【作者】 任佳丽；

【导师】 何凤姣；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 博士

【摘要】 由微生物感染引发的疾病已经严重威胁到人类的健康。结核病是由结核分枝杆菌病原体感染引发的重大传染性疾病,曾经是人类的第一大杀手,随着抗菌素的广泛应用,上世纪六七十年代,结核病的危害直线下降,人类颇有“大获全胜”的感觉。在上世纪末,结核病却又卷土重来,疫情直线回升,大有重新找回“第一杀手”位置之势,而最令人担心的,莫过于耐药性结核病病例的大量出现。要控制耐药菌株的发展,最直接而有效的方法是进行结核分枝杆菌早期快速检测。结核分枝杆菌由于其增代时间长,生长缓慢,比其它微生物更难实现快速检测。快速全自动微生物培养系统对于临床病原菌感染的早期诊断、抗生素的合理运用、延缓病原菌的耐药性都起到了十分重要的作用。而目前临床使用得较多的几款全自动微生物培养系统包括BACTEC 460 TB和BACTEC MGIT 960。BACTEC 460 TB存在放射性污染、仪器操作和维护困难。BACTEC MGIT 960系统所使用的荧光物质易分解造成假阴性。此外,以上仪器本身造价就高,其配套试剂需依赖进口,因此其推广受到经济条件的限制,对于结核病发生率高的贫穷国家或发展中国家是可望而不可及的。串联式压电传感器的主体部分是由压电石英晶体和一对插入溶液中的平行板金属对电极串联组成,该传感器对电极间的电参数变化有灵敏的响应。由于其操作简便、准确、灵敏以及价格便宜而深受广大分析化学者的欢迎。本论文在本室前期工作的基础上,致力于快速、灵敏、准确而造价便宜的新型串联式压电微生物传感器的研制。通过改进、优化或引入新的探头,解决了传感器受背景电参数及电极材料影响的问题,拓宽了该类传感器在生命科学中的应用,使信息的提取更加准确容易,测定更加快速方便。鉴于此开展了以下研究工作:(1)新型多通道串联式压电传感器(MSPQC)的构建及其在结核分枝杆菌减毒菌株H37Ra检测中的应用。该方法是将结核分枝杆菌产生的挥发性代谢产物导致接收液电导变化和串联式压电传感器对电导的灵敏响应结合起来进行检测。首先,设计了双管检测装置,外部是检测管,底部固定有一对电导电极,管内盛有2 ml KOH接收溶液;检测池内部是培养管,内盛有Youmans改良Proskauer-Beck培养基,结核分枝杆菌能代谢培养基中甘油和天门冬酰胺产生挥发性的NH3和CO2,代谢产物能被接收液吸收,导致接收液的电导发生变化,从而检测出结核分枝杆菌。由于双管结构的引入,解决了检测受仪器灵敏区间限制问题。我们用MSPQC成功地检测了H37Ra,在102~107 cfu?ml-1之间,菌液初始浓度的对数与检测时间(FDT)之间存在线性关系。检测限为10 cfu?ml-1。同时检测结果与全自动化的BACTEC MGIT 960进行了对比,二者的结果均通过Ziehl-Neelsen (Z-N)染色法镜检证实了H37Ra的存在。该仪器与同类产品相比还有一个最重要的优点,价格便宜(仪器约为:10~20万,试剂约为:20元;而同类产品BACTEC MGIT 960仪器:90万,试剂约为:80元)。MSPQC有望开发成为一种准确、快速而廉价的结核分枝杆菌检测仪器。(2) MSPQC用于结核分枝杆菌标准菌株H37Rv和临床菌株的检测。通过对未减毒标准菌株H37Rv与减毒菌株H37Ra在Youmans改良Proskauer-Beck培养基中的MSPQC典型曲线的对比,分析了未减毒菌株可能的代谢途径,并通过对代谢途径的推测,优化了培养基,获得了H37Rv的增强检出信号,初步证实了推测。同时筛查了最佳抑菌剂成分并成功用于临床菌株的检测,其检测结果与L-J斜面和BACTEC MGIT 960进行对比,结果表明,该仪器准确、快速、简便,有望应用于临床实验室结核分枝杆菌的检出。(3)提出MSPQC进行一线抗结核药物药敏试验的新方法。通过设定参比管,并将参比管加入的菌液浓度调节为药物管浓度的1/100,巧妙地解决了MSPQC的不完全定量问题。根据临床实验室标准机构(CLSI)的规定成功定义了菌株对于药物敏感性的快速划分标准,并在此基础上将临床结核分枝杆菌对异烟肼、链霉素、乙胺丁醇和利福平四种一线药物的敏感性进行了实际划分,其结果与琼脂比例法(APM)和BACTEC MGIT 960方法进行了比较,该法快速、操作简便,还可以实时监测,从而为结核分枝杆菌药敏研究及药物治疗提供有效手段。(4)叉指阵列电极串联式压电传感器(IDE-SPQC)的构建及其在微生物培养检测中的应用。IDE-SPQC是直接将叉指阵列电极(IDE)代替平行板不锈钢对电极与压电石英晶体串联构成。本章通过理论和实验对比探讨了IDE-SPQC与串联式压电传感器(SPQC)对溶液电导和电容变化的响应以及IDE电极参数对IDE-SPQC响应灵敏度的影响。结果表明,在一定条件下,IDE-SPQC较SPQC对溶液电参数的变化有更灵敏的响应。当使用IDE-SPQC对微生物进行检测时,其线性范围为10~106 cfu?ml-1,检测下限为10 cfu?ml-1。其结果与SPQC和平板计数法(PPC)进行了对比,该方法与SPQC和PPC有相同的准确度,但较后两者更快速。(5)聚苯胺多通道串联式压电传感器(PAn-MSPQC)的研制。将IDE修饰碱型聚苯胺/聚乙烯醇复合物制备成PAn探头与压电石英晶体串联,构建了PAn-MSPQC。其用于微生物测定的原理为:细菌在新陈代谢过程中均会产生CO2,而在一定湿度下CO2能与聚苯胺掺杂,能引起PAn探头电导的变化,实现对微生物的检出。相对于SPQC,由于探头不直接与培养基溶液接触,该传感器不受培养基电参数的影响,不需要特别合成的培养基。利用该传感器可定量地检测样本中的初始微生物浓度,其线性范围为10~106 cfu?ml-1,其检测限为10 cfu?ml-1。(6)聚吡咯多通道串联式压电传感器的制备(PPY-MSPQC)。常规的SPQC除了受仪器灵敏区间限制外,还有一点就是其电极材料难于选择。导电聚合物经掺杂导电率可提高十几个数量级,由绝缘体变为导体,有金属性,可作为电极,再加之其对于一些物质具有选择性电响应,可将其代替金属电极作为敏感探头,与压电石英晶体串联,构建新型串联式传感器。本章以聚吡咯为例,将聚吡咯化学沉积于绝缘基底上制成导电聚合物膜作为MSPQC的探头,研究其在高湿度和室温下对于NH3的响应特性。当NH3量在0.1μmol到6μmol之间时,氨量与频移之间存在线性关系,该方法的最低检测限为0.1μmol。该传感器有望用于需在高湿度、室温下进行NH3测量的体系。(7) PPY-MSPQC用于L-天门冬酰胺酶活性的测定。基于L-天门冬酰胺酶催化L-天门冬酰胺底物水解产生NH3,通过使用PPY-MSPQC测定NH3,从而实现酶活性的测定。使用PPY-MSPQC筛选了L-天门冬酰胺酶活性测定的最佳条件;测定了酶动力学参数;研究了该酶对不同底物L-天门冬酰胺、D-天门冬酰胺以及L-谷氨酰胺的不同催化活性;测定了不同酶浓度,该方法的线性范围是0.01～0.1 U?ml-1,其检测下限为0.01 U?ml-1。其结果与奈氏试剂法进行了对比,该方法准确、无需使用有毒的试剂且能实现实时在线检测。该方法有望用于产L-天门冬酰胺酶微生物的筛选及产氨酶活性的测定。更多还原

【Abstract】 The infection caused by microorganisms has threatened humans’health. Tuberculosis (TB) is the most important infection disease which caused by Mycobacterium tuberculosis. It has been the largest killer for human. In the 1960s, its harmfulness decreased with antibiotics’wide applications. However, the re-emergence of TB, especially resistant strains, results in a major health care problem. The development of a rapid and efficient automated microorganism culture system for the identification of TB is of extreme important for patient treatment and protection of health-care professionals. Rapid Culture detection techniques have been used in clinic, such as BACTEC 460 TB and BACTEC MGIT 960. However, the former has the radioactive damage and the latter need to use fluorescent reagent which is easily decomposed. In addition, all above systems are rather expensive for both instruments and reagents. So, their wide use was limited. Millions of people in developing countries die each year from TB because of expensive diagnostic cost. More accurate, more rapid and cheaper detection system is urgently needed in developing countries.The key section of series piezoelectric quartz crystal (SPQC) is composed of an AT cut, 9 MHz piezoelectric quartz crystal and a pair of parallel-plate metal electrodes inserted into liquid solution in series. It has sensitive response to the electric parameter changes (such as conductance and capacitance) between the electrodes. SPQC has attracted many analysts because of its sensitive response, accurate, low cost and easy operation. This dissertation is devoted to the study of novel microorganism piezoelectric quartz crystal sensors, especially TB sensor. The main work of this thesis could be summarized as follows:(1) A new multi-channel series piezoelectric quartz crystal sensor system (MSPQC) was developed for rapid growth and detection of Mycobacterium tuberculosis H37Ra. The system was used to detect H37Ra based on combination the volatile metabolic products during the growth of M.tuberculosis with SPQC sentive response to solution conductance. Firstly, the double tubes configuration was proposed. The metabolic products, diffusing from the medium into the KOH absorbing solution, resulted in the conductance change of the absorbing solution detected by the MSPQC sensitively. The frequency shift versus time response curves were recorded by self-developed software. Frequency detection time (FDT) corresponding to–100 Hz in frequency shift value was used as a parameter to quantitatively determine M.tuberculosis H37Ra (an avirulent strain). As for H37Ra, the FDT had a linear relationship with the logarithm of its initial concentration in the range of 102~107 colony forming units (cfu)?ml-1 (R=-0.998) and the detection limit was low to 10 cfu?ml-1. 4% NaOH solution that can kill contaminating microorganisms and make M.tuberculosis alive was used as pretreatment reagent to provide the selectivity to this method. Comparing tests were also carried out by using BACTEC? MGIT? 960 and conventional Lowenstein-Jensen (L-J) slants. The results showed that the proposed system was quicker than BACTEC? MGIT? 960 and it is also cheaper and will be widely used in TB tests in the world.(2) Multi-channel series piezoelectric quartz crystal (MSPQC) was developed for rapid growth and detection of H37Rv and clinic strains. The typical response curve of H37Rv growth in Youmans medium was compared with that of H37Ra. From their difference in profile and frequency response values, their different nitrogen metabolic path was speculated. Base on above speculation, the culture medium was adjusted, and excellent frequency signal was obtained. The speculation was verified. Simultaneously, the optimum antibacterial reagents were investigated and the proposed method was used to detect 40 strains clinic mycobacteria successfully. The results were compared with L-J slants and BACTEC MGIT 960. It demonstrated that the proposed method is rapid, accurate and easy operation and it could be popularized in clinical microbiology laboratories.(3) A new antitubercule susceptibility testing method based on multi-channel series piezoelectric quartz crystal (MSPQC) was proposed. Semiquantitative results could be achieved with the 1% threshold as determinant of resistance through adjusting the inoculum concentration in reference tube (1/100 in drug tube). This method was used to test susceptibility of clinical mycobacterium tuberculosis isolates against isoniazd, rifampin, ethambutol hydrochloride and streptomycin. Comparing tests were run at the same time by the agar proportion method (APM) and BACTEC MGIT 960 method. The experimental results showed that MSPQC method had a good agreement with the reference methods. Compared with those methods, the MSPQC method is simple, rapid, and convenient to perform.(4) A new interdigital electrode- series piezoelectric quartz crystal (IDE-SPQC) sensor was proposed here. It was constructed using an interdigital electrode (IDE) connection with a piezoelectric quartz crystal in series. The response was studied theoretically and experimentally. Proposed sensor was more sensitive than series piezoelectric quartz crystal sensor (SPQC) to changes in electrical parameters of solution in certain conditions. Factors affecting the response of the IDE-PQC were discussed in detail. Proposed method was used to detect Pseudomonas aeruginosa. The logarithm of the initial concentration of P. aeruginosa had a linear relationship with frequency detection time (FDT) in the range of 10 ~106 cfu?ml-1. The detection limit could be as low as 10 cfu?ml-1. Pour plate counts (PPC) and SPQC methods were used for comparing with proposed method. Escherichia coli and Staphylococcus aureus were also detected successfully with proposed method.(5) A new base-type polyaniline/poly vinyl alcohol piezoelectric quartz crystal (PAn-MSPQC) system for rapid detection of bacteria population was proposed. In this system, interdigital electrode coated with a base-type polyaniline/poly vinyl alcohol composite film was used as probe (PAn) in place of steel electrode in multi-channel series piezoelectric quartz crystal (MSPQC) system. The probe was directly connected to the piezoelectric detection system. B-PAn/PVA film can react with CO2 produced by the growth of bacteria in culture medium, which caused the film electrical properties change. This change can be sensitively monitored by piezoelectric detection system. The superiority of using proposed probe instead of steel electrode were those: firstly, it was more sensitive to bacteria growth response, and secondly, it didn’t require a specially formulated culture medium which was demanded in MSPQC system. Just like MSPQC, proposed system can be used to detect bacteria population. When used to quantitatively determine P.aeruginosa, the frequency detection time (FDT) obtained in frequency shift curve had a linear relationship with the logarithm values of initial concentration of P.aeruginosa in the range of 10~106 cfu?ml-1. The detection limit was 10 cfu?ml-1.(6) A new polypyrrole multi-channel series piezoelectric quartz crystal system (PPY-MSPQC) was proposed for NH3 detection at high humidity and room temperature. The significant electrical properties, stability characteristics, good processing of conducting polymers make them ideal candidates to act as a soft electrode material; the conductivity changes of the polymers by a doping or dedoping reaction due to introduction of ion make them to act as a sensing film. Polypyrrole, chemically deposited on surface of polyimide, had sensitive respose to low concentration NH3. In this paper, polypyrrole/polyamide was used as probe (see Fig.1 A (2)) instead of stainless steel electrodes connection with piezoelectric quartz crystal in series and a new PPY-MSPQC system was constructed for detecting NH3. From 0.1μmol to 6μmol, there is a linear relationship between the frequency shifts (Y) and the ammonia amounts (X) shown in Y=8.4464+102.99X (R=0.998), the detection limit is 0.1μmol. This method can be used to detect NH3 at high humidity and room temperature, such as amidase activity assay.(7) A PPY-MSPQC method for L-asparaginase activity assay. The PPY-MSPQC was used to assay L-asparaginase activity that can catalyze the hydrolysis of L-asparagine into L-asparatic acid and NH3. The optimum enzyme activity assay conditions were investigated. The kinetic parameter was calculated by changing the substrate concentration. The catalyzing activity of L-asparaginase to different substrates was also studied. The proposed method can be used to detect enzyme concentration quantitatively in the range of 0.01～0.1 U?ml-1. The results were compared with classic Nessleration’s method. Some advantages are found, such as real time and nontossxic. It is expected to use in sceening L-asparaginase producing microorganisms and detecting amidase activity.更多还原