Novel Piezoelectric Microorganism Sensor And Its Application For Clinic Microorganisms

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.