【作者】 杨丹婷；

【导师】 应义斌； R.Niessner； C.Haisch；

【作者基本信息】 浙江大学 ， 生物系统工程， 2013， 博士

【摘要】 “食品污染物”是指非特意在食品中添加,但能够在生产(包括谷物栽培、动物饲养和兽药使用)、制造、加工、调制、处理、填充、包装、运输和包藏等过程中产生,或是由于环境污染带入到食品中的对人类健康有危害的任何物质。它包括化学污染物和微生物污染物两大类。氨基甲酸乙酯是属于食品加工处理过程中的一种化学污染物,是发酵食品特别是酒精饮料中自然形成的一种物质,对人类健康具有致癌的潜在威胁；大肠杆菌是食源性微生物污染物非常重要的一个分支,某些致病菌菌株能够在一定程度上危害人的身体健康,使人致病甚至致死。本课题采用表面增强拉曼技术,并结合纳米技术、量子力学理论、光学、分子生物学、化学计量学等各领域的最新研究成果,探索建立了用于酒精饮料中氨基甲酸乙酯(EC)和水溶液中大肠杆菌(E.coli)快速检测的一系列免标记SERS新方法。本文的主要研究结果和结论如下：(1)建立了一种基于密度泛函理论和表面增强拉曼光谱技术的氨基甲酸乙酯快速检测方法。首先基于密度泛函理论(DFT)对氨基甲酸乙酯(EC)分子的空间几何结构进行了优化,其次结合拉曼光谱(RS)和表面增强拉曼光谱(SERS)技术,对EC分子的拉曼光谱和SERS光谱的特征峰进行了解析,对分子振动模式进行了峰的归属；再次通过分子静电势(MEP)对EC分子与SERS活性基底银纳米颗粒表面的反应吸附方式进行了研究；最后结合理论计算和试验研究的结果,研究了基于SERS技术的免标记方法对水溶液中氨基甲酸乙酯定性和定量检测的可行性,进而对EC定量检测分析的特征峰进行了优化选择,探索了此方法的检测限。结果表明：①基于B3LYP/6-31G (d)和aug-cc-pVTZ水平基组的DFT理论计算得到的EC分子的拉曼光谱峰与试验结果相符合。EC分子的拉曼特征峰为396,512,672,854,996,1076,1127,1150,1273,1346,1440,1457,1622,1688,2934,2965,2991,和3414cm-1,主要归属于羰基,C-C键,C-H键和N-H键振动。②根据SERS表面选择原则,结合MEP3D等值面图和EC的SERS特征峰振动模式归属,可以推断EC分子是通过酰胺基团和C-C键与SERS活性基底Ag NPs的表面吸附作用的。因酰胺基团的拉曼增强效果并不强,推断酰胺基团是以平行模式与Ag NPs表面作用,而C-C键的增强效果是最强的,推断其是以垂直模式与Ag NPs表面作用。③归属于us(CC)+co(CH)振动模式的1006cm-1处的拉曼峰是EC分子增强效应最强的SERS峰,在不同浓度下峰形及强度测试重复性最好,被用来作为水溶液中EC分子定量检测的特征峰。结果显示在1×10-6M到1×10-3M的浓度范围内,EC的拉曼峰强与其浓度成线性关系,相关系数为0.9253,检测限为2×10-7M(17.8μg/L)。此方法研究了EC晶体的拉曼光谱图,为物质鉴定的拉曼谱图库提供参考。证实了SERS技术在EC定性和定量检测中的可行性,结果表明基于此方法得到的定量分析检测限远低于各国设定的标准值,为EC的快速检测手段提供了新思路。(2)建立了一种基于Au@Ag NPs表面增强拉曼光谱技术的氨基甲酸乙酯定量检测方法。首先通过种子生长法在Au核表面合成不同Ag壳厚度的银壳包被金核的纳米颗粒(Au@Ag NPs),比较不同Ag壳厚度的纳米颗粒在不同激光波长下的SERS效果,选取最优Au@Ag NPs作为SERS活性基底进行EC检测。再次,基于EC分子SERS特征峰归属的研究结果,选择最佳定量特征峰对水溶液和酒精溶液中的不同浓度EC进行定量检测,并探索了此方法对酒精溶液中EC的定量检测限。结果表明：①通过染料分子罗丹明6G(R6G)和待测物EC对不同Ag壳厚度(1-11nm)的Au@Ag NPs在不同激发波长下SERS增强效果的表征,7nmAg壳厚度的Au@Ag NPs在633nm激发波长下展现出最强的SERS效应。②通过对不同浓度(1×10-9M到1×10-4M)EC水溶液的检测,得知以Au@Ag NPs为SERS活性基底得到的EC分子三个主要SERS特征峰1003cm-1,1083cm-1,和1117cm-1被显著增强,它们分别归属于us(CC)+ω(CH),δ(NH)+νs(OC)+δ(CH2)和δ(NH2+CH3)的振动模式；结果表明,EC的拉曼强度与其浓度在1×10-9M到1×10-7M的浓度范围内具有线性关系,其R2值分别为0.9682,0.9428,和0.9679；其中以1003cm-1处的SERS峰重复性最高,得到的线性关系最好,被选作为酒精溶液中EC定量检测的特征峰。③通过在三种酒精饮料(伏特加,白朗姆,和水果酒Obstler)中添加不同浓度的EC,验证基于以上方法对EC进行定量检测的可行性。结果表明与以上结果相同,在1×10-9M到1×10-7M的浓度范围内,酒精溶液中EC的拉曼强度与其浓度成线性关系,R2值分别为0.8065,0.9106,和0.9283,检测限分别为9.0×10-9M(0.8μg/L),1.3×10-7M(11.6μg/L),和7.8×10-8M(6.9μg/L)。整个检测过程只需15分钟,且不需要任何复杂的样品前处理。结果表明基于Au@AgNPs的表面增强拉曼光谱方法能够快速地检测酒精饮料中的痕量EC,检测限远远低于各国设定的标准值,这为开发实际酒精样品中EC的快速检测方法和手段提供了研究基础。(3)建立了一种基于免标记表面增强拉曼光谱技术的水溶液中大肠杆菌快速检测方法。选取E.coli DSM1116菌株作为大肠杆菌检测模型,使其自由生长在Ag NP胶体溶液中,优化其培养条件(震荡速度、培养时间、培养温度),选取较优条件培养的大肠杆菌进行检测。首先探索了基于较优培养条件SERS技术快速检测大肠杆菌以及鉴别三种不同大肠杆菌菌株E. coli DSM498/1116/5695的可行性；其次结合拉曼成像方法,探索了此方法对单个细菌和不同浓度大肠杆菌检测的可行性。结果表明：①E.coli DSM1116在Ag NP胶体溶液中以100rpm震荡速度,37℃的条件培养3h后,获得SERS检测的较优培养条件。通过计算其最强峰732cm-1处的拉曼信号,其(浓度1×108cells/mL)拉曼强度可达最大为18000cps。②基于较优培养条件建立的SERS方法与简单的混合方法相比,不仅大肠杆菌的出峰位置增多,重复性提高,且拉曼强度提高到3.5倍。③Ag NPs-E. coli混合溶液由最初的黄绿色变为鲜明的草绿色,在紫外-可见光吸收光谱图中出现500-700nm新的宽吸收峰；通过TEM结果观察,Ag NPs紧密吸附在细菌表面；通过对E.coli的SERS峰归属研究,658、732和1330cm-1三处峰的出现表明Ag NPs与细菌的细胞壁之间能够紧密结合。这表明细菌和Ag NPs之间可能形成了某种复合物。④基于较优培养条件的SERS方法对三种大肠杆菌菌株E. coli DSM498/1116/5695进行检测和鉴别。随机各取15条SERS光谱进行归一化处理,结合判别分析的化学计量学方法建立分类模型,结果表明三种大肠杆菌菌株可以被成功区分出来。⑤基于较优培养条件的SERS方法可以成功的检测出单个大肠杆菌的拉曼峰,其出峰位置与溶液中细菌的出峰位置相同,这为SERS拉曼成像的实施提供了试验基础。⑥获得在较优培养条件下的不同浓度的大肠杆菌,分别对其进行拉曼成像分析。结果表明,SERS成像能够检测到大肠杆菌的最低浓度为1×105cells/mL。结果表明,基于较优培养条件的大肠杆菌免标记SERS方法与之前报道的简单混合方法相比,SERS光谱重复性更好,检测的灵敏度更高,实现了不同浓度大肠杆菌的检测。(4)构建了一种基于免标记表面增强拉曼光谱技术快速抓取固定细菌的芯片。通过对玻璃片进行聚氧乙烯二胺修饰,使玻璃片表面带氨基,然后使用稀盐酸对玻片进行质子化处理使其带正电,因细菌带负电,根据静电吸附的原理,此种方法构建的芯片可以实现快速抓取固定细菌的目的。此芯片用细菌培养0.5h后继而用Ag NPs溶液孵育0.5h,用去离子水清洗后对细菌进行SERS检测。结合判别分析的化学计量学方法,探索了基于此芯片对三种不同的UTI细菌(CFT, PSAE,和PRM1)鉴别分类的可行性。结果表明：①细菌能够被牢固的抓紧在修饰的芯片上,与Ag NP胶体溶液混合孵育后,能够在5min内获得重复性较好的细菌SERS光谱。②结合判别分析的化学计量学方法进行建模,结果表明此芯片能够成功区分三种不同的UTI细菌。本方法构建的芯片,可携带,成本低,能够快速抓取固定和检测细菌,可以作为一种用于细菌快速检测的芯片。更多还原

【Abstract】 Food contaminants are substances that have not been intentionally added to food. But they may be present in food as a result of the various stages of its production (including cereal cultivation, animal grooming, and veterinary drug use), processing, treatment, packaging, transport, or holding. They also might result from environmental contamination. Such contamination, including chemical contaminant and microorganism contaminant, generally has a negative impact on the quality of food and may imply a risk to human health. Ethyl carbamate (EC) is one kind of chemical contaminant, which is produced in food processing. It is a by-product naturally formed in fermented foodstaff especially in alcoholic beverages, posing a potential threaten to human health, which can cause cancer. E.coli is one of the most important species in foodborne pathogens, some of which can lead people to illness even to death.Adopting surface-enhanced Raman spectroscopy (SERS), combined with latest research results of nanotechnology, quantum physics, optics, molecular biology, and chemometrics, this project, aimed to develop a series of new methods for rapid detection of ethyl carbamate and bacteria in beverages.The main contents and results are summarized as follows.(1) A rapid method for ethyl carbamate detection based on density functional theory (DFT) and surface-enhanced Raman spectroscopy (SERS) was established. Firstly, the optimized geometry structure of EC molecule was operated based on DFT calculation. Secondly, the combination of DFT, Raman spectroscopy (RS), and SERS was used to analyze the bands of EC molecule and assign the Raman vibrational modes of EC. In addition, the EC adsorption geometry on the SERS-substrate silver nanoparticle surface was discussed via molecular electrostatic potential (MEP). Based on theoretical and experimental results, the feasibility of SERS technique for qualitative and quantitative detection of EC in aqueous solution was investigated. The characteristic peak for quantitative detection was then chosen, and the limit of detection based on this method was found in the study. The results indicated that:①The calculated results of EC molecule based on DFT using B3LYP/6-31G (d) and aug-cc-pVTZ basis level were found to be in good agreement with experimental Raman data. The characteristic Raman bands observed for EC molecule are396,512,672,854,996,1076,1127,1150,1273,1346,1440,1457,1622,1688,2934,2965,2991, and3414cm-1, which are mostly assigned to carbonyl group, C-C, C-H and N-H stretching and deformation vibarations.②According to the surface selection rules of SERS, MEP3D contour map and SERS vibrational modes were used to discuss the EC molecule adsorption geometry on the silver nanoparticle surface, which indicated that the EC molecule may be absorbed on the silver nanoparticle surface via amide group, C-C and C-H vibrational bands. The bands assigned to the amide group which existed in the SERS spectrum were not strongly enhanced however. It is supposed that the EC molecule arranged in a way that the amide group stayed almost parallel to the surface. The C-C bond became oriented perpendicular to the surface and the vibrations specific to this bond were highly enhanced.③The Raman band at1006cm-1assigned to νs(CC)+ω(CH) was the highest and best reproducible peak in SERS spectrum of EC, which could be chosen as the characteristic peak for quantitative detection of EC in aqueous solution. It was found that Raman signal had a good linear relationship with EC concentration ranging from1×10-6M to1×10-3M with a corresponding correlation coefficient0.9253. The limit of detection (LOD) is2×10-7M (17.8μg/L). The method established here provided reference for Raman fingerprint bank, furthermore confirmed the feasibility of SERS technique for qualitative and quantitative detection of EC. The LOD obtained was lower than the standard value set up in every country, which opened a new possibility of EC rapid evaluation.(2) A quantitative method for ethyl carbamatc detection based on Au@Ag NPs-dependent SERS technique was contructed. Au@Ag NPs with different thicknesses of Ag shell were synthesized through seed-growing method. The performance of Au@Ag NPs with different Ag shell thicknesses at different laser was evaluated. The Au@Ag NPs with optimized condition was used as SERS amplifier to detect EC with different concentrations in aqueous solution. Based on the assignment result of SERS characteristic peaks of EC molecule, the optimized peak was chosen for quantitative analysis of EC in aqueous solution and alcoholic beverages. The LOD of EC in alcoholic beverages was investigated based on this method. The results indicated that:①The SERS performance of Au@Ag NPs with different thicknesses of Ag shell at different wavelength lasers was evaluated by a dye molecule rhodamine6G (R6G) and EC molecule, which indicated Au@Ag NPs with7-nm Ag shell thickness at633nm laser possessed the best SERS effect.②The SERS method based on Au@Ag NPs was used to detect EC with different concentrations (from1×10-9M to1×10-4M) in aqueous solution. It was found that three characteristic bands at1003cm-1,1083cm-1, and1117cm-1, assigned to vibrational modes of us(CC)+ω(CH),δ(NH)+νs(OC)+δ(CH2), and δ(NH2+CH3) respectively, were strongly enhanced. The linear relationship between SERS signal and EC concentration was found in the range of1×10-9M to1×10-7M, with a good value of R2(0.9682,0.9428, and0.9679). The band at1003cm-1represented the best reproducibility and linear relationship, could be chosen as the characteristic peak for quantitative analysis of EC in alcoholic beverages.③EC solution with different concentrations spiked into three kinds of alcoholic beverages (Vodka, White rum, and a fruit snack Obstler) were detected by SERS method. A linear relationship between SERS signal and EC concentration was found from1×10-9M to1×10-7M, with a good value of R2(0.8065,0.9106, and0.9283, respectively). LOD of9.0×10-9M (0.8μg/L),1.3×10-7M (11.6), and7.8×10-8M (6.9μg/L) for such three alcoholic beverages was obtained. The total assay time of the method was only15min, and only a small volume was required in a real-world sample without any complicated sample preparation. The result indicated that this sensitive Au@Ag NPs-dependent SERS method could successfully fulfill the detection of trace amount of EC in alcoholic beverages, which may offer an intersting alternative for simple, rapid assessment of EC in the alcoholic beverage industry.(3) A rapid label-free SERS detection method of E.coli in aqueous solution was established. In this case, E.coli DSM1116was chosen as model E.coli bacteria for the study. The bacteria were cultivated in silver nanoparticle colloids with different growing conditions including shaking speed, cultivated time, and cultivated temperature. An optimized cultivated condition was picked up to help construct a rapid SERS method. The feasibility of rapid detection of E.coli and classification of three E.coli strains (E. coli DSM498/1116/5695) based on SERS was investigated. Finally, a SERS mapping method for single bacterium and bacteria with different concentrations was construced. The results indicated that:①When E.coli DSM1116grew in Ag NP colloids for3h with100rpm shaking speed at37℃, the optimized cultivated condition was obtained. Raman signal at732cm-1of bacteria with1×108cells/mL concentration could be reached to the largest value of18000cps.②Compared to previous reported simply mixing method, the SERS reproducibility of bacteria was improved, and the Raman signal was raised to3.5fold.③The color of Ag NP colloids was obviously observed from yellow-green to grass-green. Meanwhile, a new broad absorbance band from500to700nm in UV-vis spectrum was shown. A close-linked structure seen in TEM image indicated that a new complex compound between Ag NPs and bacteria was supposed to form. According to the SERS assignment, the appearance of three bands at658,732and1330cm-1indicated a close adsorption between the Ag NPs and cell wall of bacteria.④Fifteen SERS spectra for three different E.coli strains (E. coli DSM498/1116/5695) were obtained, normalized and used for the establishment of the classified model using discriminant analysis (DA) method. The three E.coli strains could be successfully discriminated.⑤A single bacterium could be successfully spotted and detected by SERS method, with the same Raman shift of bacteria in aqueous solution, which provided experimental foundation for SERS mapping.⑥Different concentrations of bacteria were detected by SERS method with Raman mapping. The lowest concentration can be obtained at1×105cells/mL. The method can provide higher sensitity and reproduciblity of SERS spectra of E. coli compared to previous reported simply mixing method.(4) A rapid grasping bacteria chip based on label-free SERS was established. The normal glass slide was modified with diamino-PEG method to make the surface covered with diamino group. The diamino-PEG-glass slide was then protonized with HC1which could enable the glass slide with NH3+. As the bacteria possess negetive charge and the glass slide gives a positve one, the NH3+modified glass slide can trap and grasp bacteria due to the electrostatic adsorption principle. The modified glass slide was incubed with bacteria for half an hour followed by Ag NP colloids for another half an hour. Then bacteria were detected by SERS based on such chip. In addition, the feasibility of discriminating three different UT1bacteria was investigated using this method. The results indicated that:①The rapid grasping chip possessed positive charge can tightly adsorb negative-charged bacteria via electrostatic adsorption principle. When the chip was incubated with concentrated Ag NP colloid, good reproducible SERS spectra of bacteria can be obtained with recognizable bacteria characteristic peaks in5min.②Three kinds of bacteria (CFT, PSAE, and PRM1) extracted from urinary tract infection (UTI) can be discriminated using SERS and DA method. The rapid grasping bacteria chip based on SERS is easily prepared and disposed, which can provide a rapid portable detection method of bacteria.更多还原