基于适配体功能化时间分辨荧光纳米探针的真菌毒素检测方法研究
发布时间：2021-11-10 17:11
食品安全问题是各国政府和科学家面临的主要挑战之一。真菌毒素是由真菌产生的有毒次生代谢产物,能够导致人类和动物死亡。目前,人们已从农业科学,环境科学、免疫学、药理学和医学等多方面对真菌毒素展开了认真的研究。由于色谱法和传统免疫分析法的不足,所以需要开发创新和稳定的方法来对真菌毒素进行适宜、灵敏和快速的检测。正因如此,各国都在研制和发展一次性适配体传感器/生物传感器,用以识别真菌毒素。近年来,研究者们开发了多种针对各类真菌毒素的荧光适配体传感器,而时间分辨荧光技术与适配体相结合的应用才刚刚出现。时间分辨荧光技术（TRF）可以有效消除背景荧光,具有较高的噪比。通过设置一定的延迟时间,可以区分样品中所含荧光物质的荧光寿命,从而提高检测的灵敏度。基于此,本研究中,合成了几种时间分辨的荧光纳米材料并对其进行表征,并结合多种纳米材料（如磁性纳米材料、二硫化钨、石墨烯氮化碳纳米片）及相应技术（时间分辨荧光技术、磁分离技术、滚环扩增技术）等,构建了一系列快速,简单,灵敏,高效的适配体传感器用于检测多种真菌毒素。首先,基于已报道的玉米赤霉烯酮（ZEN）适配体序列,构建了一种新颖、高灵敏、强亲和性的时间分辨...
【文章来源】：江南大学江苏省 211工程院校 教育部直属院校
【文章页数】：148 页
【学位级别】：博士
【文章目录】：
ACKNOWLEDGEMENTS
DEDICATION
LIST OF ABBREVIATIONS
Abstract
摘要
Chapter 1 General introduction and review of literature
1.1 Mycotoxins: General information
1.1.1 Zearalenone (ZEN)
1.1.2 Fumonisin
1.1.3 Ochratoxin A
1.1.4 Trichothecenes A (T-2 toxin)
1.1.5 Aflatoxin
1.2 Natural co-occurrence of mycotoxins
1.3 Conventional analytical techniques for mycotoxin detection
1.3.1 Chromatographic techniques
1.3.2 Immunological methods
1.4 Aptasensors for mycotoxins detection
1.4.1 Elements of aptasensors
1.4.1.1 Aptamers: General information and aptamers selected for mycotoxins
1.4.1.2 Nanomaterials
1.4.1.2.1 Quantum dots (QDs)
1.4.1.2.2 Magnetic nanoparticles
1.4.1.2.3 Silica nanoparticles (Si-NPs)
1.4.1.2.4 Carbon nanomaterial
1.4.1.2.5 Metal nanomaterial
1.4.1.2.6 Up conversion nanoparticles (UCNPs)
1.5 Nanoparticles based aptasensors for mycotoxins detection
1.5.1 Colorimetric sensors
1.5.2 Electrochemical sensors
1.5.3 Fluorometric sensors
1.6 Time-resolved fluorescence technique (TRFL)
1.7 Time-gated luminescence technique (TGL)
1.8 Principle of time-resolved fluorescence technique
1.9 Ln~(3+)-doped fluorescent inorganic nanoparticles for time-resolved biosensing
1.10 Ln~(3+)-doped silica nanoparticles for time-resolved biosensing
1.11 Other techniques used in this research
1.11.1 Magnetic separation technique
1.11.2 Rolling circle amplification (RCA)
1.11.2.1 Rolling circle amplification process
1.11.2.2 Advantages of RCA
1.11.2.3 RCA-based fluorescence detection assay
1.11.2.4 RCA based fluorescence detection assay for mycotoxin
1.12 Other 2D nanomaterials used in this research
1.12.1 Tungsten disulfide nanomaterial (WS2)
1.12.2 Graphitic carbon nitride (g-C_3N_4)
1.13 Importance of research topic
1.14 Main objectives of the research
1.15 Technical route of the research
1.16 References
CHAPTER 2: A novel bioassay based on aptamer-functionalized magnetic nanoparticle for the detection of zearalenone using time resolved-fluorescence Na YF_4: Ce/Tb nanoparticles as signalprobe
2.1 Introduction
2.2 Materials and methods
2.2.1 Materials and apparatus
2.2.2 Preparation of amine-functionalized Fe3O4 magnetic nanoparticles
2.2.3 Preparation of Na YF_4: Ce/Tb nanoparticles
2.2.4 Synthesis of signal probe assembly and ZEN measurement
2.2.5 Sample preparation and measurement
2.3 Results and discussion
2.3.1 Principle-based on TRFL-NPs and MNPs for detection of ZEN
2.3.2 Characterization of the prepared Na YF_4: Ce/Tb and amine-functionalized magneticnanoparticles
2.3.3 Characteristics of avidin-conjugated TRFL-NPs and MNPs
2.3.4 Optimization for assay
2.3.4.1 Aptamers concentration (n M)
2.3.4.2 Volume of apt-MNPs (μL)
2.3.5 Analytical performance
2.3.6 Specificity assay
2.3.7 Analytical applications
2.4 Conclusions
2.5 References
CHAPTER 3: Simultaneous Detection of Fumonisin B1 and Ochratoxin A using Dual-Colour Time Resolved Luminescent Nanoparticles (Na YF_4: Ce, Tb and NH_2-Eu/DPA@Si O_2) asLabels
3.1 Introduction
3.2 Materials and methods
3.2.1 Reagents
3.2.2 Apparatus
3.2.3 Synthesis of amine-functionalized Fe3O4 MNPs
3.2.4 Synthesis of Na YF_4: Ce, Tb NPs
3.2.5 Synthesis of NH2-Eu/DPA@Si O2 NPs
3.2.6 Synthesis of signal probe (MNPs-Apt_1/Na YF_4: Ce, Tb-c DNA1 and MNPs-Apt_2/NH_2-Eu/DPA@Si O_2-c DNA_2)
3.2.7 Procedure for simultaneous detection
3.2.8 Preparation and measurement of real sample
3.3 Results and discussion
3.3.1 Detection principle
3.3.2 Characterizations of TRF-NPs and MNPs
3.3.3 Characterizations of NPs conjugated to avidin and aptamer
3.3.4 Optimization of experimental conditions
3.3.4.1. Effect of aptamers concentrations
3.3.4.2. Effect of volume of MNPs-aptamer conjugates
3.3.4.3. Effect of incubation time
3.3.5 Analytical performance
3.3.6 Specificity evaluation
3.3.7 Analytical applications
3.4 Conclusion
3.5 References
CHAPTER 4: A“turn on”aptasensor for simultaneous detection of zearalenone, trichothecenes Aand aflatoxin B1 mycotoxins using WS2 as a quencher for time-resolved fluorescence
4.1 Introduction
4.2 Materials and methods
4.2.1 Reagents
4.2.2 Instrumentation
4.2.3 Preparation of amine functionalized Ln~(3+) doped KYF_4 NPs
4.2.4 Preparation of aptamer modified KYF_4: Ln~(3+) multicolor bioprobe
4.2.5 Procedure for simultaneous detection of mycotoxins
4.2.6 Sample preparation
4.3 Results and discussion
4.3.1 Detection scheme
4.3.2 Characterization of Ln~(3+) doped KYF_4 NPs and WS2
4.3.3 Characterization of NPs conjugated to avidin and aptamers
4.3.4 Optimization of developed aptasensor
4.3.5 Analytical performance
4.3.6 Selectivity of assay
4.3.7 Real food sample analysis
4.4 Conclusion
4.5 References
CHAPTER 5: Fluorescence detection of aflatoxin M_1 using rolling circle amplification and carbonnitride nanosheet as quencher
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials and reagents
5.2.2 Instrumentation
5.2.3 Synthesis of KYF_4: Eu~(+3) NPs
5.2.4 Synthesis of signal probe (KYF_4: Eu~(+3)-c DNA)
5.2.5 Preparation of ultrathin g-C_3N_4 nanosheets
5.2.6 Procedure for circularization of rolling circle template (RCT) DNA
5.2.7 Procedure for rolling circle amplification (RCA) reaction
5.2.8 Procedure for fluorescence measurement of RCA product hybridized with signal probeand g-C_3N_4
5.2.9 Analytical procedure
5.2.10 Sample preparation
5.3. Result and discussion
5.3.1. Sensing principle
5.3.2. Characterization of KYF_4: Eu~(+3) NPs and ultrathin g-C_3N_4 nanosheet
5.3.3 Characteristics of signal probe (TRFNPs-c DNA) avidin and c DNA
5.3.4 Characterization of circularization of rolling circle template (RCT) DNA
5.3.5 Characterization of rolling circle amplification (RCA) product
5.3.6 Optimization of experimental conditions
5.3.7 Analytical performance
5.3.8 Specificity and selectivity
5.3.9 Analytical application in milk sample
5.4 Conclusion
5.5 References
Chapter 6: General conclusion and recommendation
General conclusion
Key innovations
Recommendations
List of Publications
【参考文献】：
期刊论文
[1]玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立[J]. 王元凯,王君,王雨晨,陈志飞,严亚贤,郝倩雯,李树清,于翠,杨翠云,孙建和. 微生物学通报. 2011(12)
[2]应用生物素-链霉亲和素的酶联免疫吸附法检测谷物中的玉米赤霉烯酮[J]. 马智鸿,黄飚,屠蔷,高蕾,张珏,王柯. 安徽农业科学. 2009(15)
本文编号：3487624
【文章来源】：江南大学江苏省 211工程院校 教育部直属院校
【文章页数】：148 页
【学位级别】：博士
【文章目录】：
ACKNOWLEDGEMENTS
DEDICATION
LIST OF ABBREVIATIONS
Abstract
摘要
Chapter 1 General introduction and review of literature
1.1 Mycotoxins: General information
1.1.1 Zearalenone (ZEN)
1.1.2 Fumonisin
1.1.3 Ochratoxin A
1.1.4 Trichothecenes A (T-2 toxin)
1.1.5 Aflatoxin
1.2 Natural co-occurrence of mycotoxins
1.3 Conventional analytical techniques for mycotoxin detection
1.3.1 Chromatographic techniques
1.3.2 Immunological methods
1.4 Aptasensors for mycotoxins detection
1.4.1 Elements of aptasensors
1.4.1.1 Aptamers: General information and aptamers selected for mycotoxins
1.4.1.2 Nanomaterials
1.4.1.2.1 Quantum dots (QDs)
1.4.1.2.2 Magnetic nanoparticles
1.4.1.2.3 Silica nanoparticles (Si-NPs)
1.4.1.2.4 Carbon nanomaterial
1.4.1.2.5 Metal nanomaterial
1.4.1.2.6 Up conversion nanoparticles (UCNPs)
1.5 Nanoparticles based aptasensors for mycotoxins detection
1.5.1 Colorimetric sensors
1.5.2 Electrochemical sensors
1.5.3 Fluorometric sensors
1.6 Time-resolved fluorescence technique (TRFL)
1.7 Time-gated luminescence technique (TGL)
1.8 Principle of time-resolved fluorescence technique
1.9 Ln~(3+)-doped fluorescent inorganic nanoparticles for time-resolved biosensing
1.10 Ln~(3+)-doped silica nanoparticles for time-resolved biosensing
1.11 Other techniques used in this research
1.11.1 Magnetic separation technique
1.11.2 Rolling circle amplification (RCA)
1.11.2.1 Rolling circle amplification process
1.11.2.2 Advantages of RCA
1.11.2.3 RCA-based fluorescence detection assay
1.11.2.4 RCA based fluorescence detection assay for mycotoxin
1.12 Other 2D nanomaterials used in this research
1.12.1 Tungsten disulfide nanomaterial (WS2)
1.12.2 Graphitic carbon nitride (g-C_3N_4)
1.13 Importance of research topic
1.14 Main objectives of the research
1.15 Technical route of the research
1.16 References
CHAPTER 2: A novel bioassay based on aptamer-functionalized magnetic nanoparticle for the detection of zearalenone using time resolved-fluorescence Na YF_4: Ce/Tb nanoparticles as signalprobe
2.1 Introduction
2.2 Materials and methods
2.2.1 Materials and apparatus
2.2.2 Preparation of amine-functionalized Fe3O4 magnetic nanoparticles
2.2.3 Preparation of Na YF_4: Ce/Tb nanoparticles
2.2.4 Synthesis of signal probe assembly and ZEN measurement
2.2.5 Sample preparation and measurement
2.3 Results and discussion
2.3.1 Principle-based on TRFL-NPs and MNPs for detection of ZEN
2.3.2 Characterization of the prepared Na YF_4: Ce/Tb and amine-functionalized magneticnanoparticles
2.3.3 Characteristics of avidin-conjugated TRFL-NPs and MNPs
2.3.4 Optimization for assay
2.3.4.1 Aptamers concentration (n M)
2.3.4.2 Volume of apt-MNPs (μL)
2.3.5 Analytical performance
2.3.6 Specificity assay
2.3.7 Analytical applications
2.4 Conclusions
2.5 References
CHAPTER 3: Simultaneous Detection of Fumonisin B1 and Ochratoxin A using Dual-Colour Time Resolved Luminescent Nanoparticles (Na YF_4: Ce, Tb and NH_2-Eu/DPA@Si O_2) asLabels
3.1 Introduction
3.2 Materials and methods
3.2.1 Reagents
3.2.2 Apparatus
3.2.3 Synthesis of amine-functionalized Fe3O4 MNPs
3.2.4 Synthesis of Na YF_4: Ce, Tb NPs
3.2.5 Synthesis of NH2-Eu/DPA@Si O2 NPs
3.2.6 Synthesis of signal probe (MNPs-Apt_1/Na YF_4: Ce, Tb-c DNA1 and MNPs-Apt_2/NH_2-Eu/DPA@Si O_2-c DNA_2)
3.2.7 Procedure for simultaneous detection
3.2.8 Preparation and measurement of real sample
3.3 Results and discussion
3.3.1 Detection principle
3.3.2 Characterizations of TRF-NPs and MNPs
3.3.3 Characterizations of NPs conjugated to avidin and aptamer
3.3.4 Optimization of experimental conditions
3.3.4.1. Effect of aptamers concentrations
3.3.4.2. Effect of volume of MNPs-aptamer conjugates
3.3.4.3. Effect of incubation time
3.3.5 Analytical performance
3.3.6 Specificity evaluation
3.3.7 Analytical applications
3.4 Conclusion
3.5 References
CHAPTER 4: A“turn on”aptasensor for simultaneous detection of zearalenone, trichothecenes Aand aflatoxin B1 mycotoxins using WS2 as a quencher for time-resolved fluorescence
4.1 Introduction
4.2 Materials and methods
4.2.1 Reagents
4.2.2 Instrumentation
4.2.3 Preparation of amine functionalized Ln~(3+) doped KYF_4 NPs
4.2.4 Preparation of aptamer modified KYF_4: Ln~(3+) multicolor bioprobe
4.2.5 Procedure for simultaneous detection of mycotoxins
4.2.6 Sample preparation
4.3 Results and discussion
4.3.1 Detection scheme
4.3.2 Characterization of Ln~(3+) doped KYF_4 NPs and WS2
4.3.3 Characterization of NPs conjugated to avidin and aptamers
4.3.4 Optimization of developed aptasensor
4.3.5 Analytical performance
4.3.6 Selectivity of assay
4.3.7 Real food sample analysis
4.4 Conclusion
4.5 References
CHAPTER 5: Fluorescence detection of aflatoxin M_1 using rolling circle amplification and carbonnitride nanosheet as quencher
5.1 Introduction
5.2 Materials and methods
5.2.1 Materials and reagents
5.2.2 Instrumentation
5.2.3 Synthesis of KYF_4: Eu~(+3) NPs
5.2.4 Synthesis of signal probe (KYF_4: Eu~(+3)-c DNA)
5.2.5 Preparation of ultrathin g-C_3N_4 nanosheets
5.2.6 Procedure for circularization of rolling circle template (RCT) DNA
5.2.7 Procedure for rolling circle amplification (RCA) reaction
5.2.8 Procedure for fluorescence measurement of RCA product hybridized with signal probeand g-C_3N_4
5.2.9 Analytical procedure
5.2.10 Sample preparation
5.3. Result and discussion
5.3.1. Sensing principle
5.3.2. Characterization of KYF_4: Eu~(+3) NPs and ultrathin g-C_3N_4 nanosheet
5.3.3 Characteristics of signal probe (TRFNPs-c DNA) avidin and c DNA
5.3.4 Characterization of circularization of rolling circle template (RCT) DNA
5.3.5 Characterization of rolling circle amplification (RCA) product
5.3.6 Optimization of experimental conditions
5.3.7 Analytical performance
5.3.8 Specificity and selectivity
5.3.9 Analytical application in milk sample
5.4 Conclusion
5.5 References
Chapter 6: General conclusion and recommendation
General conclusion
Key innovations
Recommendations
List of Publications
【参考文献】：
期刊论文
[1]玉米赤霉烯酮单克隆抗体的制备及间接竞争ELISA检测方法的建立[J]. 王元凯,王君,王雨晨,陈志飞,严亚贤,郝倩雯,李树清,于翠,杨翠云,孙建和. 微生物学通报. 2011(12)
[2]应用生物素-链霉亲和素的酶联免疫吸附法检测谷物中的玉米赤霉烯酮[J]. 马智鸿,黄飚,屠蔷,高蕾,张珏,王柯. 安徽农业科学. 2009(15)
本文编号：3487624
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