This protocol uses visible violet light irradiation to induce flavin mononucleotide photolysis. This produces a large amount of free radicals, which inhibit pathogenic bacteria such as Staphylococcus aureus and E.coli. We have tested flavin mononucleotide as an appropriate photosensitizer.
In this protocol, we use visible violet light which is safe and doesn't require expensive equipment. This method can be used for the therapy of injured skin or infected subcutaneous tissues by inserting an optical fiber for illumination. It can also be applied to the sanitation practice in food industry.
Demonstrating the procedure will be Tang-Yu Chen, a graduate student from my laboratory To begin, mount six light-emitting diodes of 12 volts each on the inside of an opaque plastic cup. Take a glass test tube with a diameter of 13 millimeters and a height of 100 millimeters and add the reactants to the tube. Secure the tubes at the top of the cup and place this setup at a constant temperature of 25 three degrees Celsius.
Monitor and record the temperature of test units throughout the photolytic reactions by an infrared thermometer. Next, prepare a 0.1-millimolar FMN solution in 100-millimolar potassium phosphate buffer at pH 7.8. Expose three milliliters of FMN samples each to blue, green, red, or violet light for five minutes.
In parallel, keep three milliliters of FMN solution in the dark as control. Place the sample into a UV-visible spectrophotometer and record the absorbance of irradiated samples in the 250-to 750-nanometer range. To prepare for the detection of superoxide radicals, first add 109.3 milligrams of L-methionine to 73.3 milliliters of 100-millimolar phosphate buffer at pH 7.8.
Into the solution, add 10 milligrams of NBT powder and 1.5 milliliters of 0.117-millimolar FMN. Expose one milliliter of the reaction solution to blue or violet LED light for one to five minutes. Record the absorbance of 516 nanometers to detect formazan, which is produced by the photochemical reaction of NBT.
Inoculate a colony of Staphylococcus aureus into 10 milliliters of lysogeny broth in a 15-milliliter screw-capped test tube. Grow the culture at 37 degrees Celsius for 16 hours in a shaker. Transfer 0.5 milliliters of the culture to a 1.5-milliliter centrifuge tube.
Dilute the culture to an optical density of 0.5 at 600 nanometers by adding sterilized water. Then, transfer 0.5 milliliters of the culture to a 1.5-milliliter centrifuge tube. Centrifuge at 14, 000 G for 10 minutes and decant the supernatant.
Add one milliliter of FMN buffered solution to the cell pellet and vortex. For irradiated control, add one milliliter of phosphate buffer. Transfer one milliliter of bacterial cell solution containing 30-micromolar FMN and phosphate buffer into glass tubes.
Place them for a radiation under violet light at 10 watts per meter square for 30 minutes. Then, transfer one milliliter of bacterial cell solution containing 30-60-and 120-micromolar FMN and phosphate buffer in glass tubes. Irradiate with blue light for 120 minutes at 20 watts per meter square.
Also, irradiate a tube containing one milliliter of 120-micromolar FMN for 60 minutes. After irradiation, add 0.2 milliliters of the reaction solution onto a luria agar plate. Spread the solution using an L-shaped glass rod and incubate overnight at 37 degrees Celsius.
On the next day, calculate the viable plate count and inactivation rate of Staphylococcus aureus. Prepare the staphylococcus aureus samples as described earlier to obtain a cell pellet. To 75 milliliters of phosphate buffer add 0.1093 grams of L-methionine, 0.1 gram of NBT, and 25 milliliters of FMN solution.
Add one milliliter of each reactant solution with varying FMN concentrations to the cell pellets and vortex. Irradiate the solutions under violet light at 10 watts per meter square for 10 minutes. Centrifuge the mixture at 14, 000 G for 10 minutes and decant the supernatant.
Resuspend the pellet in one milliliter of dimethyl sulfoxide to extract the reduced NBT. Record the absorbance at 560 nanometers. Irradiation of FMN with green and red light showed no effect on the peaks at 372 and 444 nanometers compared to dark control, whereas the reduction in absorbance of FMN at 444 nanometers was observed under blue and violet light, indicating increased photolysis.
Detection of superoxide radicals formed during FMN photolysis under blue or violet light was done using NBT reduction. The photochemical effect of FMN was dependent on the irradiation time. FMN photolysis using blue or violet light irradiation resulted in significant inactivation of Staphylococcus aureus.
Dose-dependent inactivation was observed for blue light irradiation, with 97%inactivation achieved for 120-micromolar FMN. Under violet light, an inactivation rate of 96%was achieved for staphylococcus aureus, with 30-micromolar FMN showing higher efficacy for bacterial inactivation. NBT reduction showed that the photolytic effect of FMN under violet light in Staphylococcus aureus was proportional to FMN concentration.
In untreated conditions, a lower abundance of superoxide radicals was indicated by low absorbance at 560 nanometers, whereas higher absorbance values in FMN treatment showed abundant production of superoxide radicals, resulting in increased photochemical effect. It is critical to have a good LED light source. The spectral width of the violet light regime must be narrow to avoid any overlap with UV absorption, as UV radiation may be hazardous.
Following the procedure, other chemical compounds such as tetracycline, doxycycline, catechin, and can be tested for photolysis similar to FMN.
