恩诺沙星和玉米赤霉烯酮新型纳米材料标记免疫检测方法.docx
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1、摘 要食品中有毒有害污染物威胁着人类的健康,也是导致食品安全问题出现的重要因素之一。免疫分析方法因其高灵敏度、高通量、高特异性、低检测成本及操作快捷简便等优异特性而被广泛应用于食品中痕量污染物的快速分析。以纳米材料为探针元件的新型纳米免疫传感分析方法为进一步提高检测灵敏度及拓宽应用范围提供了必要的支持。本论文以零维半导体量子点(QDs)、金纳米粒子(AuNPs)和二维黑磷纳米片(BPNSs)-贵金属纳米复合物(BP-Au、BP-Pt、BP-Au-Pt)等不同维度的纳米材料为研究对象,探索了其独特的光,热,吸附和催化性能,并依次为免疫传感分析探针元件,构建了4类新型纳米免疫分析方法,从而实现了对
2、食品中小分子污染物的快速,高度灵敏的检测。具体内容如下:1. 量子点荧光及荧光淬灭纳米免疫层析传感分析方法的研究以最大发射波长(ex)为585 nm的橘红色CdTe/ZnS羧基水溶性QDs作为荧光探针元件,通过化学共价结合分别与玉米赤霉烯酮(ZEN)单克隆抗体(ZEN-Ab)或抗恩诺沙星(ENR)多克隆抗体(ENR-Ab)偶联制备免疫探针,构建了基于QDs荧光信号的半定量免疫层析传感分析方法(QICS)。并选用ex=525 nm的绿色QDs与鸡卵白蛋白(OVA)偶联制备作为荧光供体探针,选用最大吸收波长(Ab)为520 nm的AuNPs分别与ZEN-Ab或ENR-Ab结合制备荧光受体探针,构建
3、了基于QDs/AuNPs淬灭体系的荧光淬灭半定量免疫层析传感分析方法(A-FICS)。在用于ZEN的检测时,QICS及A-FICS分别实现了PBS缓冲液中1、0.25 g L-1 ZEN的检出以及谷物样品中20、5 g L-1 ZEN的检出。在用于ENR的检测时,QICS及A-FICS同样分别实现了PBS缓冲液中1、0.25 g L-1ENR的检出,且实现了动物源性食品中5、1.25 g L-1 ENR的检出。两种方法均可以在30 min内实现对食品中污染物的快速检测,且A-FICSs具有更高的灵敏度。相比于市售酶联免疫吸附(ELISA)试剂盒,ICSs具有灵敏度高、快捷及简便等优点,适用于动
4、物源性食品及谷物样品中食品污染物的现场可视化快速检测。2. 黑磷-金纳米复合物新型荧光淬灭免疫层析传感分析方法研究利用BPNSs的化学不稳定性合成了Ab=518 nm的BPNSs-AuNPs复合物(BP-Au),通过密度泛函理论(DFT)探索了BP-Au复合材料的形成机理,并通过荧光分析测定了BP-Au复合材料对QDs的荧光猝灭率。BP-Au与ZEN-Ab偶联制备新型荧光淬灭探针。选用ex=525 nm的QDs与OVA偶联作为荧光供体探针,构建了基于QDs/BP-Au淬灭体系的荧光淬灭半定量免疫层析分析方法(B-FICS)。B-FICS对ZEN的检测限为0.1 g L-1,对谷物样品进行检测,
5、检测限为2 g kg-1。B-FICS的灵敏度较A-FICS提高了2.5倍,证明对于QDs的淬灭而言,BP-Au比AuNPs具有更强的性能和应用潜力。B-FICS的成功构建为谷物中ZEN的快速,高灵敏度检测提供了一种新颖的方法,并为BPNSs在免疫传感分析及食品安全检测中的应用提供了新思路。3. 黑磷-金纳米复合物高灵敏度光热免疫层析传感分析方法研究除光吸收性能外BP-Au具有优异的光热性能。相较于BPNSs,BP-Au的光热转换率()提高了12.9%。借助BP-Au良好的光热性能及物理吸附性能,将ENR-Ab与其偶联制备了新型光热传感探针。以此构建了基于BP-Au的光热定量免疫层析分析方法(
6、PT-ICS)。该方法通过对T线处温度变化的分析实现了ENR的高灵敏定量检测分析,其检测限为0.023 g L-1,线性范围为0.03-10 g L-1。对动物源性食品样品进行检测,检测范围为0.45150 g kg-1,回收率为72.6-126.2。该PT-ICS较可视化半定量ICS和ELISA试剂盒的测定结果更为灵敏,为动物源性食品中ENR残留的快速、高灵敏定量检测提供了新策略,并有望用于其他食品污染物的高度灵敏检测。4. 以黑磷-铂纳米复合物为纳米酶探针的免疫吸附比色及光热传感分析技术研究在BPNSs表面原位还原超小铂纳米粒子(PtNPs),形成BP -Pt纳米酶。基于其良好的类过氧化物
7、酶性质和物理吸附性质,制备了新型的非酶信号探针用以替代辣根过氧化物酶(HRP)标记二抗,并建立了一步法非酶免疫吸附分析方法(NISS)用于高灵敏度检测ENR,LOD为0.005gL-1,灵敏度为0.068gL-1(R2=0.994)。此外,基于TMB催化氧化产物(oxTMB)在808 nm处的良好光热性能,建立了光热免疫吸附测定(PT-NISS)。PT-NISS的检测结果与NISS一致。另外,NISS和PT-NISS在动物衍生食品样品中的ENR回收率分别为75.1-124.1和71.2-119.1。检测结果与市售ELISA试剂盒一致。NISS和PT-NISS为检测食品样品中的残留污染物提供了更
8、为快速和有希望的策略,并有望用于检测其他高度敏感的生物大分子。关键词:食品污染物;二维黑磷纳米片;光热传感;纳米酶;量子点;荧光猝灭;免疫分析ABSTRACTFood contamination threatens human health and is one of the important factors leading to food safety issues. Immunoassays are widely used for the rapid analysis of trace contaminants in food due to their high specificity,
9、 high sensitivity, high throughput, low detection cost, and fast and easy operation. The new nano-immunoassays using nano-materials as probe elements provides necessary support for further improving the sensitivity and broadening the application range. In this thesis, nano-materials with different d
10、imensions such as zero-dimensional semiconductor quantum dots (QDs), gold nanoparticles (AuNPs) and two-dimensional black phosphorus nanosheets (BPNSs) -precious metal nanocomposites (BP-Au, BP-Pt, BP-Au-Pt) are taken as research object, and their unique light, heat, adsorption and catalytic propert
11、ies are explored. Using the above nanomaterials as probe elements for immunosensing analysis, five new types of nanoimmunoassays have been established, thereby achieving rapid and highly sensitive detection of small molecular contaminants in food. The details are as follows:1. Quantum dot fluorescen
12、ce and fluorescence quenching nanoimmunochromatographic sensing assaysIn this section, orange-red CdTe/ZnS carboxyl water-soluble QDs with a maximum emission wavelength of 585 nm are used as fluorescent probe elements. They are chemically covalently bound to zearalenone (ZEN) monoclonal antibody (ZE
13、N-Ab) or anti- Enrofloxacin (ENR) polyclonal antibody (ENR-Ab) was conjugated to prepare immunoprobe, and fluorescent semi-quantitative immunochromatographic sensing assay (QICS) was established. In addition, green QDs with maximum emission wavelength of 525 nm was coupled with chicken ovalbumin (OV
14、A) as fluorescent donor probes, and AuNPs with maximum absorption wavelength of 520 nm was combined with ZEN-Ab or ENR-Ab as fluorescent receptor probes. A fluorescence quenching semi-quantitative immunochromatographic sensing assay (A-FICS) based on QDs/AuNPs quenching system was constructed. When
15、used for the detection of ZEN, QICS and A-FICS respectively achieved the detection limits (LODs) of 1 and 0.25 g L-1 ZEN in PBS buffer and 20 and 5 g L-1 ZEN in cereal samples. When used for ENR detection, QICS and A-FICS achieved LODs of 1 and 0.25 g L-1ENR in PBS buffer and 5 and 1.25 g L-1 ENR in
16、 animal-derived food samples. Both assays achieved rapid detection of contaminants in food within 30 minutes, and A-FICSs has higher sensitivity. Compared with commercial ELISA kits, ICSs have the advantages of high sensitivity, quickness, and simple operation. They are suitable for the on-site visu
17、alization and rapid detection of food contaminants in cereal and animal-derived food samples.2. A novel fluorescence quenching immunochromatographic sensing method based on black phosphorus-Au nanocomposites as acceptor probeIn this section, the chemical instability of BPNSs was used to synthesize a
18、 BPNSs-AuNPs nanocomposites (BP-Au) with a maximum absorption wavelength of 518 nm. The formation mechanism of BP-Au composites was explored by density functional theory (DFT). The fluorescence quenching rate of QDs by BP-Au composites was measured by fluorescence analysis. Furthermore, BP-Au was co
19、upled with ZEN-Ab as novel fluorescence acceptor probe, and QDs with a maximum emission wavelength of 525 nm was coupled with OVA as fluorescent donor probe. A fluorescence quenching semi-quantitative immunochromatographic analysis method (B-FICS) based on the QDs/BP-Au quenching system was construc
20、ted. B-FICS has LOD of 0.1 g L-1 for ZEN in PBS buffer and 2 g kg-1 in cereal samples. The sensitivity of B-FICS is 2.5 times higher than that of A-FICS, which proves that for quenching QDs, BP-Au has stronger performance and application potential than AuNPs. The successful construction of B-FICS pr
21、ovided a novel method for rapid and high-sensitivity detection of ZEN in cereals, and provided new ideas for the application of two-dimensional BPNSs in food safety testing.3. High sensitivity photothermal immunochromatographic sensing assay based on black phosphorus-Au nanocomposite as photothermal
22、 probeA novel photothermal sensing probe was established by coupling BP-Au with ENR-Ab through physical adsorption, and a photothermal quantitative immunochromatographic analysis method (PT-ICS) based on the good photothermal performance of BP-Au was constructed. The LOD of PT-ICS for ENR is 0.023 g
23、 L-1 in PBS buffer, and the linear range is 0.03-10 g L-1. When detection animal derived food samples, the detection range of 0.45150 g kg-1 and a recovery rate of 72.6% -126.2% was achieved. The PT-ICS is more sensitive than semi-quantitative ICSs and the ELISA kit, which provides a new strategy fo
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