生物功能化粒子/核酸探针技术快速检测病原菌及ATP的研究
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摘要
病原菌引起的感染性疾病是近年威胁人类健康和造成社会恐慌的主要因素,同时也是造成人类死亡的重要原因。检测任务的紧迫性与检测样品的复杂性给传统病原菌检测方法提出了严峻的挑战,而传统的检测方法由于在特异性、灵敏度、操作简便性、快捷性等方面存在缺陷,难以满足日益发展的病原菌快速检测的需要。因此发展病原菌的快速、灵敏和准确的检测方法具有十分重要的意义。纳米材料有着独特的表面效应、小尺寸效应、量子尺寸效应和宏观隧道效应,在其表面进行各种修饰获得的各种功能化纳米材料与传统检测方法相结合,衍生出具有高通量、高灵敏、快速检测的方法；核酸探针技术是目前分子生物学中应用最广泛的技术之一,是定性或定量检测特异RNA或DNA序列的有力工具。本论文以三种纳米粒子和核酸探针技术为基础,结合新型的检测工具,发展了新的灵敏度高、特异性好的病原菌及ATP分子检测技术。本论文主要包括以下几个研究内容：
(1)构建了基于免疫磁珠(Immunomolecular magnetic beads, IMBs)/表面等离子共振技术(surface plasmon resonance, SPR)的G群链球菌(Group G Streptococcus, GGS)快速分离检测体系。首先通过磁珠(magnetic beads, MBs)表面的不同功能基团与抗体分子共价结合得到三种功能化IMBs.实验考察了三种MBs与抗体的结合能力,结果表明三种磁珠均能很好地结合抗体分子,醛基化的磁珠结合能力最佳；实验还针对免疫磁珠分离链球菌的效果进行了考察,结果也表明三种免疫磁珠都能特异地分离样品中GGS,分离效果亦以醛基化的免疫磁珠效果最好。为了对IMBs分离的GGS进行定量分析,本研究联合了SPR技术。基于抗体分子与GGS的相互作用,在数十分钟内可以对菌浓度处于1.0×10'CFU/ml-1.0×108 CFU/ml范围内的GGS进行实时快速检测。与其它传统分离方法相比,本研究所采取的分离方法要简便和快速得多,能在一小时之内快速地分离得到目标菌。
(2)基于核酸适配体-ATP复合物与绿色荧光染料Eva GreenTM作用,建立了荧光信号检测腺嘌呤核苷三磷酸(adenosine-triphosphate, ATP)新方法。体系优化了核酸适配体与ATP结合反应的实验条件。实验结果表明,核酸适配体与ATP反应的最适温度为12℃；pH值为7.5时,荧光检测结果最灵敏；在最优化的实验条件下,随着ATP浓度的减小,所测得的荧光信号与空白对照的变化值变小。由检测结果可知,当ATP浓度从10-6mol/L到10_2mol/L变化,对应的荧光强度随着浓度的减小而增大,呈现一定的线性关系。因此可作为该范围浓度的ATP定量检测的依据。体系还对该方法的特异性作出了探讨,将ATP同类型分子CTP、GTP、UTP作为对照,根据检测出的结果可知该核酸适配体对与ATP分子的检测具有较强的特异性。相较于其他的检测方法,本研究所建立的基于核酸适配体-ATP特异识别的荧光检测技术,对于ATP分子的检测,不仅反应体系特异性高,并且实验操作简单,耗时少,成本低。可见,一本研究为之后的分子识别和检测提供了更为坚实的基础。
(3)利用量子点(QD)与金黄色葡萄球菌特异性序列制备了一种功能化QD-DNA探针,基于此探针荧光能量共振转移,建立了一种用于高效检测金黄色葡萄球菌16s rDNA的方法。本研究详细研究了氨基修饰的DNA与羧基修饰的QD不同比值、不同靶序列浓度等条件对荧光强度与荧光能量共振转移效率的影响,实验结果表明,荧光强度在羧基修饰的QD与氨基修饰的DNA比值为1：80时达到最大值,荧光能量共振转移效率在靶序列为60nM时趋向于稳定并在80nM时效率最高。在最佳实验条件下,检测系统最低可检测至20nM的金黄色葡萄球菌16s rDNA序列,其灵敏度已达到荧光标记的分子信标、DNA传感器、电化学线性探针等方法检测下限。实验结果亦证实该方法具有较强特异性。总之,此方法具有操作简单、耗时较短、灵敏度及特异性较强等特点,为金黄色葡萄球菌的检测开辟了一条新的道路。
(4)以核酸适配子与配体的特异性识别为基础,以金纳米粒子溶液在不同状态下颜色的变化为表现方式,建立了一种金黄色葡萄球菌快速简捷的、肉眼可见的检测方法。采用氯金酸柠檬酸三钠水相还原法,制备出粒径均一,分散性较好的GNPs。将金黄色葡萄球菌的ssDNA适配子经巯基修饰后与GNPs孵育可得其检测探针并成功地用此探针进行了快速检测。
Diseases caused by pathogen infection are the major problems that threaten human health and lead to the recent social panic and the major cause of human death. The complexity of sample and the urgency of pathogenic bacteria posed a severe challenge for the traditional detection methods. But the traditional analytical tools and methods are difficult to meet the growing demands of rapid detection in the deficiencies of the sensitivity, specificity, simple operation, and rapidity. Therefore, it is very significant to develop a rapid, sensitive and accurate detection method towards pathogenic bacteria. Functional nanomaterials are well-suited for a wide range of biological applications because of their unique physical and chemical properties. As one of the most widely used molecular biology technologies, gene probe technology is a powerful tool for the qualitative or quantitative detection of specific RNA or DNA sequences. A new high sensitive and specifical detection method for pathogenic bacteria and their biomolecules was well constructed based on the nano-materials and gene probe technology in this study.
This paper includes the following four reseaches:
(1) Three functional immunomagnetic beads (IMBs) were constructed through the different functional groups on the surfaces of magnetic beads (MBs) covalently binding to antibodies, respectively. Their binding properties to antibody were also analysed. The results showed that all of the beads have high affinity, and ones with aldehyde groups are best among them. The effect of Group G Streptococcus (GGS) seperated by IMB was discussed. It was demonstrated that the three kinds of IMBs could separate GGS from samples, and the separation effects of IMBs with aldehydes are actually best. Continually, SPR technology based on the interaction of antibody and streptococci was used in order to quantitatively analyze GGS. It can successfully detect Strep, rapidly and its detected limit is 1.0x107CFU/ml. This method is more simple and rapid compared with traditional means.
(2) A simple, efficient fluorescence detection technology for ATP was developed. Using DNA-intercalating dye Eva GreenTM to react with aptamer-ATP complex, the method can be quickly used to detect ATP based on its significant fluorescence signal changes. Some affection on the detection has been discussed, such as temperature, concentration and reaction time. It was shown that the best temperature and pH were 12℃and pH7.5, respectivily. The detection limitation of ATP could be 10-6 M under the optimized conditions.
(3) Fluorescence resonance energy transfer (FRET) system has been utilized in order to find a fast and brief detection method for Staphyloccocus aureus, in which quantum dot was used as donors and organic fluorophore dyes as acceptors to inspect S. aureus specific 16s RNA. The result showed that the best fluorescent effect can been achieved when the ratio of carboxyl-modif ied QD to the amino-modified DNA is 1:80. The FRET efficiency would be steady when target DNA is 60nm and reach to its peak when target DNA is 80nm. Furthermore, this developed system is so sensitive that it can detect 20nm 16s rDNA. This work clearly indicated that FRET system is more sensitive and specific than other detect methods for S. aureus, in which quantum dot as.donors and organic fluorophore dyes as acceptors. Of course, it is possible to detect other bio-macromolecules with low density such as RNA and proteins in future.
(4) A rapid, visible detection method for S. aureus was established based on the specific identification between aptamer and ligand. The results were displayed through the color changes of gold nanoparticles (GNPs) in different conditions. GNPs with uniform size and good dispersion were synthesized with chemical reduction method. The probe for S. aureus detection has been constructed based on the combination of sulphur-modified aptamer and GNPs and the batctera has been successfully detected.
(1)构建了基于免疫磁珠(Immunomolecular magnetic beads, IMBs)/表面等离子共振技术(surface plasmon resonance, SPR)的G群链球菌(Group G Streptococcus, GGS)快速分离检测体系。首先通过磁珠(magnetic beads, MBs)表面的不同功能基团与抗体分子共价结合得到三种功能化IMBs.实验考察了三种MBs与抗体的结合能力,结果表明三种磁珠均能很好地结合抗体分子,醛基化的磁珠结合能力最佳；实验还针对免疫磁珠分离链球菌的效果进行了考察,结果也表明三种免疫磁珠都能特异地分离样品中GGS,分离效果亦以醛基化的免疫磁珠效果最好。为了对IMBs分离的GGS进行定量分析,本研究联合了SPR技术。基于抗体分子与GGS的相互作用,在数十分钟内可以对菌浓度处于1.0×10'CFU/ml-1.0×108 CFU/ml范围内的GGS进行实时快速检测。与其它传统分离方法相比,本研究所采取的分离方法要简便和快速得多,能在一小时之内快速地分离得到目标菌。
(2)基于核酸适配体-ATP复合物与绿色荧光染料Eva GreenTM作用,建立了荧光信号检测腺嘌呤核苷三磷酸(adenosine-triphosphate, ATP)新方法。体系优化了核酸适配体与ATP结合反应的实验条件。实验结果表明,核酸适配体与ATP反应的最适温度为12℃；pH值为7.5时,荧光检测结果最灵敏；在最优化的实验条件下,随着ATP浓度的减小,所测得的荧光信号与空白对照的变化值变小。由检测结果可知,当ATP浓度从10-6mol/L到10_2mol/L变化,对应的荧光强度随着浓度的减小而增大,呈现一定的线性关系。因此可作为该范围浓度的ATP定量检测的依据。体系还对该方法的特异性作出了探讨,将ATP同类型分子CTP、GTP、UTP作为对照,根据检测出的结果可知该核酸适配体对与ATP分子的检测具有较强的特异性。相较于其他的检测方法,本研究所建立的基于核酸适配体-ATP特异识别的荧光检测技术,对于ATP分子的检测,不仅反应体系特异性高,并且实验操作简单,耗时少,成本低。可见,一本研究为之后的分子识别和检测提供了更为坚实的基础。
(3)利用量子点(QD)与金黄色葡萄球菌特异性序列制备了一种功能化QD-DNA探针,基于此探针荧光能量共振转移,建立了一种用于高效检测金黄色葡萄球菌16s rDNA的方法。本研究详细研究了氨基修饰的DNA与羧基修饰的QD不同比值、不同靶序列浓度等条件对荧光强度与荧光能量共振转移效率的影响,实验结果表明,荧光强度在羧基修饰的QD与氨基修饰的DNA比值为1：80时达到最大值,荧光能量共振转移效率在靶序列为60nM时趋向于稳定并在80nM时效率最高。在最佳实验条件下,检测系统最低可检测至20nM的金黄色葡萄球菌16s rDNA序列,其灵敏度已达到荧光标记的分子信标、DNA传感器、电化学线性探针等方法检测下限。实验结果亦证实该方法具有较强特异性。总之,此方法具有操作简单、耗时较短、灵敏度及特异性较强等特点,为金黄色葡萄球菌的检测开辟了一条新的道路。
(4)以核酸适配子与配体的特异性识别为基础,以金纳米粒子溶液在不同状态下颜色的变化为表现方式,建立了一种金黄色葡萄球菌快速简捷的、肉眼可见的检测方法。采用氯金酸柠檬酸三钠水相还原法,制备出粒径均一,分散性较好的GNPs。将金黄色葡萄球菌的ssDNA适配子经巯基修饰后与GNPs孵育可得其检测探针并成功地用此探针进行了快速检测。
Diseases caused by pathogen infection are the major problems that threaten human health and lead to the recent social panic and the major cause of human death. The complexity of sample and the urgency of pathogenic bacteria posed a severe challenge for the traditional detection methods. But the traditional analytical tools and methods are difficult to meet the growing demands of rapid detection in the deficiencies of the sensitivity, specificity, simple operation, and rapidity. Therefore, it is very significant to develop a rapid, sensitive and accurate detection method towards pathogenic bacteria. Functional nanomaterials are well-suited for a wide range of biological applications because of their unique physical and chemical properties. As one of the most widely used molecular biology technologies, gene probe technology is a powerful tool for the qualitative or quantitative detection of specific RNA or DNA sequences. A new high sensitive and specifical detection method for pathogenic bacteria and their biomolecules was well constructed based on the nano-materials and gene probe technology in this study.
This paper includes the following four reseaches:
(1) Three functional immunomagnetic beads (IMBs) were constructed through the different functional groups on the surfaces of magnetic beads (MBs) covalently binding to antibodies, respectively. Their binding properties to antibody were also analysed. The results showed that all of the beads have high affinity, and ones with aldehyde groups are best among them. The effect of Group G Streptococcus (GGS) seperated by IMB was discussed. It was demonstrated that the three kinds of IMBs could separate GGS from samples, and the separation effects of IMBs with aldehydes are actually best. Continually, SPR technology based on the interaction of antibody and streptococci was used in order to quantitatively analyze GGS. It can successfully detect Strep, rapidly and its detected limit is 1.0x107CFU/ml. This method is more simple and rapid compared with traditional means.
(2) A simple, efficient fluorescence detection technology for ATP was developed. Using DNA-intercalating dye Eva GreenTM to react with aptamer-ATP complex, the method can be quickly used to detect ATP based on its significant fluorescence signal changes. Some affection on the detection has been discussed, such as temperature, concentration and reaction time. It was shown that the best temperature and pH were 12℃and pH7.5, respectivily. The detection limitation of ATP could be 10-6 M under the optimized conditions.
(3) Fluorescence resonance energy transfer (FRET) system has been utilized in order to find a fast and brief detection method for Staphyloccocus aureus, in which quantum dot was used as donors and organic fluorophore dyes as acceptors to inspect S. aureus specific 16s RNA. The result showed that the best fluorescent effect can been achieved when the ratio of carboxyl-modif ied QD to the amino-modified DNA is 1:80. The FRET efficiency would be steady when target DNA is 60nm and reach to its peak when target DNA is 80nm. Furthermore, this developed system is so sensitive that it can detect 20nm 16s rDNA. This work clearly indicated that FRET system is more sensitive and specific than other detect methods for S. aureus, in which quantum dot as.donors and organic fluorophore dyes as acceptors. Of course, it is possible to detect other bio-macromolecules with low density such as RNA and proteins in future.
(4) A rapid, visible detection method for S. aureus was established based on the specific identification between aptamer and ligand. The results were displayed through the color changes of gold nanoparticles (GNPs) in different conditions. GNPs with uniform size and good dispersion were synthesized with chemical reduction method. The probe for S. aureus detection has been constructed based on the combination of sulphur-modified aptamer and GNPs and the batctera has been successfully detected.
引文
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