基于上轉(zhuǎn)換發(fā)光納米探針的赭曲霉毒素A檢測(cè)方法研究
發(fā)布時(shí)間：2018-09-18 15:20
【摘要】：由真菌毒素引起的食品安全問(wèn)題一直受到人們的重視。赭曲霉毒素A是世界范圍內(nèi)公認(rèn)的污染較廣、毒性較強(qiáng)的真菌毒素之一,嚴(yán)重危害食品安全和人類(lèi)健康。目前針對(duì)赭曲霉毒素A的檢測(cè)方法大多需要專(zhuān)門(mén)的儀器設(shè)備和操作人員,既需要較為昂貴的設(shè)備投入、人員培訓(xùn),又不能滿(mǎn)足現(xiàn)場(chǎng)快速檢測(cè)的需要;雖然有一些快速檢測(cè)的方法,但是受到環(huán)境等外界因素的影響,往往不能進(jìn)行準(zhǔn)確快速的檢測(cè)。因此,研究和建立快速、低廉和穩(wěn)定的赭曲霉毒素A檢測(cè)方法對(duì)于食品安全有著重要意義。上轉(zhuǎn)換發(fā)光納米材料是利用稀土元素亞穩(wěn)態(tài)的能級(jí),能夠吸收長(zhǎng)波近紅外光子,發(fā)射短波紫外可見(jiàn)光子的新型發(fā)光材料,具有窄帶發(fā)射、大Stokes位移、光化學(xué)性質(zhì)穩(wěn)定、沒(méi)有生物背景干擾、信噪比高、不存在光漂白效應(yīng)等優(yōu)點(diǎn),在生物分析檢測(cè)中有著巨大的應(yīng)用前景。但同時(shí)也存在生物兼容性差、量子產(chǎn)率低等問(wèn)題。本研究采用高溫?zé)峤夥ㄖ苽渖限D(zhuǎn)換發(fā)光納米材料,采用適配體作為特異性識(shí)別分子,結(jié)合磁分離技術(shù)和發(fā)光共振能量轉(zhuǎn)移技術(shù),成功構(gòu)建了具有高度特異性和靈敏性的赭曲霉毒素A適配體傳感器。主要研究工作包括:1構(gòu)建了基于近紅外上轉(zhuǎn)換發(fā)光標(biāo)記和磁分離技術(shù)的赭曲霉毒素A適配體傳感器。課題組前期研究表明,上轉(zhuǎn)換發(fā)光可以實(shí)現(xiàn)可區(qū)分單峰發(fā)射,于是在嘗試拓展上轉(zhuǎn)換光譜單峰發(fā)射的設(shè)想下,本實(shí)驗(yàn)制備了在近紅外光區(qū)804 nm具有強(qiáng)發(fā)射峰的NaYF4:Yb0.2,Tm0.02上轉(zhuǎn)換發(fā)光納米材料作為發(fā)光標(biāo)記物,將其修飾赭曲霉毒素A適配體后制成apt-UCNPs納米探針;將磁性納米材料修飾適配體互補(bǔ)鏈后,制備成cDNA-MNPs納米探針,結(jié)合磁分離技術(shù),構(gòu)建了近紅外上轉(zhuǎn)換發(fā)光標(biāo)記和磁分離的OTA適配體傳感器。在最優(yōu)實(shí)驗(yàn)條件下,OTA在0.01-100 ng/mL范圍內(nèi)與近紅外發(fā)光值變化量具有良好的線性關(guān)系(R2=0.9958),該法檢測(cè)限為0.005 ng/mL,并應(yīng)用于實(shí)際樣品檢測(cè),通過(guò)加標(biāo)回收測(cè)試和特異性研究,表明該傳感器可以用于實(shí)際樣品檢測(cè)。2構(gòu)建了基于上轉(zhuǎn)換發(fā)光納米材料和金納米棒發(fā)光共振能量轉(zhuǎn)移檢測(cè)赭曲霉毒素A適配體傳感器。金納米棒由于其可調(diào)的縱向等離子峰一直受到很多研究者的關(guān)注,我們?cè)O(shè)想調(diào)節(jié)金納米棒縱向等離子峰使其與Er摻雜上轉(zhuǎn)換發(fā)光光譜重疊,構(gòu)建基于發(fā)光共振能量轉(zhuǎn)移檢測(cè)OTA的均相體系。本實(shí)驗(yàn)分別制備Er摻雜上轉(zhuǎn)換發(fā)光納米材料,修飾適配體后作為能量供體探針;制備長(zhǎng)徑比為2.5左右的金納米棒,修飾互補(bǔ)鏈后制成能量受體探針,構(gòu)建了發(fā)光共振能量轉(zhuǎn)移OTA適配體傳感器。通過(guò)優(yōu)化兩種探針用量、雜交時(shí)間和孵育時(shí)間等,在最優(yōu)條件下,OTA在0.05-100 ng/mL范圍內(nèi)與上轉(zhuǎn)換發(fā)光信號(hào)的恢復(fù)量有良好的線性關(guān)系(R2=0.9951),方法檢測(cè)限為0.027 ng/mL,通過(guò)加標(biāo)回收和特異性分析,表明該傳感器可以用于實(shí)際食品樣品檢測(cè),將上轉(zhuǎn)換發(fā)光納米材料實(shí)現(xiàn)從非均相到均相的應(yīng)用。3構(gòu)建了基于核殼型上轉(zhuǎn)換發(fā)光納米材料和氧化石墨烯發(fā)光共振能量轉(zhuǎn)移檢測(cè)赭曲霉毒素A適配體傳感器。上轉(zhuǎn)換發(fā)光納米材料的低量子產(chǎn)率一直是限制其得到更大范圍應(yīng)用的重要原因,通過(guò)層層包覆(Layer By Layer)策略,制備了NaYF4:Yb0.18,Er0.02@NaYF4核殼型上轉(zhuǎn)換發(fā)光納米材料,通過(guò)發(fā)光光譜表征,證明其比未包裹NaYF4外殼的NaYF4:Yb0.18,Er0.02上轉(zhuǎn)換發(fā)光納米材料具有較高的量子產(chǎn)率,發(fā)光強(qiáng)度是后者的2.74倍,將其修飾適配體后制成核殼型能量供體探針;選用GO作為猝滅劑,構(gòu)建樂(lè)發(fā)光共振能量轉(zhuǎn)移OTA適配體傳感器。在最優(yōu)實(shí)驗(yàn)條件下,OTA在0.001-250 ng/mL范圍內(nèi)與上轉(zhuǎn)換發(fā)光保留值有良好的線性關(guān)系(R2=0.9928),該法檢測(cè)限為0.001 ng/mL。通過(guò)加標(biāo)回收和特異性分析,表明該傳感器可以用于實(shí)際食品樣品檢測(cè)。與傳統(tǒng)ELISA方法相比無(wú)顯著性差異(R2=0.9991,P0.0001),本實(shí)驗(yàn)改善了上轉(zhuǎn)換發(fā)光納米材料的表面缺陷,實(shí)現(xiàn)了發(fā)光增強(qiáng),并使方法的檢測(cè)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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：TS207.3;R155.5
，
本文編號(hào)：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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：TS207.3;R155.5
，
本文編號(hào)：2248320
本文鏈接：http://sikaile.net/yixuelunwen/yufangyixuelunwen/2248320.html
最近更新
教材專(zhuān)著
