高特异性碳纳米生物传感器快速检测沙门氏菌的研究
摘要
食源性病原菌是当今世界最为普遍的问题之一。微生物食品中毒可以由细菌、病毒、真菌以及原生生物引起,其中绝大部分的细菌性感染都是由沙门氏菌引起的。目前,由沙门氏菌所引发的禽蛋肉产品的污染以及食物中毒事件已成为制约国民经济发展、危害人类健康的严重食品安全问题。随着人们对食品安全问题关注度的不断增长,对发展快速、准确、灵敏、高效的食品安全检测技术提出了更高的要求。然而目前的传统检测方法存在耗时耗力、价格昂贵、操作繁琐、灵敏度不高、特异性不强等局限性,已不能满足沙门氏菌快速检测技术发展的新要求。因此,建立一种简单、高效、快速、灵敏的检测手段来监控沙门氏菌就显得尤为重要。碳纳米管基于其独特的化学、电学、光学、力学性质在生物传感器以及药物传递等方面有着非常广泛的应用前景。尤其是近些年来,碳纳米管作为一种广谱性的荧光猝灭剂已经越来越受到人们的关注,成为最具发展前景的纳米材料之一。本论文在对碳纳米管性质研究的基础上,结合分子探针构建了基于生物功能化的碳纳米管生物传感器对沙门氏菌进行检测；此外,一种类似于碳纳米管的碳材料—石墨烯也被应用于检测沙门氏菌中ATP的含量。结果显示,所构建的传感器具有灵敏度高、特异性强、稳定性好等优势。最后,鉴于沙门氏菌形成生物菌膜后难以清除和难以治疗的现状,本论文筛选出一种抑制型适配体用以控制沙门氏菌菌膜形成,从而拓展了适配体的应用范围。基于以上情况,具体研究内容如下：
(1)检测含毒力岛SSeC基因的鼠伤寒沙门氏菌。利用碳纳米管作为载体和荧光猝灭剂,分子信标作为识别元件,建立了一种全新的快速检测含毒力岛SSeC基因的鼠伤寒沙门氏菌的方法。在本检测方法中,羧基修饰的碳纳米管与一端氨基修饰的分子信标在EDC/NHS的作用下脱水结合形成生物功能化的碳纳米管。在没有靶标的情况下,由于DNA与碳纳米管的双重猝灭效应,荧光分子柔红霉素发生荧光猝灭；当加入靶标后,由于靶标与环状结构结合导致分子信标结构崩解,插入茎状结构中的柔红霉素释放出来,使得荧光值增强。而加入错配碱基序列,柔红霉素荧光值增强的信噪比远低于靶标。实验结果表明,通过检测柔红霉素荧光值的增强可以对含有SSeC基因的鼠伤寒沙门氏菌进行定量检测,而且此方法具有较高的灵敏性和特异性。在本检测体系中,靶标的浓度与柔红霉素荧光值的增强在0.2-0.7μM的范围内呈良好的线性关系,并且目标DNA的检测下限为50nM。
(2)检测甲型副伤寒沙门氏菌的研究。通过酸水解法提取甲型副伤寒的鞭毛蛋白,并以其为靶标分子,经过14轮的SELEX筛选,筛选出能够高亲和性、高特异性结合鞭毛蛋白的适配体群。利用DNAMAN软件分析其一级结构的相似性并进行二级结构的预测,根据二级结构的相似性以及一级结构的重复性,将所选的适配体分为4个家族。从4个家族中各挑选出1条适配体并对其进行亲和力测试,通过比较Kd值,最终筛选出一条最佳的适配体。将该适配体截去部分引物序列并修饰上FITC,同时在另一端结合上一段DNA酶序列,并将该探针非共价结合到碳纳米管上,从而设计出一条能够通过双重信号检测甲型副伤寒沙门氏菌的功能化核酸探针。实验结果表明,通过检测荧光值的增加以及吸光度的增加可以对牛奶样品中的甲型副伤寒沙门氏菌进行定量检测,在本检测体系中,用荧光信号以及吸光度所测出的最低检测下限分别为105CFU/mL和106CFU/mL。该方法同时利用两种信号来检测靶标物质,极大的提高了检测的准确性和可靠性,降低了假阳性信号产生的可能性。这一检测方法不仅可以用于检测甲型副伤寒沙门氏菌,也为其它细菌以及其它物质的快速检测提供了一种新的思路。
(3)基于氧化石墨烯的非标记性荧光适配体传感器快速检测ATP的研究。实验首先证明了该方法的可行性,结果表明,在只加入信号探针互补链的情况下,荧光信号并不会增强；只有在同时加入信号探针互补链和ATP的条件下荧光信号才会增强。由此证明,荧光信号的增强只与加入ATP的多少有关,因此该体系可以用来定量检测ATP。由检测结果可知,当ATP的浓度从1nM到800nM变化,对应的荧光强度随浓度的增加而增加,其检测下限可达1nM,而且F/FO在10nM到700nM之间呈良好的线性关系。体系还对检测ATP的特异性作出了探讨,将ATP的类似物UTP、GTP、CTP作为对照,由检测结果可知该适配体传感器对ATP分子的检测具有很强的特异性。相比于其它传统的检测方法,该分子识别技术对ATP的检测,不仅灵敏度高,特异性强,而且操作简单,成本低廉,稳定性好,为其它检测方法的开发和利用打下了坚实的基础。
(4)适配体控制细菌菌膜形成的研究。以猪霍乱沙门氏菌为靶标,经过14轮的SELEX筛选,筛选出能够特异性识别猪霍乱沙门氏菌的适配体群,并在第十轮和第十一轮分别用甲型副伤寒沙门氏菌和鼠伤寒沙门氏菌进行反筛,以提高筛选的特异性。经亲和力检测后,确定一条最佳的适配体,将该适配体生物素修饰后并结合链酶亲和素修饰磁珠用以垂钓猪霍乱沙门氏菌表面标记分子。经质谱鉴定后,该适配体特异性结合的靶标为鞭毛蛋白。由鞭毛介导的趋向运动和起始粘附在早期细菌生物膜的形成中至关重要,基于以上事实,本研究开发出能抑制鞭毛运动进而控制细菌生物膜形成的功能化适配体。实验结果表明,经抑制性实验、荧光倒置显微镜和原子力显微镜观察,该适配体能很好的抑制早期生物菌膜的形成,更重要的是,它能提高抗生素清除生物菌膜的效率。因此,该方法不仅拓展了适配体应用的范围,而且在减少抗生素的使用,降低耐药性菌株产生方面也有着重要的意义。
Pathogenicity aroused by food-borne pathogen is one of the most common problems in the world today. Microbial food poisoning can be caused by bacteria, viruses, fungi and protists, in which most of bacterial infections are caused by Salmonella. Currently, eggs'and meat products' contaminations and food poisoning resulted from Salmonella have become a serious food safety issues that posed the adverse influence on economic development and human health. With the increasing attention to food safety problem, higher requirements were proposed for the development of food safety detective techniques that are fast, accurate, sensitive and efficient. However, the traditional detection methods have many limitations, such as time-consuming, expensive, complicated operation, low sensitivity and specificity, which can not satisfy the new requirement of rapid detection of Salmonella. Therefore, it is particularly important to establish simple, efficient, rapid and sensitive detection methods for Salmonella supervision. Carbon nanotubes have many application prospects in biosensors, drug delivery and other fields because of their unique chemical, electrical, and mechanical properties. Especially in recent years, they, as a broad-spectrum fluorescent quencher, have been gained more and more attention, becoming one of the most promising nano-materials. In this paper, upon studying their properties, biosensors were constructed for Salmonella detection based on the bio-functionalized carbon nanotubes. In addition, graphene, another carbon material similar to them, had been also applied in the detection of ATP in Salmonella. It was indicated that these sensors have high sensitivity, specificity, stability and other advantages. Finally, given the status that the Salmonella's biofilm is difficult to remove and treat after its formation, an aptamer was well screened out for controlling the biofilm. Based on the above descriptions, the main contents list as follows:
(1) Detection of S. typhimurium carrying SSeC gene located in pathogenicity island. A novel method has been fabricated to detect the bacteria carrying SSeC gene by using carbon nanotubes as a carrier and fluorescent quencher, molecular beacon as recognition element. In this detection, amino modified molecular beacons were convalently coupled to carboxyl modified carbon nanotubes through EDC/Sulfo-NHS chemistry, generating bio-functionalized carbon nanotubes. In the absence of target, the fluorescence of daunorubicin was fairly week as result of dual fluorescence quenching. On the contrary, the daunorubicin was competed from the beacon due to the target-induced formation of rigid structure between the loop structure of the MB and the target sequence, which resulted in a decrease in the effect of dual fluorescence quenching, thereby the fluorescence intensity increased substantially. The quantity of the target was achieved by fluorescence increment. However, the signal to noise ratio was much lower than the target'when the mismatches were added. These results clearly demonstrated a high selectivity and specificity for the biosensor. The experimental results showed that the recovery of fluorescence of daunorubicin is proportion to the concentration of the target DNA with the range0.2-0.7μM and the low detection limit is50nM.
(2) The research for S. paratyphi A. In this work, the flagellin protein were obtained from S. paratyphi A by acid hydrolysis, and then single-stranded DNA aptamers that are highly specific to enterotoxigenic Salmonella paratyphi A were obtained from an enriched oligonucleotide pool using14rounds'SELEX to target the flagellin protein. DNAMAN software was used to analyze the similarity of their primary structure and secondary structure prediction, the selected aptamers can be divided into four families according to the similarity of secondary structure and repeatability of primary structure. By comparing the Kd values of typical aptamers picked out of the four families, an optimal aptamer was ultimately screened out. A probe containing the DNAzyme sequence and FITC-labeled truncated aptamer sequence wrapped onto carbon nanotubes was employed as a dual probe for S. paratyphi A detection.The results displayed that the milk spiked with S. paratyphi A could be quantified by fluorescence and absorbance increment, and the low detection limits increasing to105CFU/mL by fluorescence and106CFU/mL by spectrophotometry. The combination of fluorescence and spectrophotometry offers an accurate, reliable and non false-positive way for detecting Salmonella paratyphi A and has potential for detecting other pathogens and materials.
(3) Label-free fluorescent aptasensor based on a graphene oxide self-assembled probe for the determination of adenosine triphosphate. The feasibility of this approach was first demonstrated that the fluorescence intensity of probe/graphene oxide did not increase when the cDNA was mixed only, while the fluorescence enhancement occurred only under ATP present. Therefore, the enhancement of the fluorescence was only related to the number of added ATP, making it can be used to detect ATP quantitatively. The fluorescence intensity considerably increased with the increasing concentration of ATP from0to800nM, which enabled ATP to be detected in a linear range of10-700nM with a low limit of lnM. In addition, GTP, CTP and UTP, analogues of ATP, were selected to verify the selectivity of this approach. The results indicated that only ATP can induce substantial fluorescence enhancement rather than the analogues, which implied that this aptasensor could detect ATP with high specificity. Compared to other conventional detection methods, the molecular recognition technology used for ATP detection was not only high sensitivity and specificity, but also simple, low cost and good stability, which paved the way for the development and use of other detection methods.
(4) The study of controlling the biofilm formation by aptamer. Single-stranded DNA aptamers with high specificity to S. choleraesuis were obtained from an enriched oligonucleotide pool using14rounds'SELEX procedures. During the SELEX, S. paratyphi A and S. typhimurium were used as counter-selection targets at round10and11to improve its affinity. After the Kd was measured, an optimal aptamer was ultimately screened out, and the selected biotinylated aptamer-conjugated with streptavidin-modified magnetic beads, were then used to capture their binding targets on the bacteria. After mass spectrometry, the flagellin was ultimately identified as target captured by the aptamer. As motility and initial attachment mediated by flagella plays a critical role in biofilm formation, a functionalized aptamer was developed to control their movement, reducing the biofilm formation. Inhibition experiments, inverted microscope and atomic force microscope observation demonstrated that the selected aptamer was able to control the biofilm formation, more importantly, it could promote inhibitory effect of antibiotic against biofilm formation. Consequently, this method not only expanded the application of aptamer, but also reduced the use of antibiotics, which made it sense in decreasing drug resistance strains.
(1)检测含毒力岛SSeC基因的鼠伤寒沙门氏菌。利用碳纳米管作为载体和荧光猝灭剂,分子信标作为识别元件,建立了一种全新的快速检测含毒力岛SSeC基因的鼠伤寒沙门氏菌的方法。在本检测方法中,羧基修饰的碳纳米管与一端氨基修饰的分子信标在EDC/NHS的作用下脱水结合形成生物功能化的碳纳米管。在没有靶标的情况下,由于DNA与碳纳米管的双重猝灭效应,荧光分子柔红霉素发生荧光猝灭；当加入靶标后,由于靶标与环状结构结合导致分子信标结构崩解,插入茎状结构中的柔红霉素释放出来,使得荧光值增强。而加入错配碱基序列,柔红霉素荧光值增强的信噪比远低于靶标。实验结果表明,通过检测柔红霉素荧光值的增强可以对含有SSeC基因的鼠伤寒沙门氏菌进行定量检测,而且此方法具有较高的灵敏性和特异性。在本检测体系中,靶标的浓度与柔红霉素荧光值的增强在0.2-0.7μM的范围内呈良好的线性关系,并且目标DNA的检测下限为50nM。
(2)检测甲型副伤寒沙门氏菌的研究。通过酸水解法提取甲型副伤寒的鞭毛蛋白,并以其为靶标分子,经过14轮的SELEX筛选,筛选出能够高亲和性、高特异性结合鞭毛蛋白的适配体群。利用DNAMAN软件分析其一级结构的相似性并进行二级结构的预测,根据二级结构的相似性以及一级结构的重复性,将所选的适配体分为4个家族。从4个家族中各挑选出1条适配体并对其进行亲和力测试,通过比较Kd值,最终筛选出一条最佳的适配体。将该适配体截去部分引物序列并修饰上FITC,同时在另一端结合上一段DNA酶序列,并将该探针非共价结合到碳纳米管上,从而设计出一条能够通过双重信号检测甲型副伤寒沙门氏菌的功能化核酸探针。实验结果表明,通过检测荧光值的增加以及吸光度的增加可以对牛奶样品中的甲型副伤寒沙门氏菌进行定量检测,在本检测体系中,用荧光信号以及吸光度所测出的最低检测下限分别为105CFU/mL和106CFU/mL。该方法同时利用两种信号来检测靶标物质,极大的提高了检测的准确性和可靠性,降低了假阳性信号产生的可能性。这一检测方法不仅可以用于检测甲型副伤寒沙门氏菌,也为其它细菌以及其它物质的快速检测提供了一种新的思路。
(3)基于氧化石墨烯的非标记性荧光适配体传感器快速检测ATP的研究。实验首先证明了该方法的可行性,结果表明,在只加入信号探针互补链的情况下,荧光信号并不会增强；只有在同时加入信号探针互补链和ATP的条件下荧光信号才会增强。由此证明,荧光信号的增强只与加入ATP的多少有关,因此该体系可以用来定量检测ATP。由检测结果可知,当ATP的浓度从1nM到800nM变化,对应的荧光强度随浓度的增加而增加,其检测下限可达1nM,而且F/FO在10nM到700nM之间呈良好的线性关系。体系还对检测ATP的特异性作出了探讨,将ATP的类似物UTP、GTP、CTP作为对照,由检测结果可知该适配体传感器对ATP分子的检测具有很强的特异性。相比于其它传统的检测方法,该分子识别技术对ATP的检测,不仅灵敏度高,特异性强,而且操作简单,成本低廉,稳定性好,为其它检测方法的开发和利用打下了坚实的基础。
(4)适配体控制细菌菌膜形成的研究。以猪霍乱沙门氏菌为靶标,经过14轮的SELEX筛选,筛选出能够特异性识别猪霍乱沙门氏菌的适配体群,并在第十轮和第十一轮分别用甲型副伤寒沙门氏菌和鼠伤寒沙门氏菌进行反筛,以提高筛选的特异性。经亲和力检测后,确定一条最佳的适配体,将该适配体生物素修饰后并结合链酶亲和素修饰磁珠用以垂钓猪霍乱沙门氏菌表面标记分子。经质谱鉴定后,该适配体特异性结合的靶标为鞭毛蛋白。由鞭毛介导的趋向运动和起始粘附在早期细菌生物膜的形成中至关重要,基于以上事实,本研究开发出能抑制鞭毛运动进而控制细菌生物膜形成的功能化适配体。实验结果表明,经抑制性实验、荧光倒置显微镜和原子力显微镜观察,该适配体能很好的抑制早期生物菌膜的形成,更重要的是,它能提高抗生素清除生物菌膜的效率。因此,该方法不仅拓展了适配体应用的范围,而且在减少抗生素的使用,降低耐药性菌株产生方面也有着重要的意义。
Pathogenicity aroused by food-borne pathogen is one of the most common problems in the world today. Microbial food poisoning can be caused by bacteria, viruses, fungi and protists, in which most of bacterial infections are caused by Salmonella. Currently, eggs'and meat products' contaminations and food poisoning resulted from Salmonella have become a serious food safety issues that posed the adverse influence on economic development and human health. With the increasing attention to food safety problem, higher requirements were proposed for the development of food safety detective techniques that are fast, accurate, sensitive and efficient. However, the traditional detection methods have many limitations, such as time-consuming, expensive, complicated operation, low sensitivity and specificity, which can not satisfy the new requirement of rapid detection of Salmonella. Therefore, it is particularly important to establish simple, efficient, rapid and sensitive detection methods for Salmonella supervision. Carbon nanotubes have many application prospects in biosensors, drug delivery and other fields because of their unique chemical, electrical, and mechanical properties. Especially in recent years, they, as a broad-spectrum fluorescent quencher, have been gained more and more attention, becoming one of the most promising nano-materials. In this paper, upon studying their properties, biosensors were constructed for Salmonella detection based on the bio-functionalized carbon nanotubes. In addition, graphene, another carbon material similar to them, had been also applied in the detection of ATP in Salmonella. It was indicated that these sensors have high sensitivity, specificity, stability and other advantages. Finally, given the status that the Salmonella's biofilm is difficult to remove and treat after its formation, an aptamer was well screened out for controlling the biofilm. Based on the above descriptions, the main contents list as follows:
(1) Detection of S. typhimurium carrying SSeC gene located in pathogenicity island. A novel method has been fabricated to detect the bacteria carrying SSeC gene by using carbon nanotubes as a carrier and fluorescent quencher, molecular beacon as recognition element. In this detection, amino modified molecular beacons were convalently coupled to carboxyl modified carbon nanotubes through EDC/Sulfo-NHS chemistry, generating bio-functionalized carbon nanotubes. In the absence of target, the fluorescence of daunorubicin was fairly week as result of dual fluorescence quenching. On the contrary, the daunorubicin was competed from the beacon due to the target-induced formation of rigid structure between the loop structure of the MB and the target sequence, which resulted in a decrease in the effect of dual fluorescence quenching, thereby the fluorescence intensity increased substantially. The quantity of the target was achieved by fluorescence increment. However, the signal to noise ratio was much lower than the target'when the mismatches were added. These results clearly demonstrated a high selectivity and specificity for the biosensor. The experimental results showed that the recovery of fluorescence of daunorubicin is proportion to the concentration of the target DNA with the range0.2-0.7μM and the low detection limit is50nM.
(2) The research for S. paratyphi A. In this work, the flagellin protein were obtained from S. paratyphi A by acid hydrolysis, and then single-stranded DNA aptamers that are highly specific to enterotoxigenic Salmonella paratyphi A were obtained from an enriched oligonucleotide pool using14rounds'SELEX to target the flagellin protein. DNAMAN software was used to analyze the similarity of their primary structure and secondary structure prediction, the selected aptamers can be divided into four families according to the similarity of secondary structure and repeatability of primary structure. By comparing the Kd values of typical aptamers picked out of the four families, an optimal aptamer was ultimately screened out. A probe containing the DNAzyme sequence and FITC-labeled truncated aptamer sequence wrapped onto carbon nanotubes was employed as a dual probe for S. paratyphi A detection.The results displayed that the milk spiked with S. paratyphi A could be quantified by fluorescence and absorbance increment, and the low detection limits increasing to105CFU/mL by fluorescence and106CFU/mL by spectrophotometry. The combination of fluorescence and spectrophotometry offers an accurate, reliable and non false-positive way for detecting Salmonella paratyphi A and has potential for detecting other pathogens and materials.
(3) Label-free fluorescent aptasensor based on a graphene oxide self-assembled probe for the determination of adenosine triphosphate. The feasibility of this approach was first demonstrated that the fluorescence intensity of probe/graphene oxide did not increase when the cDNA was mixed only, while the fluorescence enhancement occurred only under ATP present. Therefore, the enhancement of the fluorescence was only related to the number of added ATP, making it can be used to detect ATP quantitatively. The fluorescence intensity considerably increased with the increasing concentration of ATP from0to800nM, which enabled ATP to be detected in a linear range of10-700nM with a low limit of lnM. In addition, GTP, CTP and UTP, analogues of ATP, were selected to verify the selectivity of this approach. The results indicated that only ATP can induce substantial fluorescence enhancement rather than the analogues, which implied that this aptasensor could detect ATP with high specificity. Compared to other conventional detection methods, the molecular recognition technology used for ATP detection was not only high sensitivity and specificity, but also simple, low cost and good stability, which paved the way for the development and use of other detection methods.
(4) The study of controlling the biofilm formation by aptamer. Single-stranded DNA aptamers with high specificity to S. choleraesuis were obtained from an enriched oligonucleotide pool using14rounds'SELEX procedures. During the SELEX, S. paratyphi A and S. typhimurium were used as counter-selection targets at round10and11to improve its affinity. After the Kd was measured, an optimal aptamer was ultimately screened out, and the selected biotinylated aptamer-conjugated with streptavidin-modified magnetic beads, were then used to capture their binding targets on the bacteria. After mass spectrometry, the flagellin was ultimately identified as target captured by the aptamer. As motility and initial attachment mediated by flagella plays a critical role in biofilm formation, a functionalized aptamer was developed to control their movement, reducing the biofilm formation. Inhibition experiments, inverted microscope and atomic force microscope observation demonstrated that the selected aptamer was able to control the biofilm formation, more importantly, it could promote inhibitory effect of antibiotic against biofilm formation. Consequently, this method not only expanded the application of aptamer, but also reduced the use of antibiotics, which made it sense in decreasing drug resistance strains.
引文
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