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

Summary

A 405 nm blue light LED device demonstrates antimicrobial efficacy against a broad range of wound pathogens when evaluated on a collagen-based synthetic skin model. This simplified in vitro approach offers a reproducible and ethically viable alternative for early-phase evaluation of light-based antimicrobial therapies.

Abstract

Conventional wound management is increasingly challenged by the rise of antimicrobial resistance and the cytotoxic side effects of traditional agents. Phototherapy, particularly using blue light (BL), offers a promising non-invasive and non-contact alternative. This study evaluated the antimicrobial efficacy and safety of a 405 nm BL-LED device against a broad spectrum of medically important wound pathogens using a collagen-based synthetic skin model, emphasizing the novelty of applying this platform for light-based antimicrobial testing. This synthetic skin provides a more realistic environment, mimicking the structure and topography of human skin. Light uniformity mapping showed consistent irradiance across a 10 cm diameter (30.14 ± 0.78 mW/cm²). The device achieved a substantial fluence-dependent microbial log reduction of up to 3.5 at 27 J/cm², demonstrating efficacy considerably higher than that reported in similar studies. Its broad-spectrum activity was confirmed against ESKAPE pathogens and Candida albicans, a significant advantage for managing polymicrobial wound infections. A key finding was the differential susceptibility among species, with Klebsiella pneumoniae exhibiting the highest susceptibility while Staphylococcus aureus proved most resistant. This is likely due to variations in microbial envelope structure and photosensitive molecule content, which affect the reactive oxygen species (ROS). Optical transmission experiments and surface versus embedded-colony analyses revealed no statistical significance, suggesting the potential application of BL to inhibit pathogens beneath the skin. Infrared spectroscopy confirmed that BL exposure, even at high fluences (108 J/cm²), caused no detectable chemical or conformational degradation of the collagen matrix, unlike the damaging effects observed with germicidal UV-C control. Finally, a unique evaluation in an aerosol chamber demonstrated that BL treatment reduced microbial deposition on surfaces by over 95%, highlighting its potential for mitigating nosocomial infections. These findings demonstrate the suitability of skin-mimicking substrates as an accessible alternative for evaluating BL in preclinical testing, thereby eliminating the need for animal models.

Introduction

Wound infections pose a persistent challenge and significant burden on the global healthcare system and affect the quality of life of patients. Wound healing is often complicated by microbial infections that delay the closure of cutaneous wounds, resulting in lesions and chronic wounds predominantly in elderly and diabetic populations. Conventional wound management strategies rely on antibiotics, antiseptics, chemicals, and natural bioactive agents that primarily suppress wound-causing organisms. While these traditional methods are effective in speeding up the healing process by reducing inflammatory cells and enhancing the number of fibroblasts¹, they are associated with many side effects. Many topical antimicrobials used in wound dressings are generally not skin-friendly and may lead to undesirable effects such as skin dehydration, tissue irritation, and cytotoxic effects on cells required for the wound healing process². Antimicrobial resistance (AMR), which is widely reported due to the overuse of antimicrobials³, mainly systemic antibiotic use, threatens the long-term viability of existing pharmacological treatments. Considering these limitations of existing clinical practice, the need for safer and sustainable alternatives for wound care management is emphasized, particularly the non-contact and pain-free methods like phototherapy.

Phototherapy, harnessing the visible spectrum of light, has progressively gained attention as a promising non-invasive and non-pharmacological intervention for various dermatological and therapeutic applications. Its applications extend from general skin rejuvenation⁴ and cosmetic enhancements to targeted treatments for conditions like acne and anti-aging therapies. Among the visible wavelengths (400-760 nm), blue light, typically ranging from 400 nm to 480 nm, has garnered substantial scientific and clinical interest due to its multifaceted therapeutic potential⁵. In contrast to ultraviolet (UV) radiation, particularly the UV-C band (100-280 nm), which is widely employed for surface disinfection due to its germicidal properties but carries well-documented risks of DNA damage, carcinogenesis, and skin cancer, blue light is classified as non-ionizing radiation. This fundamental difference renders blue light inherently safer for human tissues⁵, as it does not possess the energy to directly damage cellular DNA or induce mutagenic effects.

The antimicrobial activity of blue light has been reported against both Gram-positive⁶ and Gram-negative⁷ bacteria. Some clinical trials have been conducted to validate the use of blue light therapy against common infections or disorders⁸, for example, the suppression of Propionibacterium acne that causes acne vulgaris^9, and the treatment for stomach infections¹⁰ caused by Helicobacter pylori. However, clinical trials investigating the antimicrobial efficacy of wound infections are not reported. A growing number of recent in vitro studies demonstrate that violet-blue light (400-420 nm) is capable of inactivating multiple ESKAPE pathogens while maintaining minimal cytotoxicity to mammalian cells¹¹. Recently, blue light within the 410-430 nm range has been shown to modulate fibroblast activity and collagen deposition, promote cell proliferation, regulate inflammatory responses, and generate reactive oxygen species (ROS) via endogenous porphyrin excitation that disrupts bacterial cells¹², making it a promising candidate for wound-healing applications.

However, the complexity of wound healing, determined by multiple variables including individual variance, type of wounds, and physiological and pre-existing pathological conditions, poses significant challenges for research. Direct in vivo testing of wound therapies remains complicated due to biological variability, ethical considerations, and the complexity of wound environments. Animal models, though commonly used, often fail to accurately represent human skin physiology and are limited by stringent regulatory requirements¹³. Wound healing studies involving clinical trials are rare and often rarely representative due to small sample size, necessitating large statistical samples, robust controls, and stringent ethical approvals. To overcome these barriers, in vitro models that replicate human skin properties have emerged as effective platforms for initial screening¹⁴. Collagen-based synthetic skin systems, such as VITRO-Skin, mimic human skin in pH, moisture retention, and surface texture, and are ideal for testing external interventions under controlled conditions¹⁵. Although several studies have shown broad-spectrum antimicrobial effects of blue light, most have used broth cultures, agar plates, or animal models, with limited application to skin-like substrates.

Building upon the demonstrated potential of blue light and the advantages of in vitro models, the current study aims to test the efficacy of a Light Emitting Diode (LED) device operating at a peak wavelength of 405 nm for suppressing common wound pathogens. We introduce a novel evaluation methodology for assessing wound healing efficacy based on a simplified microbiome model applied to collagen-based synthetic skin. It also compares antimicrobial efficacy and the impact on the skin topography of Light Emitting Diode-Generated Blue Light (LED-BL) against germicidal UV-C fluence. Although this method lacks the complexity of biological systems, it adopts a pragmatic approach that allows for highly controlled and reproducible experiments that circumvent the variability and ethical complexities associated with in vivo studies and clinical trials, thereby accelerating the preliminary evaluation and initial screening of similar novel therapeutic interventions. The central innovation of this work lies in applying a collagen-based skin model as a reproducible, controllable platform for evaluating blue light antimicrobial efficacy, accompanied by optical penetration, uniformity, and FTIR-based safety analyses.

Protocol

1. Preparation of synthetic skin samples

A collagen-based synthetic skin model (VITRO-Skin) sheet was cut into rectangular swatches measuring 5.0 cm × 2.5 cm under aseptic conditions in a biosafety cabinet. Each swatch was placed into a sterile Petri dish and stored at 4 °C until use. Prior to inoculation, the samples were equilibrated to room temperature and hydrated in a humidity chamber according to the manufacturer's instructions to mimic the hydration level of human skin.

2. Preparation of microbial cultures

American Type Culture Collection (ATCC) reference strains, including Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Enterobacter aerogenes (ATCC 13048), Pseudomonas aeruginosa (ATCC 27853), Acinetobacter baumannii (ATCC 19606), Klebsiella pneumoniae (ATCC 13883), and Candida albicans (ATCC 10231), were used. Single colonies were inoculated into 5 mL Tryptic Soy Broth (TSB) under aseptic conditions. Bacterial cultures were incubated at 35 °C for 24 h, and fungal cultures were incubated at 22 °C for 5 days. Following incubation, turbidity was adjusted to 0.5 McFarland Standard (approximately 1.5 × 10⁸ CFU/mL) using sterile Phosphate Buffered Saline (PBS) for antimicrobial assays.

3. Inoculation of synthetic skin models

Each hydrated synthetic skin section was inoculated with 100 µL of microbial suspension and evenly spread using a sterile disposable spreader. The inoculated surfaces were air-dried for 15 min in a sterile chamber under ambient laboratory conditions (22-24 °C, 60%-70% RH). Samples were assigned to three groups: untreated control (Group A), light source (blue light LED, 405 nm) irradiation (Group B), and UV-C LED (265 nm) irradiation (Group C). Figure 1 shows the schematic representation of the experiments in evaluating the antimicrobial efficacy of the light source using the synthetic skin model.

4. Light source configuration and irradiation

A 405 nm blue-light LED device was mounted on an aluminum heat sink to prevent heat accumulation and ensure thermally safe irradiation of synthetic skin swatches (Figure 2). Irradiance was measured at the exact treatment plane using a calibrated optical power meter, with the probe height adjusted to account for sensor thickness. The working irradiance was standardized to 30 mW/cm² by adjusting the throw distance of the LED.

Illumination uniformity across the treatment field was verified by measuring irradiance at 16 evenly spaced points, yielding an average of 30.14 ± 0.78 mW/cm² (Figure 3). For baseline comparison of germicidal effects, UV-C treatments were performed using a 265 nm LED source delivering 3 mW/cm² at a 20-cm distance for 2 min.

Fluence was calculated as irradiance × exposure time and corresponded to 27 J/cm² for blue light (30 mW/cm² × 900 s) and 3.6 J/cm² for UV-C.

For antimicrobial assays, hydrated synthetic skin swatches inoculated with microbial suspensions were placed aseptically into sterile Petri dishes (lids removed) and positioned directly under the blue-light LED. Samples assigned to the irradiation group (Group B) were exposed continuously until the target fluence was reached (15 min). The 15-min exposure duration (27 J/cm²) was selected based on preliminary time-kill experiments, which demonstrated consistent antimicrobial efficacy across species while representing a practical exposure duration for potential clinical or point-of-care applications. The temperature of each swatch was monitored every 5 min using a non-contact infrared thermometer to confirm that no significant heating occurred during illumination. Temperature monitoring revealed no measurable increase in surface temperature (>2 °C) during blue-light irradiation, confirming that the observed antimicrobial effects were non-thermal.

5. Antimicrobial efficacy of blue light LED and UV-C against wound pathogens

Following treatment, each synthetic skin swatch was transferred into a sterile 15 mL tube containing 9.0 mL PBS (pH 7.4). The tubes were vortexed gently for 1 min to dislodge surface-adherent microbes. Serial ten-fold dilutions were prepared, and 100 µL aliquots were plated on Tryptic Soy Agar (TSA) for bacteria and Sabouraud Dextrose Agar (SDA) for fungi. Plates were incubated (bacteria at 35 °C for 24 h; fungi at 22 °C for 5 days), and colony-forming units (CFUs) were enumerated. Microbial reduction was quantified using the following equations:

Log Reduction = log₁₀(A) - log₁₀(B)

Percent Reduction = ((A - B)/A) × 100

Where A = CFU count from control; B = CFU count from treated samples.

Negative controls consisted of media-only plates and PBS to verify sterility, while untreated inoculated swatches were used for comparing the treatment efficacy. Positive control plates consisted of inoculated ATCC microbial suspensions to confirm culture viability. All experimental steps were performed under aseptic conditions.

6. Time-dependent antimicrobial efficacy of blue light irradiation

To assess the fluence-dependent effect, blue light exposure durations were set to 2 min, 5 min, 10 min, 15 min, 20 min, and 30 min. Antimicrobial activity was evaluated by CFU enumeration as above, allowing analysis of dose-dependent relationships.

7. Blue light LED efficacy in an aerosolized chamber simulating nosocomial conditions

A customized acrylic aerosol chamber, under controlled environmental conditions, was used to simulate nosocomial airborne exposure (Figure 4). Aerosolized microbial suspensions (~1.5 x 10⁶ CFU/mL) were introduced using a nebulizer, and synthetic skin swatches were exposed for 30 min. The blue light was directed at the swatches throughout exposure (10 µW/cm², 20 cm distance). In the control experiment, identical exposure conditions were applied, but the BL-LED device remained turned off. Microbial cell densities within the aerosol chamber were monitored using fall-out activity measurements, in which the agar plates were placed inside the chamber prior to exposure. Viable microbial cells adhered to the synthetic skin swatches (2 cm x 2 cm) were quantified using standard plate count analysis as described above. This low irradiance (10 µW/cm²) was selected to mimic real-world ambient lighting conditions in healthcare settings, where continuous high-intensity irradiation is not feasible.

8. Optical transmission and embedded-colony testing

1. Optical transmission through synthetic skin substrate
   To quantify blue-light penetration through synthetic skin, irradiance was measured using a calibrated optical power meter positioned at the treatment plane. BL intensity (405 nm, 30 mW/cm²) was recorded with and without hydrated synthetic skin swatches placed horizontally between the LED and the detector. Transmission (%) was calculated as the ratio of transmitted irradiance (with skin) to incident irradiance (no skin).
   Transmission in aqueous conditions was evaluated using a UV-Viz spectrophotometer. A hydrated synthetic skin strip was inserted vertically into a standard polystyrene cuvette filled with deionized water. The cuvette filled with water alone served as the blank. Absorbance at 405 nm was recorded, and percent transmission was calculated using %T = 10^(-A) × 100.
2. Functional validation using embedded-colony assay
   Surface- and embedded-colony microbial reduction assays were performed as a functional correlate of transmission, where swatches were inoculated on one surface but irradiated from the opposite (bottom) side to simulate microbes residing beneath the skin surface. CFUs were quantified as described above in the antimicrobial efficacy section.

9. FT-IR spectroscopy for synthetic skin degradation after blue light and UV-C exposure

To assess potential chemical and structural changes to the collagen-based synthetic skin matrix due to light exposure, Fourier Transform Infrared (FT-IR) spectroscopy was performed. Synthetic skin swatches (2 cm × 2 cm) were hydrated per manufacturer instructions, identically to swatches used in the microbial reduction assays. Hydrated samples were exposed to either 405 nm blue light (30 mW/cm²) or 265 nm UV-C (3 mW/cm²) for 15 min (standard exposure, 27 J/cm²) or 60 min (worst-case stress condition). After irradiation, samples were gently blotted to remove surface moisture and immediately analyzed by FT-IR.

10. ATR-FTIR measurement

Spectra were acquired using an IR spectrophotometer equipped with a compatible analysis software. All samples were analyzed using the Attenuated Total Reflectance (ATR) iD7 module. To isolate spectral changes attributable to irradiation, (1) Direct spectral comparison was performed between untreated controls and irradiated samples. (2) Baseline-subtracted comparisons were performed by using the untreated control swatch as the background reference before scanning the irradiated samples, allowing visualization of only the new or altered peaks.

This dual approach reduces topographical noise and improves the detection of subtle changes in amide and fingerprint regions. Hydration level, swatch thickness, and orientation were standardized across all samples to reduce spectral variability. Each exposure condition (control, BL-15 min, BL-60 min, UV-15 min, UV-60 min) was scanned in triplicate.

11. Statistical analysis

All experiments were performed using independent biological triplicates unless otherwise stated. Data are presented as mean ± standard deviation (SD) or as mean with 95% confidence intervals (CI), as indicated in the figure legends. Normality of data distribution was assessed using the Shapiro-Wilk test. Pairwise comparisons between two groups (e.g., blue light vs. UV-C, surface vs. embedded colonies) were performed using unpaired two-tailed Student's t-tests. For comparisons involving more than two conditions or time points, one-way analysis of variance (ANOVA) followed by appropriate post-hoc testing was applied. The specific statistical test used for each comparison is indicated in the corresponding figure legend. A p-value <0.05 was considered statistically significant.

Results

Antimicrobial efficacy of blue light LED and UV-C against wound pathogens
The blue light LED device (405 nm), operated at an intensity of 30 mW/cm² for 15 min, demonstrated significant antimicrobial efficacy against all tested wound pathogens on synthetic skin models (Figure 5). Among the tested species, Klebsiella pneumoniae showed the highest susceptibility to blue light, with a mean log reduction of 3.4 in viable cell counts. Conversely, Staphylococcus au...

Discussion

This study demonstrates the significant antimicrobial potential of a 405 nm Blue Light LED (BL-LED) device against a panel of clinically relevant wound pathogens tested on collagen-based synthetic skin models. The BL-LED exposure at a fluence of 27 J/cm² achieved up to a 3.5-log reduction in microbial load (Figure 6), exceeding the antimicrobial performance reported in several earlier studies^6,7,17

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acinetobacter baumannii	ATCC The Global Bioresource Center	ATCC 19606
Blue Light - Light Emiiting Diode	Violumas	N/A	BL-LED is a prototype thus does not have a Catalog number
Candida albicans	ATCC The Global Bioresource Center	ATCC 10231
Enterobacter aerogenes	ATCC The Global Bioresource Center	ATCC 13048
Escherichia coli	ATCC The Global Bioresource Center	ATCC 25922
Fourier Transform InfraRed  (FT-IR) Spectrophotometer Nicolet iS5	Thermo Scientific	IQLAADGAAGFAHDMAZA	OMNIC Software is used for Analysis
FT-IR Nicolet iS5 iD1 Direct Beam Transmission Module (Base Adaptor)	Thermo Scientific	IQLAADGAAGFAJAMAYX
FT-IR Nicolet iS5 iD7 Diamond Attenuated Internal Refraction Module	Thermo Scientific	IQLAADGAAGFAJAMBFN
Incubator	Percival Scientific	9330.01.06L
IR theromometer	Thermo Fisher Scientific	06-664-254
Klebsiella pneumonia	ATCC The Global Bioresource Center	ATCC 13883
Phosphate-Buffered Saline	Thermo Fisher Scientific	AM9624	PBS Catalog number (Thermo Fisher Scientific)
Pseudomonas aeruginosa	ATCC The Global Bioresource Center	ATCC 27853
Sabouraud Dextrose Agar	BD Difco	DF0109-17-1	SAB Catalog number (BD Difco)
Staphylococcus aureus	ATCC The Global Bioresource Center	ATCC 25923
Tryptic Soy Agar	BD Difco	DF0369-17-6	TSA Catalog number (BD Difco)
Tryptic Soy Broth	BD Difco	DF0370-17-3	TSB Catalog number (BD Difco)
UV-LED	Violumas	N/A	UV-LED is a prototype thus does not have a Catalog number
UV-VIZ Spectrophometer (Genesys 10 UV Scanning)	Thermo Scientific	335907P
Vitro-Skin	MEDELINK	SKU24000	Vitro-Skin