This paper intended to develop an automatic multi-channel luminometer/ fluorometer equipped with a sonificator, which is simple to operate and can detect DNA, ATP, and protein toxins in a short time by breaking cell membranes and using fluorescence and luminescence reaction on site. With the gram-ne...
This paper intended to develop an automatic multi-channel luminometer/ fluorometer equipped with a sonificator, which is simple to operate and can detect DNA, ATP, and protein toxins in a short time by breaking cell membranes and using fluorescence and luminescence reaction on site. With the gram-negative bacteria, Escherichia coli, the gram-positive bacteria, Bacillus globigii and Streptococcus epidermidis, the spore, Bacillus anthracis Δ-sterne, and protein toxin, BSA, this paper established the optimum conditions for the sonificator to be built on the automatic multi-channel luminometer/fluorometer. * A thesis for the degree of Doctor in February 2007. It was found that the deeper the probe tip of the sonificator sank in the sample solution, the higher energy was approved for the sample solution and the greater the effect of breaking the cells became. The larger diameter the probe tip of the sonificator had, the greater the effect of breaking the cells became. sonification with 13 ㎜ diameter probe tip for 20 seconds was found most efficient. In the gram-negative bacteria, sonification was done better than in the gram-positive bacteria, and in the Bacillus globigii, sonification was done better than in the Streptococcus epidermidis. Under the selected optimum sonification conditions, the detection limit for all the Escherichia coli, Bacillus globigii, and Streptococcus epidermidis was 5×105 CFU/㎖. The Bacillus anthracis Δ Sterne testing group to which zirconia bead was added and sonification was done, showed higher fluorescence value than the testing group to which no zirconia bead was added and sonification was done, which means that addition of zirconia bead played an important role in disruption of the spore. In the testing group to which zirconia bead was added, it was possible to detect the spore at 106 CFU/㎖ concentration. In the testing group to which no zirconia bead was added, it was possible to detect the spore at the concentration of 5×107 CFU/㎖. Protein should be measured within 1 minute after the reaction starts at the concentration of more than 0.7 X fluorescence sample solution, and it should be measured within 2～3 minutes after the reaction starts at the concentration of less than 0.3 X fluorescence sample solution. One of the best-known collisional quenchers is molecular oxygen, which quenches almost all known fluorophores. The oxygen in air which is dissolved in the solution, causes collisional quenching. Therefore, if sonification or heat treatment is given, the oxygen is removed and the quenching effect is remarkably reduced and accordingly, the fluorescence value goes up. In the BSA sample solution of less than 1 ㎍/㎖, BSA of 0.125 ㎍/㎖ could be detected better through sonification, which tells that at the low concentration of protein, sonification is more advantageous. The correlation between ATP concentration and luminescence quantity showed a linear relationship from 0.5 nM to 50000 nM of ATP. Within 30 seconds, it increased to the maximum and then, went down slowly. As the time of sonification increased, the luminescence amount of ATP went down. When measuring ATP of microorganisms, it was found as the best condition to do sonification for 20 seconds with 13 ㎜ probe tip. Under the selected optimum sonification conditions, the detection limit of Escherichia coli, Bacillus globigii, and Streptococcus epidermidis that the probe tip could detect was 5×105 CFU/㎖. On the basis of the research result, this paper designed and produced an automatic multi-channel luminometer/fluorometer equipped with a sonificator to detect microorganisms as well as protein toxins in a short time. The sonificator using a probe tip of 13 ㎜ diameter is designed in order for the probe tip to sink 18 ㎜ deep in the sample solution. The automatic multi-channel luminometer/fluorometer consists of two fluorescence channels, which are used to detect DNA of microorganisms and protein toxins, and one luminometer channel, which is used to detect APT of microorganisms. It can detect three types of biological substances at the same time and quickly find out biological agents because of its enhancement of reliability of fluorescence and luminescence reaction by automation.
This paper intended to develop an automatic multi-channel luminometer/ fluorometer equipped with a sonificator, which is simple to operate and can detect DNA, ATP, and protein toxins in a short time by breaking cell membranes and using fluorescence and luminescence reaction on site. With the gram-negative bacteria, Escherichia coli, the gram-positive bacteria, Bacillus globigii and Streptococcus epidermidis, the spore, Bacillus anthracis Δ-sterne, and protein toxin, BSA, this paper established the optimum conditions for the sonificator to be built on the automatic multi-channel luminometer/fluorometer. * A thesis for the degree of Doctor in February 2007. It was found that the deeper the probe tip of the sonificator sank in the sample solution, the higher energy was approved for the sample solution and the greater the effect of breaking the cells became. The larger diameter the probe tip of the sonificator had, the greater the effect of breaking the cells became. sonification with 13 ㎜ diameter probe tip for 20 seconds was found most efficient. In the gram-negative bacteria, sonification was done better than in the gram-positive bacteria, and in the Bacillus globigii, sonification was done better than in the Streptococcus epidermidis. Under the selected optimum sonification conditions, the detection limit for all the Escherichia coli, Bacillus globigii, and Streptococcus epidermidis was 5×105 CFU/㎖. The Bacillus anthracis Δ Sterne testing group to which zirconia bead was added and sonification was done, showed higher fluorescence value than the testing group to which no zirconia bead was added and sonification was done, which means that addition of zirconia bead played an important role in disruption of the spore. In the testing group to which zirconia bead was added, it was possible to detect the spore at 106 CFU/㎖ concentration. In the testing group to which no zirconia bead was added, it was possible to detect the spore at the concentration of 5×107 CFU/㎖. Protein should be measured within 1 minute after the reaction starts at the concentration of more than 0.7 X fluorescence sample solution, and it should be measured within 2～3 minutes after the reaction starts at the concentration of less than 0.3 X fluorescence sample solution. One of the best-known collisional quenchers is molecular oxygen, which quenches almost all known fluorophores. The oxygen in air which is dissolved in the solution, causes collisional quenching. Therefore, if sonification or heat treatment is given, the oxygen is removed and the quenching effect is remarkably reduced and accordingly, the fluorescence value goes up. In the BSA sample solution of less than 1 ㎍/㎖, BSA of 0.125 ㎍/㎖ could be detected better through sonification, which tells that at the low concentration of protein, sonification is more advantageous. The correlation between ATP concentration and luminescence quantity showed a linear relationship from 0.5 nM to 50000 nM of ATP. Within 30 seconds, it increased to the maximum and then, went down slowly. As the time of sonification increased, the luminescence amount of ATP went down. When measuring ATP of microorganisms, it was found as the best condition to do sonification for 20 seconds with 13 ㎜ probe tip. Under the selected optimum sonification conditions, the detection limit of Escherichia coli, Bacillus globigii, and Streptococcus epidermidis that the probe tip could detect was 5×105 CFU/㎖. On the basis of the research result, this paper designed and produced an automatic multi-channel luminometer/fluorometer equipped with a sonificator to detect microorganisms as well as protein toxins in a short time. The sonificator using a probe tip of 13 ㎜ diameter is designed in order for the probe tip to sink 18 ㎜ deep in the sample solution. The automatic multi-channel luminometer/fluorometer consists of two fluorescence channels, which are used to detect DNA of microorganisms and protein toxins, and one luminometer channel, which is used to detect APT of microorganisms. It can detect three types of biological substances at the same time and quickly find out biological agents because of its enhancement of reliability of fluorescence and luminescence reaction by automation.
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