基于上转换发光纳米探针的赭曲霉毒素A检测方法研究
发布时间：2018-09-18 15:20
【摘要】：由真菌毒素引起的食品安全问题一直受到人们的重视。赭曲霉毒素A是世界范围内公认的污染较广、毒性较强的真菌毒素之一,严重危害食品安全和人类健康。目前针对赭曲霉毒素A的检测方法大多需要专门的仪器设备和操作人员,既需要较为昂贵的设备投入、人员培训,又不能满足现场快速检测的需要;虽然有一些快速检测的方法,但是受到环境等外界因素的影响,往往不能进行准确快速的检测。因此,研究和建立快速、低廉和稳定的赭曲霉毒素A检测方法对于食品安全有着重要意义。上转换发光纳米材料是利用稀土元素亚稳态的能级,能够吸收长波近红外光子,发射短波紫外可见光子的新型发光材料,具有窄带发射、大Stokes位移、光化学性质稳定、没有生物背景干扰、信噪比高、不存在光漂白效应等优点,在生物分析检测中有着巨大的应用前景。但同时也存在生物兼容性差、量子产率低等问题。本研究采用高温热解法制备上转换发光纳米材料,采用适配体作为特异性识别分子,结合磁分离技术和发光共振能量转移技术,成功构建了具有高度特异性和灵敏性的赭曲霉毒素A适配体传感器。主要研究工作包括:1构建了基于近红外上转换发光标记和磁分离技术的赭曲霉毒素A适配体传感器。课题组前期研究表明,上转换发光可以实现可区分单峰发射,于是在尝试拓展上转换光谱单峰发射的设想下,本实验制备了在近红外光区804 nm具有强发射峰的NaYF4:Yb0.2,Tm0.02上转换发光纳米材料作为发光标记物,将其修饰赭曲霉毒素A适配体后制成apt-UCNPs纳米探针;将磁性纳米材料修饰适配体互补链后,制备成cDNA-MNPs纳米探针,结合磁分离技术,构建了近红外上转换发光标记和磁分离的OTA适配体传感器。在最优实验条件下,OTA在0.01-100 ng/mL范围内与近红外发光值变化量具有良好的线性关系(R2=0.9958),该法检测限为0.005 ng/mL,并应用于实际样品检测,通过加标回收测试和特异性研究,表明该传感器可以用于实际样品检测。2构建了基于上转换发光纳米材料和金纳米棒发光共振能量转移检测赭曲霉毒素A适配体传感器。金纳米棒由于其可调的纵向等离子峰一直受到很多研究者的关注,我们设想调节金纳米棒纵向等离子峰使其与Er掺杂上转换发光光谱重叠,构建基于发光共振能量转移检测OTA的均相体系。本实验分别制备Er掺杂上转换发光纳米材料,修饰适配体后作为能量供体探针;制备长径比为2.5左右的金纳米棒,修饰互补链后制成能量受体探针,构建了发光共振能量转移OTA适配体传感器。通过优化两种探针用量、杂交时间和孵育时间等,在最优条件下,OTA在0.05-100 ng/mL范围内与上转换发光信号的恢复量有良好的线性关系(R2=0.9951),方法检测限为0.027 ng/mL,通过加标回收和特异性分析,表明该传感器可以用于实际食品样品检测,将上转换发光纳米材料实现从非均相到均相的应用。3构建了基于核壳型上转换发光纳米材料和氧化石墨烯发光共振能量转移检测赭曲霉毒素A适配体传感器。上转换发光纳米材料的低量子产率一直是限制其得到更大范围应用的重要原因,通过层层包覆(Layer By Layer)策略,制备了NaYF4:Yb0.18,Er0.02@NaYF4核壳型上转换发光纳米材料,通过发光光谱表征,证明其比未包裹NaYF4外壳的NaYF4:Yb0.18,Er0.02上转换发光纳米材料具有较高的量子产率,发光强度是后者的2.74倍,将其修饰适配体后制成核壳型能量供体探针;选用GO作为猝灭剂,构建乐发光共振能量转移OTA适配体传感器。在最优实验条件下,OTA在0.001-250 ng/mL范围内与上转换发光保留值有良好的线性关系(R2=0.9928),该法检测限为0.001 ng/mL。通过加标回收和特异性分析,表明该传感器可以用于实际食品样品检测。与传统ELISA方法相比无显著性差异(R2=0.9991,P0.0001),本实验改善了上转换发光纳米材料的表面缺陷,实现了发光增强,并使方法的检测限有所提高。
[Abstract]:Ochratoxin A is one of the most widely recognized mycotoxins with high toxicity, which is harmful to food safety and human health. At present, the detection methods of ochratoxin A mostly need special instruments and operators. To invest more expensive equipment, personnel training, but can not meet the needs of on-site rapid detection; although there are some rapid detection methods, but by environmental and other external factors, often can not be accurate and rapid detection. Therefore, research and establish a rapid, inexpensive and stable ochratoxin A detection method for food safety Up-conversion luminescent nanomaterials are new luminescent materials which can absorb long-wave near-infrared photons and emit short-wave ultraviolet-visible photons by using metastable energy levels of rare earth elements. They have narrow-band emission, large Stokes shift, stable photochemical properties, no biological background interference, high signal-to-noise ratio, no photobleaching effect and so on. In this study, upconversion luminescent nanomaterials were prepared by pyrolysis at high temperature, aptamers were used as specific recognition molecules, magnetic separation technology and luminescent resonance energy transfer technology were combined to construct successfully. A highly specific and sensitive ochratoxin A aptamer sensor has been developed. The main research work includes: 1. An ochratoxin A aptamer sensor based on near-infrared up-conversion luminescence labeling and magnetic separation technology has been constructed. Under the assumption of single-peak emission of conversion spectroscopy, NAYF4:Yb 0.2 and Tm 0.02 upconversion luminescent nanomaterials with strong emission peaks at 804 nm in the near-infrared region were prepared as luminescent markers, and apt-UCNPs nanoprobes were prepared by modifying ochratoxin A aptamer with magnetic nanomaterials. NPs nanoprobe combined with magnetic separation technology was used to construct an OTA aptamer sensor for near infrared up-conversion luminescence labeling and magnetic separation. The biosensor can be used to detect the ochratoxin A aptamer based on up-conversion luminescent nanomaterials and gold nanorods. Gold nanorods have attracted much attention due to their adjustable longitudinal plasma peaks. A homogeneous system based on luminescence resonance energy transfer detection for OTA was constructed by adjusting the longitudinal plasma peak of gold nanorods to overlap with Er-doped up-conversion luminescence spectrum. Optimizing the dosage of two probes, hybridization time and incubation time, OTA has a good linear relationship with the recovery of up-conversion luminescence signal in the range of 0.05-100 ng/mL (R2=0.9951), and the detection limit is 0.9951. 027 ng / mL. The results of recovery and specificity analysis showed that the sensor could be used for real food samples detection, and the up-conversion luminescent nanomaterials could be used for heterogeneous to homogeneous applications. Low quantum yield of up-conversion luminescent nanomaterials has been an important reason for their wider application. NaYF4:Yb0.18, Er0.02@NaYF4 core-shell type up-conversion luminescent nanomaterials were prepared by Layer By Layer strategy. The luminescent spectra showed that the up-conversion luminescent nanomaterials were better than NaYF4:Yb0.18, Er0. 02 upconversion luminescent nanomaterials have high quantum yield, luminescent intensity is 2.74 times that of the latter, and the core-shell energy donor probe is made by modifying the aptamer. GO is chosen as quenching agent to construct an OTA aptamer sensor. Under the optimal experimental conditions, OTA and upconversion luminescence are in the range of 0.001-250ng/mL. There was a good linear relationship between the retention values (R2 = 0.9928) and the detection limit was 0.001 ng/mL. The recovery and specificity analysis showed that the sensor could be used for the detection of real food samples. The luminescence enhancement and the detection limit of the method were improved.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS207.3;R155.5
，
本文编号：2248320
[Abstract]:Ochratoxin A is one of the most widely recognized mycotoxins with high toxicity, which is harmful to food safety and human health. At present, the detection methods of ochratoxin A mostly need special instruments and operators. To invest more expensive equipment, personnel training, but can not meet the needs of on-site rapid detection; although there are some rapid detection methods, but by environmental and other external factors, often can not be accurate and rapid detection. Therefore, research and establish a rapid, inexpensive and stable ochratoxin A detection method for food safety Up-conversion luminescent nanomaterials are new luminescent materials which can absorb long-wave near-infrared photons and emit short-wave ultraviolet-visible photons by using metastable energy levels of rare earth elements. They have narrow-band emission, large Stokes shift, stable photochemical properties, no biological background interference, high signal-to-noise ratio, no photobleaching effect and so on. In this study, upconversion luminescent nanomaterials were prepared by pyrolysis at high temperature, aptamers were used as specific recognition molecules, magnetic separation technology and luminescent resonance energy transfer technology were combined to construct successfully. A highly specific and sensitive ochratoxin A aptamer sensor has been developed. The main research work includes: 1. An ochratoxin A aptamer sensor based on near-infrared up-conversion luminescence labeling and magnetic separation technology has been constructed. Under the assumption of single-peak emission of conversion spectroscopy, NAYF4:Yb 0.2 and Tm 0.02 upconversion luminescent nanomaterials with strong emission peaks at 804 nm in the near-infrared region were prepared as luminescent markers, and apt-UCNPs nanoprobes were prepared by modifying ochratoxin A aptamer with magnetic nanomaterials. NPs nanoprobe combined with magnetic separation technology was used to construct an OTA aptamer sensor for near infrared up-conversion luminescence labeling and magnetic separation. The biosensor can be used to detect the ochratoxin A aptamer based on up-conversion luminescent nanomaterials and gold nanorods. Gold nanorods have attracted much attention due to their adjustable longitudinal plasma peaks. A homogeneous system based on luminescence resonance energy transfer detection for OTA was constructed by adjusting the longitudinal plasma peak of gold nanorods to overlap with Er-doped up-conversion luminescence spectrum. Optimizing the dosage of two probes, hybridization time and incubation time, OTA has a good linear relationship with the recovery of up-conversion luminescence signal in the range of 0.05-100 ng/mL (R2=0.9951), and the detection limit is 0.9951. 027 ng / mL. The results of recovery and specificity analysis showed that the sensor could be used for real food samples detection, and the up-conversion luminescent nanomaterials could be used for heterogeneous to homogeneous applications. Low quantum yield of up-conversion luminescent nanomaterials has been an important reason for their wider application. NaYF4:Yb0.18, Er0.02@NaYF4 core-shell type up-conversion luminescent nanomaterials were prepared by Layer By Layer strategy. The luminescent spectra showed that the up-conversion luminescent nanomaterials were better than NaYF4:Yb0.18, Er0. 02 upconversion luminescent nanomaterials have high quantum yield, luminescent intensity is 2.74 times that of the latter, and the core-shell energy donor probe is made by modifying the aptamer. GO is chosen as quenching agent to construct an OTA aptamer sensor. Under the optimal experimental conditions, OTA and upconversion luminescence are in the range of 0.001-250ng/mL. There was a good linear relationship between the retention values (R2 = 0.9928) and the detection limit was 0.001 ng/mL. The recovery and specificity analysis showed that the sensor could be used for the detection of real food samples. The luminescence enhancement and the detection limit of the method were improved.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS207.3;R155.5
，
本文编号：2248320
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