1 Introduction
The total number of bacteria is detected is an important test indicator in the field of food hygiene testing, mainly to determine the extent of food contamination. Fast and accurate testing of the total number of bacteria has always been an important research topic in this field. The national standard method for detecting the total number of bacteria is the tablet counting method, and the sample is required to cultivate 48 h in nutrient agar 37 ° C, the test time is long, and the operation is cumbersome, and it is not suitable for on-site testing. The ATP biosainer detection method has the characteristics of fast detection speed, low cost, easy operation, etc., has attracted many researchers. At the same time, because ATP bioluminescence techniques use ATPs in biology as detection objects, not only include bacteria, but also food contaminants such as food residue in somatic cells, so suitable for rapid detection of health in the field of food hygiene detection.
The ATP was measured using the ATP bioluminescence technology to report the earliest report of MCELROY in 1949. Since then, ATP biofumin techniques have attracted widespread attention. Based on the analysis of predecessors, this paper designs an ATP bioluminescent detector, selects photomultiplier (H5773-02, Japan, and Song Photos) as photoelectric conversion devices, its peak response wavelength is 500 nm, and reaction The fluorescence wavelength is basically consistent, thereby improving the detection sensitivity. This detector uses auto-drawn techniques. After the addition is detected, the reaction chamber is automatically added to the detected sample cell in the reaction chamber, which avoids the interference of the external light, and effectively improves the addition. Sample precision, thereby improving the detection reliability and accuracy of the instrument. With this detector, 10-6 ~ 10-14 mol / L standard ATP samples were detected, and the correlation between output signals and sample concentration reached 0.986, which can meet the measurement requirements.
2, system design
2.1 detection principle
ATP is a three-phosphate adenosine, which is widely present in an energy substance in the biological body. Experiments have shown that under physiological conditions, the content of ATP contained in each bacterial cell is substantially the same, about 10-18 mol /. Firefly luciferase referred to invertese, is an active protein that can convert chemical energy into light energy, ie a biocatalyst. ATP releases fluorescence with fluorescein in an aerobic environment and a bivalent magnesium ion under the catalytic action of Cordycene. When fluorescein and invertese excess, released fluorescence and ATP are linearly formed in a certain range, and thus, the total number of bacteria is detected by measuring the fluorescent light to detect the ATP in the sample.
2.2 Structure Design
The structure of this detector is shown in FIG. 1, mainly including three parts, which are automatic spray units, photoelectric conversion units, and signal acquisition processing and control units. Among them, the auto-spatant unit includes three sets of peristaltic pumps (WX10, Baoding Lange constant current pumps) and kits, divided into independent three-way plus-style path, which is made of body cell lysing agents, bacterial cell lysates and fluorescein. A fluorescein enzyme light reagent. According to the test, the peristaltic pump is controlled to the sample cell in the photoelectric conversion unit under the control unit control. The photoelectric conversion unit includes detecting a sample cell and a photoelectric conversion device. In order to eliminate interference of the external light, the entire photoelectric conversion unit is sealed in the cassette. The photoelectric conversion device converts the reaction release fluorescence into an electrical signal, collects and processes the signal acquisition processing unit, and performs real-time display and storage of the test results.
2.3 circuit design
The circuit structure of the system is shown in Figure 2. The photoelectric conversion device converts the fluorescent signal into a current signal, collects and processes the signals through the micro-processing chip ADUC834 (ANALOG DEVICES, USA). The ADUC834 simultaneously acts as a control core, controls the automatic drawing unit, display, and storage unit, and can upload measurement results to the host computer via the RS232 interface to perform results analysis.
2.4 software design
The operation software of the detection instrument is written by the C language, and the software structure is shown in Figure 3, and three functional modules are shared, respectively, respectively, and the measurement, recording, and meter settings. Among them, the measurement is divided into instrument independent measurements and connected computer measurements. When the meter is independently measured, the microprocessing chip ADUC834 acquires and integrates the signals in a certain period of time, and then display the test results. According to the user selection, the test results can be uploaded to the computer; when online measurement, the ADUC834 pairs collected Do not process, but directly to the upper computer, and display the time response curve in real time, so that the user analyzes the measured data in the computer, suitable for test analysis in the laboratory. The recording submenu is divided into recording query and record deletion to operate on the record. The 24LC256 is used as an external data memory chip. This instrument design can store the test results 200, which can be displayed by the query; record deletion is divided into full deletion and all deletions by one by one; set the submenu to realize the meter calibration and multiplier voltage Adjust two functions.
3, experiments and results
3.1 Device Selection
Since the ATP biofuminous is weak, this detector selects a highly sensitive photomultiplier as a photoelectric conversion device. The fluorescence wavelength of the ATP bioluminescence reaction was scanned by the F-4500 fluorescence spectrophotometer (Japan, Hitachi), and the peak wavelength was about 550 nm. In order to ensure that the detection has higher sensitivity, the response wavelength of the selected optical multiplier should be consistent with the fluorescence wavelength. Japan's H5773-02 optical multiplier homeware has a response wavelength of 330 to 850 nm, and peak detection wavelength is substantially constant at 500 to 600 nm, so that the design requirements of this system are in line with this system.
3.2 Results and Discussion
Experiment Select Standard ATP Samples as the detection object, the concentration range of 10-6 ~ 10-14mol / L, fluorescein and fluorescein enzyme used in the experiment from the US Pliger. The experiment adopts the online measurement method, and the detector is passed from the computer. The RS232 interface is connected, and the meter signal acquisition unit acquires the fluorescent signal to the computer directly to the computer and display the test results. The measurement results are shown in Figure 4.
In the reaction, when the luminescence reagent is added, the concentration of the sample in the system is relatively high, so that a rapid reaction is released in a short time, and the fluorescence is released, and the fluorescence is strong, so the measurement results may exceed the system test range. Since the maximum output voltage of this instrument is 2500 mV, the fluorescence released in the initial period of the reaction may exceed this range, and the horizontal line of the front end is manifested in the figure. As the reaction is carried out, the concentration of the samples and reagents in the system gradually decreases, and the release fluorescence is gradually attenuated. As can be seen from the figure, the ATP of different sample concentrations is carried out with the reaction, the time in which the measurement range is different, the higher the concentration, the slower the attenuation, the longer the time, the longer the measurement range, and the shortest is short. The measured value in each of the response curves in Fig. 4 is integrated, and the output signal of each concentration sample is performed. The relationship between the output signal and the concentration is shown in Figure 5. As can be seen from the figure, the output signal increases with the concentration, and has a significant gradient, and the linear relationship coefficient of the two reaches 0.986. This result indicates that this test system can quickly detect the concentration of ATP. Since the ATP content contained in bacterial cells is basically consistent, this detector can realize the rapid detection of the total number of bacteria.
4 Conclusion
This paper designs a rapid number of bacteria based on ATP biofumin techniques for the total number of bacteria. On the basis of analyzing the existing product, the detector selects the H5773-02 photoelectric multiplier as a photoelectric conversion device, and the detection wavelength is consistent with the fluorescence wavelength released by the biometric reaction, and can effectively improve the detection sensitivity. The automatic add-fitting technique is used, and the luminescent reaction reagent is added to the dark chamber, and the interference of the external light is eliminated, and the initial signal of the reaction is obtained. This paper is tested as a detection object with a standard ATP solution of bacterial detection as a detection object. The detector was measured to the computer to obtain a time response curve of the fluorescence intensity released by each reaction. On this basis, the value within 400 S within 400 s is integrated. As the output signal of the corresponding sample concentration, the relationship curve of the output voltage and the concentration is obtained, and the linear relationship coefficient of the two reaches 0.986, which can meet the test requirements. This paper has the characteristics of detecting speed, high automation, simple operation, simple portability, etc. in the port, health supervision department, food production department, etc. in the port, health supervision department, food production sector, etc. The field has a good application prospect. Read more
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