测定B型葡萄球菌肠毒素DNA的压电生物传感器研究
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摘要
二十一世纪是生命科学的世纪。生物技术中的检测和控制及样品分析，都要借助于生物传感技术获取所需的信息。在生物传感器中，压电生物传感器(piezoelectric biosensors，PEBS)以其不需要任何标记、仪器简单、操作方便且性能稳定的优点，引起了人们浓厚的兴趣，成为生物传感器领域的研究热点之一。该类传感器利用压电石英谐振器对质量的敏感性，通过谐振器表面吸附或结合待测物后自身参数的变化实现对目标物的检测。
金黄色葡萄球菌(简称金葡菌，Staphylococcus aureus)是常见的食物中毒和医院污染的主要病原菌之一，金葡菌能产生多种体外毒性蛋白，其中最重要的是葡萄球菌肠毒素(staphylococcal enterotoxins，SES)。金葡菌肠毒素是一种可溶性耐高热蛋白，是引起食物中毒频率较高的毒素，也是生物战剂中的主要毒素。其中B型葡萄球菌肠毒素(SEB)是一种通常条件下更稳定，毒性最强的毒素，而核酸杂交技术则是分子生物学和基因工程中最常用和最基本的方法之一，因此本论文以该毒素的产毒基因为检测对象，以压电石英晶体为敏感元件，以合成的寡核苷酸探针为识别分子，构建了用于SEB基因检测的压电生物传感器。论文主要介绍了压电生物传感器与葡萄球菌肠毒素检测研究进展，同时重点介绍了体波式(QCM)和声表面波式(SAW)两种用于SEB DNA检测的压电生物传感器原理、制作方法并对其检测结果作了报告。另外，在论文的第二部分，主要对Ce(Ⅳ)在硫酸介质中氧化罗丹明B(RhB)化学发光行为进行了探讨。
第一部分 压电生物传感器与葡萄球菌肠毒素检测研究
第一章 压电生物传感器与葡萄球菌肠毒素检测研究进展
第一节 压电生物传感器研究进展
文中系统的评述了近年来压电生物传感器研究进展，其中包括压电生物传感器的基本原理和构成、压电生物传感器的分类、压电生物传感器表面修饰技术以及压电生物传感器在免疫检测、基因检测、微生物检测、与其它技术联用等方面的应用研究，同时对压电生物传感器的发展趋势做了评述。引用参考文献69篇。
第二节 金黄色葡萄球菌及其毒素检测研究进展
该部分内容主要介绍了在葡萄球菌分类与分型、金黄色葡萄球菌毒素学研究以及金黄色葡萄球菌及其毒素检测等方面的研究进展，重点对金黄色葡萄球菌及其毒素检测方法：生物学检测方法、免疫血清学方法、聚合酶链反应技术、核酸杂交技术、生物传感器技术和超抗原技术在近几年的研究进展做了评述，引用文献69篇。
火n Xia即。馆:Research onP滋oeleetr纪Biosensorsforlhe Detection ofD入月Encoding StaPhyloeoceal EnterotoxinB
第二章S戌W压电生物传感器测定B型葡萄球菌DNA的研究
本章建立了一种用于B型葡萄球菌肠毒素检测的SAW压电生物传感器。该传感器
采用以ST一切割的石英晶片为基体，以510:为波传导层的乐甫波(Love waves)装置，
利用具有分子识别功能的DNA合成探针来实现对样品中SEB DNA的检测。实验过程
中对石英晶体表面进行了硅烷化修饰，并采用Avidin一Biodin体系实现了DNA探针的
固定。实验证明该生物传感器不仅能够测定含单一SEB DNA的样品，而且对含其它葡
萄球菌肠毒素DNA的干扰样品也能准确响应，不存在干扰信号。其最低检测限为
75n留ml，线性范围是100~3 50ng/n 11。该传感器经变性再生能够重复使用3次以上，4℃
条件下能够保存3周保持活性不降低，用于实际含菌样品测试结果良好。
第三章QcM压电生物传感器用于B型葡萄球菌肠毒素DNA检测
本文确立了一种新的检测SEB DNA的方法，该方法以石英晶体微天平作为质量敏
感元件，以合成的特异性DNA探针为分子识别元件构建了适合于SEB DNA检测的
QcM压电生物传感器。探讨了作为电极的金膜表面疏基自组装修饰以及DNA探针的固
定方法，实验证明该传感器能够满足实际含SEB DNA样品的测定，对含其它葡萄球菌
肠毒素DNA的干扰样品也能准确响应且不存在千扰信号。其最低检测限为6 .ong/ml，
线性范围是10一15Ong/mh该传感器再生后能够重复使用3、次以上。
第二部分Ce(I均在硫酸介质中氧化RhB化学发光行为研究
本文对RhB一Ce(IV)化学发光体系在硫酸介质中的化学发光行为进行了研究。实验
借助流动注射化学发光仪，确立了在酸性条件下ce(Iv)氧化R五B的最佳反应条件，并
得出了该反应的动力学曲线;.同时利用荧光仪、紫外一可见分光光度计和红外光谱仪对
反应前后以及反应中反应物和生成物进行了光谱学研究，得出了较为合理的发光反应机
理。实验结果证明，Ce(IV)氧化RllB产生的化学发光是由反应过程中生成激发态的RhB
氧化产物所产生的，其发光波长在425nm左右。该研究结果确立了一种新的适合酸性条
件下的化学发光试剂，对大多数在酸性条件下进行氧化还原反应的物质实现化学发光分
析，具有重要的参考价值。
The 21th Century is the critical development period of life-sciences. The detection control and the analysis of samples in bio-techniques ultimately depend on the information from biosensor technique. Considering the sensitivity of piezoelectric quartz oscillator to quality, the piezoelectric biosensors (PEBS) that are based on the changes of oscillator's parameters due to the surface absorption associated with contaminants, have been developed. Nowadays, PEBS has become a significant research field since the technique shows the advantages of stable capability, simplicity in instrument construction and operation, and no label to be needed.
Staphylococcus aureus (S. aureus) is a major cause of bacterial food poisoning.
Staphylococcal food poisoning is resulted from the ingestion of enterotoxins preformed in
food by certain strains of S. aureus. Staphylococcal enterotoxins (SES) are categorized to a family of seven major serological types of emetic enterotoxins with heat stability. Because of its incredible toxicity and stability, Staphylococcal enterotoxin B (SEB) is considered as a potential threat as a biological weapon of mass destruction. Therefore, the determination of SEB is very important for food hygienic analysis as well as for clinical analysis. Nucleic acid hybridization technique is one of the widely-used methods in molecular biology and gene technology. The present work has developed piezoelectric biosensors used in the detection of SEB DNA by tacking the piezoelectric quarts crystal as a sensitive component while synthetic oligonucleotide probe as recognize molecule. The paper mainly introduces the development of PEBS and SES detection, and focus on the principle, constitutes and detection results of SEB DNA by using the quartz crystal microbalance (QCM) and surface acoustic waves (SAW)
piezoelectric biosensors respectively. In addition, the Chemilurm'nescence (CL) behavior at a
system of Ce(IV)-rhodamine B in sulfuric acid has also been discussed in Part Two of the
paper.
Part one The studies of piezoelectric biosensors and detection of Staphylococcal
enterotoxins
Chapter one The progresses in piezoelectric biosensors and detection of
Staphylococcal enterotoxins
Section One The progresses in piezoelectric biosensors
In this section, a review is reported with 69 references about the piezoelectric biosensors, including the principle and constitutes of PEBS, the classification of PEBS, surface modification technique in PEBS and the application of PEBS in immunoassay, detection of the gene and microorganism, coupling with other techniques, moreover, the development of PEBS was involved.
Section Two The progresses in detections of Staphylococcus aureus and staphylococcal enterotoxins
In this section, the author introduced classification, typology and toxicological study of Staphylococcus aureus in addition to relevant determination methods including PCR technique, nucleic acid hybridization technique, biosensor technique, superantigen technique. Chapter Two Detecting the gene of staphylococcal enterotoxin B using a surface acoustic wave biosensor
A kind of SAW biosensor for detecting the gene of staphylococcal enterotoxin B was developed. The Love wave device with the substrate of ST-cut quartz and the guiding layer of Si02 was used as the SAW biosensor. DNA sensor employing synthetic SEB DNA probe as a molecular recognizing component was utilized to detect SEB by measuring SEB DNA in samples. It demonstrated that biosensor used for measurement of SEB DNA, had a possible usage not only for the simple SEB DNA samples but also for the complex samples in the presence of extra enterotoxins' DNA that could not evoke a positive response. The assay sensitivity was 75ng/ml and the linear ranger was 100~350ng/ml. The fabricated DNA sensors were applied to detect a set of real samples.
Chapter Three Detection of the gene of staphylococcal enterotoxin B employing a quartz crystal microbalance biosensor
In this work, a new method t
金黄色葡萄球菌(简称金葡菌，Staphylococcus aureus)是常见的食物中毒和医院污染的主要病原菌之一，金葡菌能产生多种体外毒性蛋白，其中最重要的是葡萄球菌肠毒素(staphylococcal enterotoxins，SES)。金葡菌肠毒素是一种可溶性耐高热蛋白，是引起食物中毒频率较高的毒素，也是生物战剂中的主要毒素。其中B型葡萄球菌肠毒素(SEB)是一种通常条件下更稳定，毒性最强的毒素，而核酸杂交技术则是分子生物学和基因工程中最常用和最基本的方法之一，因此本论文以该毒素的产毒基因为检测对象，以压电石英晶体为敏感元件，以合成的寡核苷酸探针为识别分子，构建了用于SEB基因检测的压电生物传感器。论文主要介绍了压电生物传感器与葡萄球菌肠毒素检测研究进展，同时重点介绍了体波式(QCM)和声表面波式(SAW)两种用于SEB DNA检测的压电生物传感器原理、制作方法并对其检测结果作了报告。另外，在论文的第二部分，主要对Ce(Ⅳ)在硫酸介质中氧化罗丹明B(RhB)化学发光行为进行了探讨。
第一部分 压电生物传感器与葡萄球菌肠毒素检测研究
第一章 压电生物传感器与葡萄球菌肠毒素检测研究进展
第一节 压电生物传感器研究进展
文中系统的评述了近年来压电生物传感器研究进展，其中包括压电生物传感器的基本原理和构成、压电生物传感器的分类、压电生物传感器表面修饰技术以及压电生物传感器在免疫检测、基因检测、微生物检测、与其它技术联用等方面的应用研究，同时对压电生物传感器的发展趋势做了评述。引用参考文献69篇。
第二节 金黄色葡萄球菌及其毒素检测研究进展
该部分内容主要介绍了在葡萄球菌分类与分型、金黄色葡萄球菌毒素学研究以及金黄色葡萄球菌及其毒素检测等方面的研究进展，重点对金黄色葡萄球菌及其毒素检测方法：生物学检测方法、免疫血清学方法、聚合酶链反应技术、核酸杂交技术、生物传感器技术和超抗原技术在近几年的研究进展做了评述，引用文献69篇。
火n Xia即。馆:Research onP滋oeleetr纪Biosensorsforlhe Detection ofD入月Encoding StaPhyloeoceal EnterotoxinB
第二章S戌W压电生物传感器测定B型葡萄球菌DNA的研究
本章建立了一种用于B型葡萄球菌肠毒素检测的SAW压电生物传感器。该传感器
采用以ST一切割的石英晶片为基体，以510:为波传导层的乐甫波(Love waves)装置，
利用具有分子识别功能的DNA合成探针来实现对样品中SEB DNA的检测。实验过程
中对石英晶体表面进行了硅烷化修饰，并采用Avidin一Biodin体系实现了DNA探针的
固定。实验证明该生物传感器不仅能够测定含单一SEB DNA的样品，而且对含其它葡
萄球菌肠毒素DNA的干扰样品也能准确响应，不存在干扰信号。其最低检测限为
75n留ml，线性范围是100~3 50ng/n 11。该传感器经变性再生能够重复使用3次以上，4℃
条件下能够保存3周保持活性不降低，用于实际含菌样品测试结果良好。
第三章QcM压电生物传感器用于B型葡萄球菌肠毒素DNA检测
本文确立了一种新的检测SEB DNA的方法，该方法以石英晶体微天平作为质量敏
感元件，以合成的特异性DNA探针为分子识别元件构建了适合于SEB DNA检测的
QcM压电生物传感器。探讨了作为电极的金膜表面疏基自组装修饰以及DNA探针的固
定方法，实验证明该传感器能够满足实际含SEB DNA样品的测定，对含其它葡萄球菌
肠毒素DNA的干扰样品也能准确响应且不存在千扰信号。其最低检测限为6 .ong/ml，
线性范围是10一15Ong/mh该传感器再生后能够重复使用3、次以上。
第二部分Ce(I均在硫酸介质中氧化RhB化学发光行为研究
本文对RhB一Ce(IV)化学发光体系在硫酸介质中的化学发光行为进行了研究。实验
借助流动注射化学发光仪，确立了在酸性条件下ce(Iv)氧化R五B的最佳反应条件，并
得出了该反应的动力学曲线;.同时利用荧光仪、紫外一可见分光光度计和红外光谱仪对
反应前后以及反应中反应物和生成物进行了光谱学研究，得出了较为合理的发光反应机
理。实验结果证明，Ce(IV)氧化RllB产生的化学发光是由反应过程中生成激发态的RhB
氧化产物所产生的，其发光波长在425nm左右。该研究结果确立了一种新的适合酸性条
件下的化学发光试剂，对大多数在酸性条件下进行氧化还原反应的物质实现化学发光分
析，具有重要的参考价值。
The 21th Century is the critical development period of life-sciences. The detection control and the analysis of samples in bio-techniques ultimately depend on the information from biosensor technique. Considering the sensitivity of piezoelectric quartz oscillator to quality, the piezoelectric biosensors (PEBS) that are based on the changes of oscillator's parameters due to the surface absorption associated with contaminants, have been developed. Nowadays, PEBS has become a significant research field since the technique shows the advantages of stable capability, simplicity in instrument construction and operation, and no label to be needed.
Staphylococcus aureus (S. aureus) is a major cause of bacterial food poisoning.
Staphylococcal food poisoning is resulted from the ingestion of enterotoxins preformed in
food by certain strains of S. aureus. Staphylococcal enterotoxins (SES) are categorized to a family of seven major serological types of emetic enterotoxins with heat stability. Because of its incredible toxicity and stability, Staphylococcal enterotoxin B (SEB) is considered as a potential threat as a biological weapon of mass destruction. Therefore, the determination of SEB is very important for food hygienic analysis as well as for clinical analysis. Nucleic acid hybridization technique is one of the widely-used methods in molecular biology and gene technology. The present work has developed piezoelectric biosensors used in the detection of SEB DNA by tacking the piezoelectric quarts crystal as a sensitive component while synthetic oligonucleotide probe as recognize molecule. The paper mainly introduces the development of PEBS and SES detection, and focus on the principle, constitutes and detection results of SEB DNA by using the quartz crystal microbalance (QCM) and surface acoustic waves (SAW)
piezoelectric biosensors respectively. In addition, the Chemilurm'nescence (CL) behavior at a
system of Ce(IV)-rhodamine B in sulfuric acid has also been discussed in Part Two of the
paper.
Part one The studies of piezoelectric biosensors and detection of Staphylococcal
enterotoxins
Chapter one The progresses in piezoelectric biosensors and detection of
Staphylococcal enterotoxins
Section One The progresses in piezoelectric biosensors
In this section, a review is reported with 69 references about the piezoelectric biosensors, including the principle and constitutes of PEBS, the classification of PEBS, surface modification technique in PEBS and the application of PEBS in immunoassay, detection of the gene and microorganism, coupling with other techniques, moreover, the development of PEBS was involved.
Section Two The progresses in detections of Staphylococcus aureus and staphylococcal enterotoxins
In this section, the author introduced classification, typology and toxicological study of Staphylococcus aureus in addition to relevant determination methods including PCR technique, nucleic acid hybridization technique, biosensor technique, superantigen technique. Chapter Two Detecting the gene of staphylococcal enterotoxin B using a surface acoustic wave biosensor
A kind of SAW biosensor for detecting the gene of staphylococcal enterotoxin B was developed. The Love wave device with the substrate of ST-cut quartz and the guiding layer of Si02 was used as the SAW biosensor. DNA sensor employing synthetic SEB DNA probe as a molecular recognizing component was utilized to detect SEB by measuring SEB DNA in samples. It demonstrated that biosensor used for measurement of SEB DNA, had a possible usage not only for the simple SEB DNA samples but also for the complex samples in the presence of extra enterotoxins' DNA that could not evoke a positive response. The assay sensitivity was 75ng/ml and the linear ranger was 100~350ng/ml. The fabricated DNA sensors were applied to detect a set of real samples.
Chapter Three Detection of the gene of staphylococcal enterotoxin B employing a quartz crystal microbalance biosensor
In this work, a new method t
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[64] Wang Z, Cheeke J D N, Jen C K. Sensitivity analysis for Love mode acoustic gravimetric sensors. Appl. Phys. Lett., 1994,64 (22) : 2940．
[65] 高志贤，检测葡萄球菌B型肠毒素基因的压电DNA传感器研究，军事医学科学院 博士学位论文，2000，6
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[67] Freudenberg, J. Schelle, S. Beck, K. et al.. Contactless surface acoustic wave biosensor. Biosensors and Bioelectronics v 14 n 4 1999 p 423-425.
[68] Harding, G.L. Du, J. Dencher, P.R. et al.. Love wave acoustic immunosensor operating in liquid. Sensors and Actuators, A: Physical v 61 n 1-3 June 1997 p 279-286.
[69] 晋晓勇，高志贤，陈慧，压电生物传感器及其研究进展，生命的化学，2002，22(4)：385-387
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