Based on bioluminescence TBC fast detector

Yue Wei wei, hcma000653 jade, Baoshan, Jinping, new-Xia

(1. CAs electronics Institute sensing technology in the State Key Laboratory, Beijing 100080; 2. Academy, Beijing 100080)

1 Introduction

Total bacteria detection is a food hygiene inspection areas important to detect targets primarily is used to determine the extent of the contamination of food.

The total number of bacteria for fast and accurate detection has been the area important research topic. Total number of bacterial detection GB。 is flat notation requires that the sample used nutritional agar 37 ° c for 48 h count training, testing, operation, do not fit the scene detection. ATP bioluminescence method has to detect speed, low cost, easy to operate, and other characteristics, has aroused the interest of many researchers. At the same time, because ATP bioluminescence in vivo detection of ATP as objects, not only including bacteria, also including somatic, food residues, food contaminants, and thus suitable for food safety testing conducted on the health situation in the field of rapid detection.

Use the ATP bioluminescence measuring ATP earliest in 1949 Mcelroy reports.

Since then, the ATP bioluminescence technology attracted widespread attention. This article analyses the predecessors on the basis of the work, the design of an ATP bioluminescence detector, select photomultiplier tubes (H5773-02, Japan, Hamamatsu Photonics) as opto-electrical converter, its peak response for 500 nm wavelength, and responses by fluorescence wavelength basic agreement, thereby enhancing the detection sensitivity. The tester adopts automatic-like technology, in joining is test sample, in the reaction chamber and by the automatic device detection sample sample to automatically join the reaction reagent in the pool, in order both to avoid the interference of light from the outside world, but also effectively improves accuracy with-like, thereby improving the reliability of detection instruments and accuracy. Use this instrumentation to 10-6 ~ 10-14mol/L standard ATP samples were detected, the output signal and sample concentrations between 0.986, can meet the measurement requirements.

2 system design

2.1 detection principle

ATP the adenosine triphosphate is widely present in the organism of a energy substances.

Experimental proof, under physiological conditions, each bacterial cells in much the same ATP content is about 10-18mol/a. Firefly luciferase of insect light for short, is a kind of chemical energy can be transformed into the light energy of active protein, that is, biological catalysts. ATP in insect light catalytic function of enzymes, and fluorescein in aerobic environments and bivalent magnesium ions, reaction releases fluorescence. When the fluorescein and insect light of overdose cases, release of fluorescence and ATP, to a certain extent as a linear relationship, so you can measure the fluorescent intensity testing samples of ATP, to detect the total bacteria.

2.2 structure design

This instrumentation for the structure as shown in Figure 1, mainly consists of three parts, namely the automatic sampling unit, photoelectric conversion unit and signal and control unit.

Where automatic sampling unit consists of three sets of peristaltic pump (WX10, Baoding Lange constant flow pump co., Ltd.) and Kit, divided into separate three-way plus-like pathway, somatic cell lysis of, bacterium cell lysis reagent and fluorescein a luciferase luminescence reagents. Depending on the test require, peristaltic pump control unit control, photoelectric conversion units in the sample pool join corresponding reaction reagent. Photoelectric conversion units including test sample pool and opto-electrical converter. To eliminate the interference of light from the outside world, the photoelectric conversion unit is sealed in a cassette. Opto-electrical converter will response release fluorescent into electrical signals, signal acquisition and processing unit acquisition and processing, and on the test results in real time display and storage.

　　

　　

2.3 circuit design

System's circuit structure as shown in Figure 2.

Opto-electrical converter will convert the fluorescence signal current signal, through micro-processing chip ADuC834 (Analog Devices, United States) on signal acquisition and processing. ADuC834 as both the control core, the control automatically displays the pipetting unit, and storage units and measurements via RS232 interface upload first bit machine for results analysis.

　　

　　

2.4 software design

Detection instrument operating software is written by C language software structure as shown in Figure 3, is divided into three functional modules for measuring, recording and instrument settings.

Where the measurement is divided into independent measurement instrumentation and measurement connected computer. Instrument independent measurement, microchips ADuC834 on time, signal acquisition and integral handle, and then set the test results display in real time, according to the user to select the test results can also be uploaded to your computer; online measurement, the acquisition of ADuC834 on signal processing, but is directly transmitted to the host computer, and real-time display of time response curve, user-friendly computer on the measuring data, suitable for laboratory testing and analysis. Record submenu consists of records the query and delete records to the record. Used as external data storage 24LC256 chips, this instrument is designed to store test results 200, record query can be displayed for each record; records deleted by article is divided into the delete and remove all; setting submenu for instrument calibration and multiplier tube voltage adjustment of two functions.

　　

　　

3 experiments and results

3.1 device selection

Due to the ATP bioluminescence the luminous intensity is low, therefore this instrumentation of high sensitive photomultiplier tubes as opto-electrical converter.

Use f-4500 fluorescence spectrophotometer (Japan, Hitachi) for ATP bioluminescent reaction by fluorescence wavelength is scanned, the peak wavelength of about 550 nm. In order to ensure detection has a higher sensitivity, the selected response of photomultiplier tubes and fluorescence wavelength wavelength. Japan Hamamatsu photon H5773-02 PMT response wavelength to 330 ~ 850 nm wavelength, peak detection in the 500 ~ 600 nm basic remain constant, so the system design requirements.

3.2 results and discussion

ATP experiment selection criteria as a test object, the sample concentrations in the range of 10-6 ~ 10-14mol/L, experiment with fluorescein and luciferase purchased from United States Chapleau Meg Corporation.

Experimental use of online measurement, the measuring instrument and the computer. RS232 interface connected to the instrument signal acquisition unit acquisition fluorescence signal after upload directly to your computer and the test results display in real time. Measurement results are shown in Figure 4.

　　

　　

In response, adding luminescent reaction reagent, the system of the relatively high concentration samples, so that within a short time to release large amounts of fluorescence quick response, fluorescent light, so the measurement results may exceed the system test range.

Since this instrument's maximum output voltage 2500 mV, early release of fluorescence reaction may exceed the range, in the diagram to the front of the horizontal line. Along with the response, in the concentration of samples and reagents, gradually reduce the release of fluorescence decay gradually along with it. The figure shows that different sample concentrations of ATP with the response, enter the measurement range of the time, the higher the concentration, the slower the attenuation, enter the measurement range, instead of the longer. On Figure 4 in the response curve 400 s within the measurement values integral, as the concentration of the sample of the output signal. Output signal and the concentration of relationships as shown in Figure 5. As can be seen from the figure, the output signal increases with the concentration, and there is obviously a linear gradient, both of which reached 0.986 correlation coefficient. This result shows the detection system can quickly detect ATP concentration. As a result of bacterial cells in ATP in basically the same, therefore, the detector can be realized on the total number of bacteria scene fast examination.

　　

　　

4 conclusions

This article needs against bacteria detection, design an ATP bioluminescence technology based TBC fast detector.

The detector in the analysis of existing products, selected on the basis of H5773-02 photomultiplier tubes as opto-electrical converter, its detection wavelength and the releases bioluminescent reaction, fluorescent wavelengths can effectively improve the detection sensitivity. The use of automatic plus-like technology, dark Interior joined glow reaction reagents, eliminating the interference of light from the outside world, and received a response to the original signal. This article by bacteria detection of intermediate standard solution for detecting objects ATP, on the design of the measuring instrument for testing. The instrumentation and measurement, get connected to the computer the reaction to the release of fluorescence intensity response curve of time. On this basis, the response curve 400 s are integral within the value as the corresponding sample concentration of output signal, the output voltage and concentration curve, the linear correlation coefficient 0.986, can meet the test requirements. This article is designed for total bacteria detection of detector with high speed, high automation, easy operation, compact, portable, and other characteristics, in port, health authorities, food production sector, and so on, the environment, the health situation for scene fast examination area has good prospects.