Investigation of a Blue Light LED Device to Suppress Wound Pathogens Using a Collagen-Based Synthetic Skin Model

Our research investigates how a 405-nanometer blue light can safely suppress wound pathogens using a collagen-based synthetic skin model. Most studies use broth cultures, agar plates, or animal models. We introduce realistic, reproducible skin mimicking platform for phototherapy testing.

To begin, obtain the desired bacterial and fungal strains. Pick a single isolated colony and aseptically inoculate it into five milliliters of tryptic soy broth. Once all strains are inoculated, incubate the cultures.

After the desired incubation time, adjust the turbidity using sterile PBS to the 0.5 McFarland standard for antimicrobial assays. Next, take previously prepared collagen-based hydrated synthetic skin sections and inoculate each section with 100 microliters of microbial suspension. Using a sterile spreader, distribute the suspension evenly across the surface.

Then air dry the inoculated samples in a sterile chamber under ambient conditions. Assign the samples to three treatment groups for varying light exposure. For irradiation, mount the blue light LED device onto an aluminum heat sink.

Use a calibrated optical power meter to measure irradiance at the exact treatment plane. Adjust the probe height to account for sensor thickness. Now adjust the throw distance of the LED to standardize the working irradiance to 30 milliwatts per square centimeter.

Place the inoculated skin swatches from group B aseptically into sterile Petri dishes without lids. Position the dishes directly under the blue light LED and irradiate for 15 minutes to achieve a target fluence of 27 joules per square centimeter. Similarly, irradiate group C under UVC light for two minutes to achieve a target fluence of 3.6 joules per square centimeter.

Following irradiation, transfer the treated synthetic skin swatch into a sterile 15-milliliter tube containing nine milliliters of PBS at pH 7.4 and vortex it gently to dislodge surface-adherent microbes. Now prepare tenfold serial dilutions of the suspension. Plate 100 microliters from the selected dilutions onto the agar plates, and incubate the plates.

At the end of the incubation period, count the number of colonies and record the number of colony-forming units. Calculate microbial reduction using the presented equations. To simulate nosocomial airborne exposure, use a customized acrylic aerosol chamber under controlled environmental conditions.

Place sterile agar plates inside the chamber to monitor microbial fallout activity. Then position the sterile skin swatches inside the chamber. Using a nebulizer, aerosolize two milliliters of microbial suspension into the chamber and expose the swatches to continuous blue light at 10 microwatts per square centimeter from a distance of 20 centimeters for 30 minutes.

For the control, repeat identical chamber conditions with the blue light device turned off. Quantify viable counts from the swatches by standard plate count analysis. For FTIR analysis, obtain synthetic skin swatches and hydrate them as per the manufacturer's instructions.

Divide the hydrated swatches into four sets on a sterile surface. Expose two sets of swatches to 405-nanometer blue light at an intensity of 30 milliwatts per square centimeter. Expose one set for 15 minutes and the other for 60 minutes.

Similarly, expose two sets of swatches to 265-nanometer UVC light at an intensity of three milliwatts per square centimeter, with one set exposed for 15 minutes and the other for 60 minutes. After irradiation, gently blot the samples to remove surface moisture. Analyze the samples using FTIR spectroscopy to assess chemical and structural changes.

Configure the FTIR instrument in ATR mode and prepare the crystal surface for sample placement. Place the swatch onto the ATR crystal for spectral acquisition. Perform direct spectral comparisons between untreated control and irradiated samples.

Next, use the untreated control swatch as a background reference and scan the irradiated samples to obtain baseline subtracted spectra. The blue light LED device demonstrated significant antimicrobial efficacy against all tested wound pathogens on synthetic skin models. Average log reductions reveal species-dependent differences in susceptibility, with Klebsiella pneumoniae demonstrating the greatest sensitivity to blue light and Staphylococcus aureus showing comparatively lower, though still significant reduction.

UVC irradiation achieved higher microbial reductions across all tested species. Blue light and UVC irradiation demonstrated broad spectrum antimicrobial activity against clinically relevant bacterial pathogens, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. Exposure to blue light caused a time-dependent reduction in all three wound pathogens, with Escherichia coli and Candida albicans inactivating rapidly, and Staphylococcus aureus showing a slower progressive decline.

Blue light efficacy was further assessed under simulated airborne contamination conditions. Continuous exposure significantly decreased microbial deposition on synthetic skin surfaces relative to untreated controls for every species evaluated. Baseline-corrected ATR-FTIR spectra indicated that UVC treatment reduced the intensity of amide peaks and enhanced carbon-oxygen stretching vibrations, reflecting collagen breakdown.

In contrast, blue light exposure did not induce any significant spectral or chemical changes. This method evaluates phototherapy light sources by testing antimicrobial efficacy while preserving collagen integrity and safety on skin-like surfaces. The key challenge in this protocol is maintaining uniform irradiance and accurate fluence control of the light sources to ensure reproducible antimicrobial results.

This protocol can be extended to biofilm testing, oxidative stress analysis, next-generation light device optimization, and screening of similar novel therapeutic interventions.