). The method has been applied to practical samples, and compared with the plate counting method, it is proved that this method can be used for the detection of real samples. Secondly, The surface amino Fe_3O_4 magnetic nanomaterials (MNPs) and another Na Gd F_4:Eu nanomaterial (TRNPs). Na Gd F_4:Eu nanomaterials with time-resolved fluorescence properties and Na YF_4: were synthesized. Ce/Tb nanomaterials can be excited by excitation light of the same wavelength. The emission spectrum does not overlap. Two kinds of TRNPs and MNPs were modified with Salmonella typhimurium and Staphylococcus aureus aptamer respectively to form fluorescence probe and capture probe. A method for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus was developed based on fluorescence labeling and magnetic separation of two color time-resolved fluorescent nanomaterials. Under the optimum experimental conditions, the linear range of detection of Salmonella typhimurium was 10~2~10~5cfu/m L (yttrix 10213x-12208.92) 0.9922, the detection limit was 15 cfu/m / L, and the linear range of detection of Staphylococcus aureus was 100.803.20x-1933.87 cfu/m / L. The detection limit was 20 cfu/m L. The method is applied to practical samples, and the result is in good agreement with the plate counting method, which verifies the practical application of this method. Finally, the high performance fluorescence quenching properties of tungsten disulfide (WS_2) were studied by using the synthesized Na YF_4:Ce/Tb and Na Gd F_4:Eu nanomaterials. A method for simultaneous detection of Salmonella typhimurium and Staphylococcus aureus based on two-color time-resolved fluorescent nanomaterials and WS_2 fluorescence resonance energy transfer was developed. Under the optimum experimental conditions, the linear range of detection of Salmonella typhimurium was 100.59x-11372.41 cfu/m / L (YY 8670.59x-11372.41), and the detection limit was 20cfu/m L. The linear range of detection of Staphylococcus aureus was 10 ~ (2) ~ (6) cfu/m / L (YP4613.55x-5332.39) and the detection limit was 33 cfu/m / L. The method is applied to the detection of practical samples, and the results are in good agreement with the plate counting method, which verifies the practical application of this method.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3;TS207.4

【参考文献】