Study On The Key Technology Of Total Bacteria Detection System Based On Single Particle Analysis

Objective:In recent years, along with the outbreak of a variety of microbial contamination problems, generalized biosafety issues is becoming a hotspot. Air and water resources as the main media, its extensive distribution and irreplaceable importance reminds us to pay more attention to resources monitoring and protection. As one of the important indexes of microorganisms in water, the total number of bacterial can directly reflect the degree of water contamination, and in many water quality regulations stipulate the total number as a necessary testing items. To establish a rapid and accurate detection of bacterial samples of water in daily life is of great importance in monitoring and protecting quality of water resources of living water, scientific experiments, medical care, precision machining and even the prevention of bioterrorism. The traditional detection methods are still used widely in the field, especially the plate culture method as the gold standard in many countries. Lots of new testing methods have been proposed and offer many more quickly and more accurate research ideas, but in general, these methods are still in the research stage.Based on the study of the research group, this paper will lead to further research of the single-particle flow detection technology and its application in water detection of total bacteria. To accumulate more experience in the process of the system prototype is helpful for the technology promotion in special detection of water and provide new ideas for the technology application.Contents and Methods:Based on the researching of the flow cytometry principle and analyzing the foreign detection technology and equipment, we propose to start research and system prototype development about the single-particle flow detection technology. The main contents and methods include:I. Research on the sample pre-treatment methods. Through literature and experimental verification, we want to establish an accurate and feasible pre-treatment method. We compare dyeing effect of different dyes and carbon points, explore various principles of pre-treatment and by doing this to provide the necessary design parameters about the light source and optical lens.II. Design and optimization of optical module. The optical module is divided into two parts, excitation module and detection module. Through the theoretical simulation analysis, we are aim to achieve ideal detection spot by the excitation module and receive the fluorescent selectively by the detection module. Based on pre-treatment dye characteristics, we optimize the light source, the lens parameters and optical structure.III. Design and optimization of flow module. By comparison with different designs, We design to control sample liquid and sheath liquid with different electrical motor, so that in the detection zone the coaxial flow patterns can be formed by the external sheath liquid wrapped the sample liquid inside. We evaluate the effect of fluid flow system, optimize control parameters and design a more secure flow solutions.IV. Design and optimization of signal processing module. The dyed bacteria will emit fluorescent when induced by the detection spot and the fluorescence signal will be detected by the photomultiplier, after amplification, filtering and A/D conversion. The digital signal will transmit to the FPGA master control chip. We accomplish the related circuit design and parameter optimization, draw the PCB and complete the circuit debugging.V. Proposition and verification of peak processing algorithm. We make a model of the micrometer-scale sample particles, simulate the fluorescence signal intensity and explore the characteristics of the fluorescent signal. Combined with the needs of the detection system, a new signal processing method has been proposed and applied by the Verilog language. The accuracy and stability of the algorithm will be verified through the experiment, and also something helpful will be done for the algorithm optimization.VI. Design and optimization of system structure. At the beginning, with a separate submodule building program, the overall system is divide into several independent subsystems in order to realize the function rapidly. With the aim to improve the system integration, we use FPGA as the core of hardware circuits which contains center power board, signal processing board, signal control board and microcontroller board as the main four enable plug-pull circuits. We also make each independent module integrated into an organic whole and optimize system structure.Results:I. In the optical system, the original spot of the laser is about 844 * 765 um, the ellipticity is about 0.906 and the spot after XY biaxial plastic lens is 77.8 * 19.8 um. The optical spot has a good stability which changes little near the focal position.II. In the flow system, a plunger pump and an air pump is selected as power source, the design solves the "dead volume" problem of sample which introduces pollution and avoids the "pulse" problem of back flow. It also makes the sample can be detected continuously rather than limited by the volume of the tube and takes about 0.4 s to establish a stable air pressure in the sheath liquid bottle, which voltage is about 1.1 v and will be kept for a long time stably.III. The peak processing algorithm, which has been compared congruously with the existing detection methods, only need three parameters for quick peak detection and shows 5.36% relative standard deviation in 10 samples repeat testing. It is proved to be a stable algorithm and can meet the detection requirements of the system.IV. The microsphere test shows that the system works more stably when the sampling speed is between 0.5 ul/s- 1 ul/s, and according to different concentration samples the CV value is between 2-3.5, which reproves the overall system reaches a good precision.V. The staphylococcus aureus tests show that the best detection limit of the system is 103-106cfu/ml concentration range, within the detection limit the counting results have good consistency with the plate culture method, when below the detection limit the results are obviously higher than plate count method. But the results always maintain good consistency compared with the existing flow cytometer.Conclusion and Prospect:I. On the basis of the existing design, by optimizing all parts of the subsystem respectively in order to further improve the detection sensitivity of the overall system.II. Increase the fluorescence collection channels, through the current sample pretreatment method, which can realize the death bacteria and living bacteria detection respectively. By increasing the channel, the more synchronous information will improve the analysis ability in once detection.III. To optimize the peak detection algorithm, further enhance the sensitivity and multiplatform generality and improve the adaptive analysis ability.IV. To optimize the structure of the existing system and improve the system integration and portability.