压电传感器真菌鉴定中的应用及镍和柠檬酸配位研究
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摘要
一.多通道串联式压电传感器(MSPQC)具有适用对象宽,测定灵敏度高,操作简单方便,可用于实时或在位检测等优点,其应用涉及到分析化学、生命科学、环境监测、临床医学等众多领域。本文充分利用MSPQC的响应特性,并和临床念珠菌的新陈代谢特征相结合,拓宽了压电体声波传感器在临床医学中的应用,使临床念珠菌的检出、鉴定工作更加准确容易,快速方便。鉴于此开展了以下研究工作:
1.利用MSPQC和模式识别技术来早期鉴定临床常见念珠菌属病原菌。通过仔细研究MSPQC对不同念珠菌生长情况的响应发现:在相同的实验条件下,不同种的念珠菌在1-2天的培养中产生明显不同类型的频率曲线。利用这种响应曲线的差异,结合偏最小二乘技术(PLS)对不同种真菌产生的特征曲线有效区分,提出了MSPQC-PLS法早期鉴定常见念珠菌的方法。利用所提出的方法,我们成功的将从临床病人身上收集来得73株念珠菌区分成了5个种类(白色念珠菌、光滑念珠菌、热带念珠菌,克柔念珠菌,近平滑念珠菌),区分率达到97.3%。且通过模拟临床情况发现新建的MSPQC-PLS法不仅能够区分这些临床最常见的念珠菌,同时还能够在培养未经处理的临床样本的同时就鉴定出念珠菌的种类,极大的缩短了鉴定时间。
2.深入研究了临床常见念珠菌在MSPQC中出现不同响应曲线的原因。利用9M阻抗仪实时记录五种常见念珠菌生长过程中培养瓶电极两端的电参数变化情况,通过与平行在MSPQC中生长得到的频移曲线相对比得出MSPQC中真菌生长的特殊频移曲线的出现主要是由于真菌生长过程改变培养基电导变化引起的,生长过程引起的电容变化不起主导作用。再通过真菌的吸附试验证明真菌的吸附作用引起的电导变化对频移曲线也不起主导作用。利用真菌在YC培养基中不同生长阶段的显微照片我们得出真菌的多形态性所导致的新陈代谢过程的相对复杂性是真菌生长出现特殊代谢曲线的原因。
二.柠檬酸是电镀行业常用的一种弱酸配体,准确测定柠檬酸及其盐在电镀过程中的存在形式即其含量对指导电镀生产,发展电镀理论都是及其重要的,然而在电镀过程所处的高温和高离子强度背景条件下的柠檬酸及其盐的配合物组成及其稳定常数的测定一直存在空缺。pH电位滴定法是进行溶液配位化学研究最普遍运用的方法,特点是实验程序简单,计算结果准确。特别是此方法对测定体系的离子强度背景没有限制,适合电镀体系下的配位化学研究。鉴于此本文还开展了以下工作:
采用pH电位滴定法研究了电镀体系中Ni2+离子和柠檬酸配位情况。在60℃,I=4mol/L NaCl的实验条件下,测定了柠檬酸的质子化常数。实验确定了Ni~(2+)离子和柠檬酸二元体系在电镀生产条件下水溶液中主要存在的物种,并测定了其稳定常数。实验数据的处理运用计算机程序Hyperquad、Glee、Hyss处理。
The multi-channel series piezoelectric quartz crystal (MSPQC) biosensor has many advantages, such as broad sensing spectrum, high sensitivity, easy to operate, real-time and on-line measurement etc and can be widely used in many areas, such as analytical chemistry, life science, environmental monitoring, clinical medicine etc. In this thesis, new identification methods were presented base on the special response of piezoelectric sensor which related to some characteristics of clinical Candida species’growth and metabolism. These new methods were simple to operate and broaden their application in clinical medicine and life science. The new identification method for Candida species was ease to operate、rapid and accurate. The main work could be summarized as follows:
1. We have in this study investigated these Candida species using a method based on the integration of the MSPQC biosensor with pattern recognition for an early identification of the most frequent Candida species encountered in human pathology. Through the careful study of these response signals produced by different Candida species in MSPQC system, we find that under identical experimental conditions, various Candida species produce a significant different type of frequency curve in 1 days’culture. Utilizing the diversity of frequency curve and the partial least square (PLS) technique, we proposed the MSPQC-PLS method for early identification of commonly encountered Candida species. Using the method proposed, we differentiated 5 species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei) from a collection of 73 clinical strains of Candida, isolated from hospitalized patients, with a classification rate of 97.3%. This new proposed MSPQC-PLS method is simple, rapid and convenient to perform. It not only has the ability to differentiate these clinically most frequent encountered Candida species, but also can identify these Candida species at the same time with the culturing of the raw clinical materials. So the time for identification will be greatly shortened.
2. We carfully studied the cause of production of significant different type of frequency curve by various Candida species cultured in MSPQC. Using the 9M impedance instrument, the real-time electric parameters change between the two conductive stainless steel electrodes during the culture time of 5 species of Candida were recorded. Comparing to these frequency-shift curve recorded by MSPQC in parallel experiment, we conclude that the chief cause of the special frequency-shift curve is the change of the conductance between the two electrodes and the change of the capacitance caused by the growth of those Candida species is not significant. Through the adsorbtion experiment, we confirmed that the conductance change caused by the adsorbtion effect of fungi is not in dominant. The metallograph of different Candida species in different growing stage showed that the complexity of metabolism caused by the polymorphism of fungi is the chief cause of the production of special frequency curve by various Candida species.
3. Under the given conditions(60=℃,I=4mol/L NaCl),the protonation constants of ligand citric acid had been determined by pH potentiometry in aqueous solution. For the binary system Ni(II)-Citric acid,the complexes species as well as their stability constants were determinated under the same conditions as the condition mentioned above. the experimental data was calculated by employing prograins Hyperquad,Glee,and Hyss.
1.利用MSPQC和模式识别技术来早期鉴定临床常见念珠菌属病原菌。通过仔细研究MSPQC对不同念珠菌生长情况的响应发现:在相同的实验条件下,不同种的念珠菌在1-2天的培养中产生明显不同类型的频率曲线。利用这种响应曲线的差异,结合偏最小二乘技术(PLS)对不同种真菌产生的特征曲线有效区分,提出了MSPQC-PLS法早期鉴定常见念珠菌的方法。利用所提出的方法,我们成功的将从临床病人身上收集来得73株念珠菌区分成了5个种类(白色念珠菌、光滑念珠菌、热带念珠菌,克柔念珠菌,近平滑念珠菌),区分率达到97.3%。且通过模拟临床情况发现新建的MSPQC-PLS法不仅能够区分这些临床最常见的念珠菌,同时还能够在培养未经处理的临床样本的同时就鉴定出念珠菌的种类,极大的缩短了鉴定时间。
2.深入研究了临床常见念珠菌在MSPQC中出现不同响应曲线的原因。利用9M阻抗仪实时记录五种常见念珠菌生长过程中培养瓶电极两端的电参数变化情况,通过与平行在MSPQC中生长得到的频移曲线相对比得出MSPQC中真菌生长的特殊频移曲线的出现主要是由于真菌生长过程改变培养基电导变化引起的,生长过程引起的电容变化不起主导作用。再通过真菌的吸附试验证明真菌的吸附作用引起的电导变化对频移曲线也不起主导作用。利用真菌在YC培养基中不同生长阶段的显微照片我们得出真菌的多形态性所导致的新陈代谢过程的相对复杂性是真菌生长出现特殊代谢曲线的原因。
二.柠檬酸是电镀行业常用的一种弱酸配体,准确测定柠檬酸及其盐在电镀过程中的存在形式即其含量对指导电镀生产,发展电镀理论都是及其重要的,然而在电镀过程所处的高温和高离子强度背景条件下的柠檬酸及其盐的配合物组成及其稳定常数的测定一直存在空缺。pH电位滴定法是进行溶液配位化学研究最普遍运用的方法,特点是实验程序简单,计算结果准确。特别是此方法对测定体系的离子强度背景没有限制,适合电镀体系下的配位化学研究。鉴于此本文还开展了以下工作:
采用pH电位滴定法研究了电镀体系中Ni2+离子和柠檬酸配位情况。在60℃,I=4mol/L NaCl的实验条件下,测定了柠檬酸的质子化常数。实验确定了Ni~(2+)离子和柠檬酸二元体系在电镀生产条件下水溶液中主要存在的物种,并测定了其稳定常数。实验数据的处理运用计算机程序Hyperquad、Glee、Hyss处理。
The multi-channel series piezoelectric quartz crystal (MSPQC) biosensor has many advantages, such as broad sensing spectrum, high sensitivity, easy to operate, real-time and on-line measurement etc and can be widely used in many areas, such as analytical chemistry, life science, environmental monitoring, clinical medicine etc. In this thesis, new identification methods were presented base on the special response of piezoelectric sensor which related to some characteristics of clinical Candida species’growth and metabolism. These new methods were simple to operate and broaden their application in clinical medicine and life science. The new identification method for Candida species was ease to operate、rapid and accurate. The main work could be summarized as follows:
1. We have in this study investigated these Candida species using a method based on the integration of the MSPQC biosensor with pattern recognition for an early identification of the most frequent Candida species encountered in human pathology. Through the careful study of these response signals produced by different Candida species in MSPQC system, we find that under identical experimental conditions, various Candida species produce a significant different type of frequency curve in 1 days’culture. Utilizing the diversity of frequency curve and the partial least square (PLS) technique, we proposed the MSPQC-PLS method for early identification of commonly encountered Candida species. Using the method proposed, we differentiated 5 species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei) from a collection of 73 clinical strains of Candida, isolated from hospitalized patients, with a classification rate of 97.3%. This new proposed MSPQC-PLS method is simple, rapid and convenient to perform. It not only has the ability to differentiate these clinically most frequent encountered Candida species, but also can identify these Candida species at the same time with the culturing of the raw clinical materials. So the time for identification will be greatly shortened.
2. We carfully studied the cause of production of significant different type of frequency curve by various Candida species cultured in MSPQC. Using the 9M impedance instrument, the real-time electric parameters change between the two conductive stainless steel electrodes during the culture time of 5 species of Candida were recorded. Comparing to these frequency-shift curve recorded by MSPQC in parallel experiment, we conclude that the chief cause of the special frequency-shift curve is the change of the conductance between the two electrodes and the change of the capacitance caused by the growth of those Candida species is not significant. Through the adsorbtion experiment, we confirmed that the conductance change caused by the adsorbtion effect of fungi is not in dominant. The metallograph of different Candida species in different growing stage showed that the complexity of metabolism caused by the polymorphism of fungi is the chief cause of the production of special frequency curve by various Candida species.
3. Under the given conditions(60=℃,I=4mol/L NaCl),the protonation constants of ligand citric acid had been determined by pH potentiometry in aqueous solution. For the binary system Ni(II)-Citric acid,the complexes species as well as their stability constants were determinated under the same conditions as the condition mentioned above. the experimental data was calculated by employing prograins Hyperquad,Glee,and Hyss.
引文
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