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Received date: August 08, 2011; Accepted date: December 12, 2011; Published date: December 14, 2011
Citation: Saroj H, Dhote DS (2011) Design of Bioluminescence Sensor System for Rapid Detection of Bacteria in Samples without Cultivation. J Biotechnol Biomaterial 1:118. doi:10.4172/2155-952X.1000118
Copyright: © 2011 Saroj H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Based on the principle of ATP bioluminescent reaction, we design a portable bioluminescence sensor system to detect bacteria in samples rapidly, which included an optical sensing cell, a flow injection unit and a center processing unit. A fast ATP extraction technique was developed by using trichloro acetic acid as extraction reagent Experimental results showed that the intracellular ATP could be released from bacterial cells sufficiently within 5 min by TCA In the presence of Lucifer ace the reaction between ATP and lucifer5in would generate light and the luminescence intensity RLU was proportional to the amount of bacteria existed in the sample. The dynamic range of the sensor system is 10-107 CFU /ml. The linear regression coefficient was 0.976, and the detection limit was 10CFU /ml. The whole testing time needed was about 10 min. Compared with the plate count method, bacteria concentration in samples could be detected rapidly by using the portable sensor system without cultivation.
ATP; Portable; Bioluminescence sensor system; Trichloroacetic acid; Luminescence intensity
It is in the urgent need to develop a rapid method that can detect bacterial count in food In pharmaceutical, brewing and beverage companies, the plate count techniques is a classical method for evaluating microbial quality which has been used for more than a century However, this technique requires incubation periods of 24-28h and the detecting procedures are laborious and need skilled based persons.
Currently the bioluminescence assay using the firefly ATP – dependent luciferin –luciferase system become a popular method for ATP determination due to its high sensitivity and specificity. When luciferan - luciferase system is used to measure intracellular ATP, one of the most important steps is to extract ATP from intact cells. Most commonly used methods for ATP extraction include ethanol butanol boiling in buffer solutions continuous DC voltage and ultrasound. Among the ATP extraction methods mentioned the addition of ethanol and butanol would inhibit the activity of subsequent luciferase. Boiling water could accomplish two missions in one step for extracting intracellular ATP and inhibiting ATP hydrolyzing enzymes simultaneously, which may deplete ATP content in the cell extract resulting in low ATP bioluminescence, finally the yield of ATP was low and high content of cells about 104 –105 Cells/ml were required for ATP determination. Although a survey of the literature showed that the extraction efficiency of DC voltage and ultrasound methods was high, these physical extraction methods demanded special equipments and restricted their applications of DC.
Voltage and ultrasound methods were high. These physical extraction methods demanded special equipments and restricted their application onsite. To improve the extraction, we found that cellular ATP could be extracted from intact cells conveniently and efficiently by using trichloroacetic acid, Because of its strong acid nature, TCA can cause increment of the cell wall pore size and degradation of the bacterial membrane, cellular ATP could pass through cell wall easily and ATP hydrolyzing enzymes in bacterial cells could be inactivated simultaneously. After isometric neutralization, the solution could be neutralized easily without using a PH meter and TCA interfacing with the luciferin –luciferase system would be eliminated entirely. Consequently, the TCA method was simple and convenient, and suited to be used in fast evolution of microbial quality. Basing on the bioluminescence mechanism of ATP and the TCA extraction principles, a portable bioluminescence sensor system which included an optical sensing cell, a flow injection unit and a center processing unit as well as functions of data acquisition, conversion and dynamically displaying 9 is proposed and established in the presented work.
With this portable sensor system, total bacterial count in samples could be detected rapidly without cultivation. The dynamic range, detection limit, and sensitivity of the detecting method were also evaluated.
Lucifer and Lucifer’s Magnesium acetate tetra hydrate, Diathiothreitol, albumin bovine serum is used.
Construction of sensor system
A portable system which was developed to measure the light emitted by the bioluminescent reaction in our study: A center processing unit with digital / analog, analog /digital convertor interface was taken as main center of the system. The control signal was sent to a flow injection unit via the D/A interface. So that the luciferan-luciferase reagent was injected into an optical sensing cell automatically. With a weak light detector reaction was acquired and converted to electric signals. The results were dynamically displayed via a display module .In order to reducing the influence of the nature light outside the PMT and the optical sensing cell were packaged in a dark chamber.
Bacterial culture and colony counting: An aliquot of 50 μl bacterial samples was suspended into 0.5ml of sterilized Triacetate buffer solution containing 17mmol/NaCl. The culture was serially diluted by decade and an aliquot of 20 micro liter culture which was 102 and 103 diluted was spread on the selective plates Each sample was enumerated n triplicate .The plates were incubated in an inkwell instrument at 35°C for 25h Colonies on the plate were counted by a colony counter automatically to obtain the number of CFU of bacteria per ml in the sample.
ATP Measurement procedure: Series of standard ATP solution were prepared with sterilized Triacetate buffer solution. In this protocol aliquot of 30 μl of the ATP solution was added to an optical sensing cell and aliquot of 270 μl of Lucifer and lactiferous reagent was added subsequently. The luminescence patterns and luminescence intensity were recorded by using the portable sensor system simultaneously luminescence intensity was recorded as relative light unit.
Total bacterial count measurement procedure: In this protocol aliquots l of 30 μl of the bacterial samples was added to the optical sensing cell and first retreated with 30 μl of cell extracting reagent containing 0.06 mol/l TCA. After an incubation time of 5 min at 25, the extract was neutralized by addition of 30 micro liter of the neutralization reagent contain 0.06 mol /NAOH. Aliqua lit of 210 μl of Lucifer an –Lucifer’s reagent was added and the bioluminescence was immediately measured with the portable sensor system.
Selection of emission wavelength
Since the wavelength range emitted by the bioluminescence reacreaction was different with various luciferan – luferase system, first of all we obtained the wavelength scan of the bioluminescence sensor by using a fluorescence spectrometer at room temperature. In the wavelength range from 200nm to 900nm a strong peak of emission spectra appeared at 550nm, indicating that the light output bioluminescence sensor was entirely centered at 550nm. Therefore the emission wavelength of the bioluminescence reaction was selected at 550nm.
Effects of pH on bioluminescence reaction
PH Studies were performed by using above standard ATP bioluminescence procedure adopting a standard ATP solution with a concentration of 10-8 mol/l and varying the PH of the triacetate buffer through addition of concentrated TCA or NAOH solution. Measurements of PH were performed with a microprocessor PH meter. The effects of PH in the range of 4 to 10 in the ATP bioluminescence reaction were examined. Results revealed that PH was significant factor in luciferan –luciferase reactions and when PH was between 7 and 8 higher luminescence intensity was produced Strong acid or strong base would cause inactivation of luciferase. Therefore the Trist ate buffer of PH 7.8 was chosen for the following experiments.
The sensor system may be employed as a practical method to detect bacterial count in samples without cultivation. The portable device is rapid and sensitive enough to use in microbial quality control.
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