Name: inactivation of pathogens via visible-light photolysis of riboflavin-5&#8242;-phosphate
Uploaded: 2022-04-06T13:40:00
Description: Watch this Scientific Journal Video about Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5â€²-Phosphate at JoVE.com

The authors are grateful to Dr. Tak-Wah Wong and Mr. Zong-Jhe Hsieh for their support with experiments.

1. Photolysis system setup
<ol>
	<li>Mount six light-emitting diodes (LED) (DC 12 V) on the inside of an opaque plastic cup (8 cm x 7 cm) as shown in Figure 1 to establish a photolysis system31.</li>
	<li>Add reactants (see below) into the glass test tubes (13 mm in diameter and 100 mm in height) and secure the tubes at the top of the cup as shown in Figure 1. Place the experimental setup in a room with a steady tempera...

A photosensitizer increases the photochemical reaction of chemical compounds to generate ROS. Pathogenic microorganisms can be inactivated by light irradiation in the presence of photosensitizers. This study determines the aPDI of S. aureus due to ROS generated by violet light irradiation of an exogenous photosensitizer, FMN.
As shown in Figure 3, for FMN, the absorbance at 444 nm is reduced significantly after 5 min of irradiation using violet or blue li...

Riboflavin-5&#8242;-phosphate (FMN) is formed by phosphorylation at the riboflavin 5&#8242;-position of the ribityl side-chain and is required by all flavoproteins for numerous cellular processes to generate energy. It also plays the role of vitamin for some functions in the human body1. FMN is approximately 200 times more soluble in water than riboflavin2.
The antibacterial photodynamic inactivation (aPDI) of bacteria is an efficient way to control resistance to bacteria3,4 because it does not depend on the mode of bacterial resistance. Clinically, aPDI is used to treat soft tissue infections in order to decrease infection of nosocomial skin due to multi-resistant bacteria5,6,7,8,9. aPDI also produces cell death by generating reactive oxygen species (ROS). ROS, such as superoxide radicals (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />), singlet oxygen, hydroxyl radicals (&#8226;OH), and peroxyl radicals, are free radicals or molecules that contain reactive oxygen10,11,12 and are normally reactive13. Similar to DNA damage that is caused by ROS, membrane peroxidation and destruction of endothelial cells are also adverse biochemical reactions that are attributed to ROS in cells12.
The use of aPDI for pathogenic bacteria&#160;involves a visible or UV light source to inactivate microorganisms in the presence of chemical compounds, such as methylthioninium chloride14, PEI-ce6 conjugate15, porphyrin16, titanium dioxide17, toluidine blue O18, and zinc oxide nanoparticles19. Toluidine blue O and methylene blue are phenothiazinium dyes and methylene blue has toxic properties. Zinc oxide nanoparticles and UV irradiation are linked to adverse health and environmental effects. As such, the exploitation of a reliable, secure, and simple photosensitizer via photolysis under visible irradiation deserves further study.
The micronutrient, riboflavin or FMN, is not toxic and is indeed used for food manufacturing or feeding20. Both FMN and riboflavin are highly sensitive to light irradiation2. Under UV1,2,21,22,23 and blue light irradiation10,24, these two compounds achieve an excited state. The activated riboflavin or FMN that is produced by photolysis is promoted to its triplet state and ROS are generated simultaneously2,25. Kumar et al. reported that riboflavin activated by UV light selectively causes increased injury to the guanine moiety of DNA in pathogenic microorganisms26. Under irradiation by UV light, photodynamically activated riboflavin is demonstrated to promote the generation of 8-OH-dG, which is a biomarker for oxidative stress, in double-stranded DNA27. It is reported that S. aureus and E. coli are deactivated by ROS during riboflavin or FMN photolysis10,24,28. A previous study by the authors showed that the photolytic reactions involving riboflavin and FMN reduce crystal violet, a triarylmethane dye and an antibacterial agent that generates <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" />, and eliminate most of the antimicrobial capability of crystal violet28. When flavin adenine dinucleotide or FMN is irradiated by blue light, the resulting ROS produce apoptosis in HeLa cells for their poisoning in vitro29. Using photochemical treatment in the presence of riboflavin, Cui et al. inactivated lymphocytes by inhibiting their proliferation and cytokine production30.
The photolysis of riboflavin is used for the inactivation of blood pathogen by UV10,24, but blood components can be impaired under UV light irradiation30. It is also reported that platelets exposed to UV progressively enhance the performance of the activation markers P-selectin and LIMP-CD63 on their membranes. The cytotoxicity of UV and high-intensity irradiation needs to be investigated and a photosensitizer that is uncomplicated and safe during an FMN photoreaction involving visible light would be of great use.
Light of shorter wavelengths has more energy and is much more likely to cause severe damage to cells. However, in the presence of a suitable photosensitizer, irradiation with low-intensity violet light can inhibit pathogenic microorganisms. The photosensitization and the generation of <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> by FMN when irradiated with violet light is thus important to study, in order to determine the pathway by which ROS from FMN photolysis increases the inactivation of bacteria.
Antimicrobial control is a common issue and the development of new antibiotics frequently takes decades. After irradiation with violet light, photo&#173;inactivation that is intermediated by FMN can annihilate environmental pathogenic bacteria. This study presents an effective antimicrobial protocol in vitro using violet light for driving FMN photolysis and thus generating <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01.jpg" style="margin: auto;" /> for aPDI. The microbial viability of S. aureus is used to determine the feasibility of FMN-induced aPDI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blue, green and red LED lights</td>
			<td>Vita LED Technologies Co., Tainan, Taiwan</td>
			<td></td>
			<td>DC 12 V 5050</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>190186</td>
			<td></td>
		</tr>
		<tr>
			<td>Infrared thermometer</td>
			<td>Raytek Co. Santa Cruz, CA</td>
			<td>MT4</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Difco Co., NJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>1.05707</td>
			<td></td>
		</tr>
		<tr>
			<td>NBT</td>
			<td>Bio Basic, Inc. Markham, Ontario, Canada</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>China tech Co., New Taipei City, Taiwan</td>
			<td>YP30-3-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Riboflavin 5&#8242;-phosphate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>R7774</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase</td>
			<td>New England BioLabs, Inc. Ipswich, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solar power meter</td>
			<td>Tenmars Electronics Co., Taipei, Taiwan</td>
			<td>TM-207</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus subsp. aureus</td>
			<td>Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan</td>
			<td>10451</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis optical spectrometer</td>
			<td>Ocean Optics, Dunedin, FL</td>
			<td>USB4000</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrophotometer</td>
			<td>Hitachi High-Tech Science Corporation,Tokyo, Japan</td>
			<td>U-2900</td>
			<td></td>
		</tr>
		<tr>
			<td>Violet LED</td>
			<td>Long-hui Electronic Co., LTD, Dongguan, China</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inactivation of pathogens via visible-light photolysis of riboflavin-5&#8242;-phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion (<img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" />) generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive <img alt="Equation 1" src="/files/ftp_upload/63531/63531eq01v3.png" style="margin: auto;" /> species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Effect of light wavelength on FMN 
The absorbance spectra of 0.1 mM FMN that is irradiated using blue, green, red, and violet LEDs are shown in Figure 3. There are two peaks for FMN (372 nm and 444 nm) for the dark control. Green and red light have no effect because changes in the spectra are insignificant. On the other hand, the respective absorbance of FMN at 444 nm is reduced by about 19% and 34%, respectively,&#160;after blue and violet light irradiation at 10 W/m<s...

Watch this Scientific Journal Video about Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5â€²-Phosphate at JoVE.com

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Here, we present a protocol to inactivate pathogenic bacteria with reactive oxygen species produced during photolysis of flavin mononucleotide (FMN) under blue and violet light irradiation of low intensity. FMN photolysis is demonstrated to be a simple and safe method for sanitary processes.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5&#8242;-Phosphate

Riboflavin-5&#39;-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can ...

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.

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