【作者】 王冲；

【导师】 马力；

【作者基本信息】 西华大学 ， 食品科学与工程， 2021， 硕士

【摘要】 氧氟沙星（Ofloxacin,OFL）是一种合成抗菌药物,有抗菌活性强、抗菌谱广、药物动力学特征好的特点,被广泛应用于人和动物疾病防治中。由于畜牧业中对该药物的滥用,食品中OFL残留通过食物链在人体中富集,对健康造成严重影响,并增加致病菌耐药的可能。论文研究了基于核酸适配体识别的新型检测探针在OFL残留检测中的应用,结合磁分离富集技术、滚环扩增（Rolling circle amplification,RCA）技术等建立了灵敏、可靠的新型分析检测方法,为丰富食品安全技术种类和提高食品安全技术水平提供了新思路。（1）制备了表面氨基化的Fe₃O₄磁性纳米颗粒和Na YF₄:Ce/Tb时间分辨荧光纳米颗粒,采用透射电镜射、X射线衍射和傅里叶转换-红外光谱分别对其形貌、晶型成分和表面基团进行表征。以核酸适配体为识别元件,Fe₃O₄磁性纳米颗粒为分离载体,Na YF₄:Ce/Tb时间分辨荧光纳米颗粒为荧光标记物,构建了基于RCA信号放大的食品中OFL残留检测方法。在最佳条件下,方法具有较高的特异性和灵敏度,检测限为32pmol/L,线性范围为50 pmol/L～50 nmol/L,在食品样品中有较好的回收率。（2）探究了G-三联体（G-triplex,G3）与Tb³⁺之间的相互作用,并基于G3对Tb³⁺的敏化,提出了一种均相的利用RCA扩增G3序列敏化Tb³⁺发光的OFL检测方法。当OFL存在时,核酸适配体对OFL的识别而触发RCA反应,获得含有大量重复的G3序列的RCA产物,加入的Tb³⁺与RCA产物生成G3/Tb³⁺发光复合物。在最佳条件下,本方法的检测限为0.18 pmol/L,线性范围为5～1000 pmol/L,具有较高的特异性和灵敏度,在鱼肉和虾肉样品中有较好的回收率。（3）设计了一种基于核酸适配体和G3/氯化血红素（G3/hemin）的食品中OFL比色检测传感器。在该传感器中,Fe₃O₄磁性纳米颗粒做为分离载体,以G3/hemin为DNAzyme催化输出H₂O₂-ABTS的比色信号。在最佳条件下,方法的特异性良好,检测限为15.25 nmol/L,线性范围为50～300 nmol/L。该方法检测原理简单,检测时间更短,在对实际样品的加标试验中都获得了较好的回收率。本文成功构建了三种新型的用于食品中OFL残留检测的适配体传感器。这些适配体传感器分别利用磁分离富集、RCA、时间分辨荧光（Time-resolved fluorescence,TRF）和脱氧核糖酶（DNAzyme）等技术,表现出灵敏,可靠和特异的检测性能,拓展了核酸适配体的应用范围,同时也为食品安全提供了技术保障。更多还原

【Abstract】 Ofloxacin（OFL）is a synthetic antimicrobial drug.And OFL is widely used in the prevention and treatment of animal diseases because of its strong antibacterial activity,wide antibacterial spectrum and good pharmacokinetic characteristics.However,as a result of the abuse of OFL in the livestock industry,OFL residues in food accumulate in the human body through the food chain.This will cause serious damage to health and increase the resistance of pathogenic bacteria.In this thesis,the work focused on the application of novel aptamer probe in food safety detection.By combining magnetic separation technique and rolling circle amplification（RCA）technique,some sensitive and reliable aptasensors were established for the detection of OFL residues in food.This thesis provides new methods for enriching and improving food safety technology.（1）On the one hand,the amine-functionalized Fe₃O₄ magnetic nanoparticles and Na YF₄:Ce/Tb time-resolved fluorescent nanoparticles were prepared.Morphology,crystal composition and surface groups of the two kinds of nanoparticles were characterized by transmission electron microscopy（TEM）,X-ray diffraction（XRD）and fourier transform infrared（FT-IR）spectra.On the other hand,Fe₃O₄ magnetic nanoparticles were used as separation carriers,and Na YF₄:Ce/Tb time-resolved fluorescent nanoparticles were used as fluorescence source to construct an aptasensor for detecting OFL residues in food based on RCA signal amplification.The aptasensor showed high specificity and sensitivity,the limit of detection was 32 pmol/L,the linear range was 50 pmol/L～50 nmol/L,and it showed a good recovery rate in food samples.（2）The interaction between G-triplex（G3）and Tb³⁺was studied.And based on the sensitization of Tb³⁺by G3,a label-free OFL detection aptasensor using RCA to amplify G3sequences to sensitize Tb³⁺luminescence was proposed.In the presence of OFL,the recognition of OFL by aptamer triggered the RCA reaction.Since the RCA products containing a large number of repeated G3 sequences,Tb³⁺could form a G3/Tb³⁺luminescent complex with the RCA products.Under the optimum conditions,the limit of detection of this method was 0.18pmol/L,and the linear range was 5～1000 pmol/L.It showed high specificity and sensitivity,and good recovery in fish and shrimp samples.（3）A G3/hemin colorimetric assay for OFL detection based on aptamer was constructed.In this aptasensor,Fe₃O₄ magnetic nanoparticles were used as separation carrier,and G3/hemin was used as DNAzyme to catalyst the color reaction of H₂O₂-ABTS.The method showed good specificity,the limit of detection was 15.25 nmol/L,and the linear range was 50～300 nmol/L.The method and steps were simpler,the detection time was shorter,and a better recovery rate was obtained in the spike test of actual samples.In summary,three novel aptasensors for OFL detection in foods were successfully constructed.These sensors were combined with magnetic separation,RCA,time-resolved fluorescence,DNAzyme and other technologies,respectively.They showed a sensitive,reliable,and highly specific detection performance,and provided new ideas for the development of food safety technology.更多还原