Chamber design and intensity-modulated ultraviolet-C LEDs for advanced pulsed photonic disinfection

Design and development of circuitry

To ensure stable and efficient operation of the UV-C LEDs, a carefully designed circuit configuration was implemented (Figure 1). The L298P motor driver was utilized to regulate the current supplied to the LEDs, maintaining consistent power delivery and avoiding energy losses. Eight UV-C LEDs were divided into two groups, with each group connected to a separate output channel of the motor driver. The positive and negative terminals for the first group of LEDs were connected to M1+ and M1-, while those for the second group were connected to M2+ and M2-. The LEDs were operated at three distinct efficiency levels, 60 , 80, and 100 %, as recommended by the manufacturer. These levels were achieved by varying the current supplied through the motor driver. A 5 V cooling system was integrated into the circuit to prevent overheating during operation. The cooling fans were directly powered by a 5 V power supply, ensuring that the LEDs and other components remained within safe temperature limits. Additionally, a 12C-compatible LCD was connected via the 12C interface and powered using the same 5 V supply. This LCD provided a convenient way to monitor and control the operation of the LEDs, displaying real-time information about their efficiency levels and other operational parameters. To facilitate manual control and configuration, four push buttons were included in the circuit. Each button was paired with a 10 KΩ pull-down resistor to ensure stable low-state readings when the buttons were not pressed. This arrangement prevented floating values and eliminated the risk of false triggers, thus enhancing the reliability of the system. A stable 5 V power supply was used to power all components, ensuring consistent performance throughout the circuit. The motor driver played a critical role in varying the current supplied to the LEDs, allowing for uniform operation across all LEDs and achieving the desired efficiency levels of 60 , 80, and 100 %. This uniformity was essential to prevent imbalances in illumination or power consumption. To avoid unnecessary exposure causing damage to the skin, the chamber was befitted with the sensor on the door which stops the treatment as soon as the door was opened. Furthermore, the cooling fans worked effectively to dissipate heat, protecting the components and prolonging the lifespan of the system. Overall, the circuit design provided an optimized and robust solution for operating the UV-C LEDs at varying efficiency levels while maintaining system stability and component longevity.

Figure 1:

Circuitry of the chamber.

Design and development of chamber

The chamber measuring at (20 × 28.1 × 28.1 cm), was designed using CAD software and fabricated using a 3D printer (Ender3 Pro, Shenzhen, China). The chamber was designed to accommodate four cooling fans strategically positioned (two on the sides and two at the back) (Figure 2F–C). The airflow was directed outward on the opposite side to prevent internal circulation, which could lead to unnecessary external contaminants entering the chamber. To further mitigate the risk of external contamination, the entire chamber was enclosed within an isolated covering. At the center of the chamber, a tray was placed on a dedicated tray holder (Figure 2A) for optimal positioning. The LED panel was mounted on the front side of the chamber (Figure 2B), enabling various operational modes to be implemented.

Figure 2:

Design consideration of a chamber: (A) Tray holder, (B) front panel section of a chamber, (C, D and F) back and side cooling fans, (E) wiring outlet.

Fluence measurement

For the irradiance measurements, an irradiance meter (LS123, Portable UV Power Meter, China) was employed to examine the radiant output in µW-cm⁻². The sensor was placed directly at the center facing upward. At the preliminary stage while assessing different variants of LEDs, irradiance was measured at 10 and 15 cm followed by the increment of PWM from 20 % each time until it reached 100 %. Later, the chosen setup was thoroughly examined three times to validate the performance and radiant output of combined setup at 15 cm. Later, the product of radiant output and time in (s) was computed. This rigorous method was put in place to guarantee accuracy and uniformity when assessing irradiance levels throughout the experimental processes. At each distance, the irradiance (E, µW-cm⁻²) remained constant.

Where,

H is the dosage in µJ-cm⁻²,

E is the irradiance in µW-cm⁻²,

t is the total time of treatment in seconds (s).

Selection and synergistic arrangement of LEDs

The 275 nm wavelength was chosen for this investigation based on extensive evidence demonstrating its superior disinfection capability within the 275–280 nm range [21], which has been widely reported for its potent antimicrobial effects. Therefore, this study employed eight different 275 nm LED variants with varying power ratings, assessing their germicidal efficiencies at distances of 10 and 15 cm. Since LED repositioning with different orientations can significantly affect intensity through strategic and synergistic arrangements [22], this study tested various configurations and identified clustered positioning as optimal for achieving maximum irradiation across 10–15 cm distances. Furthermore, the initial phase of the study involved assessing how irradiance levels varied across different 275 nm UV-C LEDs with varying power ratings. After identifying the highest-performing LED, this unit along with seven comparable variants were selected for further evaluation. The outcome revealed distinct performance trends as the PWM (pulse width modulation) increased from 10 to 100 %. LED 1 (Figure 3a) showed exceptional performance, achieving the highest irradiance at 15 cm with a value of 244 μW/cm² at 100 % PWM. This underscored its strong efficiency over longer distances. At 10 cm, LED 1 also performed significantly better than other variants, reaching 332 μW/cm², making it one of the most consistent LEDs across the two measured distances. Its steady increase in irradiance with PWM increments reflects its ability to deliver reliable and robust light output. LED 2 (Figure 3b) exhibited outstanding results, particularly at 10 cm, where it achieved the highest irradiance among all LEDs, with a peak of 381 μW/cm² at 100 % PWM. At 15 cm, it maintained strong performance with 203 μW/cm², ranking it as one of the top performers at this distance as well. LED 3 followed a similar trend, with high irradiance values at both distances. It reached 404 μW/cm² at 10 cm and 211 μW/cm² at 15 cm, demonstrating its capacity for delivering strong light output under maximum PWM conditions.

Figure 3:

Irradiance assessment (in µW/cm²) for eight LEDs (a–h) evaluated at distances of 10 cm (purple) and 15 cm (orange), with PWM increments from 10 to 100 %.

Other LEDs, such as LED 4 (Figure 3d) and LED 5 (Figure 3e), showed competitive performance. LED 4 delivered 401 μW/cm² at 10 cm and 190 μW/cm² at 15 cm, while LED 5 reached 375 μW/cm² at 10 cm and 144 μW/cm² at 15 cm. These results highlight their ability to provide substantial irradiance, though their efficiency decreased more significantly at longer distances. LED 6 (Figure 3f), LED 7 (Figure 3g), and LED 8 exhibited lower overall irradiance values, with LED 8 (Figure 3h) showing the lowest peak irradiance at both 10 and 15 cm, with 279 μW/cm² and 168 μW/cm², respectively. The results demonstrate that as PWM increases, all LEDs exhibit proportional growth in irradiance. However, the rate of increase and the maximum achievable irradiance varied significantly between LEDs. LEDs such as LED 1, LED 2, and LED 3 emerged as superior options, delivering high irradiance values consistently at both measured distances. These findings underscore the importance of selecting specific LEDs based on application needs, particularly for scenarios requiring consistent light output over varying distances.

Among the eight LEDs, the 4 W 6565 SMD beaded LED, equipped with a quad-lens, produced the highest intensity compared to the others (Figure 3). To determine the optimal output for effective disinfection, different LED arrangements were tested. In finalized setup, four LEDs were positioned at each corner, tilted at a 60-degree angle, while the remaining four were placed in the center, all directed toward the central plate where the specimen was positioned. The electrical properties of such LEDs comprised a forward voltage (V_F) of 6.8 V (V), radiant flux of 120 mW, an average forward current (I_F) of 300 mA, a peak wavelength of 275 nm, and a viewing angle of thirty-degree. Furthermore, emission spectroscopy study was also performed using a spectrometer (HR4000-Vis-NIR, Ocean Optics, Inc, USA) to precisely identify the actual wavelength of these sources. Furthermore, during the treatment, all LEDs were powered through a motor driver, with varying current sources operating at 60 , 80, and 100 % efficiency levels.

In-vitro tests and validation

Multi-faceted assessment of bacterial disinfection

Dose dependency on bacterial suppression

To verify the anti-bacterial effectiveness of the UV irradiation, S. aureus and E. coli were treated with different PWM intensities (60 , 80, 100 %) for different treatment times (120 s, 150 s, 180 s, and 210 s) (See Figure 4). The minimum distance of 15 cm was chosen which was in accordance with the studies employing UV LED arrays and lamps [24], [25], alternating with the increment of 15 cm. Moreover, based on the recent work [26], where efficient disinfection was reported using time duration alternating with the increment of 30 s. Results show a linear correlation between increasing UV dose and bacterial suppression, with increasing PWM settings and increasing exposure over time producing greater suppression.

Figure 4:

Suppression of S. aureus and E. coli against varied UV irradiation upon employment of different irradiation efficiencies.

For S. aureus, at 100 % PWM level, a gradual increase in suppression was noted with UV doses of 29.28 mJ/cm², 40.38 mJ/cm², and 51.24 mJ/cm². This indicated the more intense effect of UV irradiation across the entire surface area as the time increased up to 210-s, however, we did not observe cell death at the edges for the 120-s exposure. Since Gram-negative bacteria are generally more vulnerable to UV irradiation than Gram-positive bacteria [27] due thick peptidoglycan wall of Gram-positive bacteria (∼80 nm) containing amino acids that can absorb UV-C light through their peptide bonds [28] compared to Gram-negative bacteria, having a much thinner peptidoglycan layer (∼8 nm) with a lower amino acid density, making them less resistant to UV-C light. The findings validated this claim as more pronounced inactivation was reported for E. coli with similar dosage in this study. In maximum exposure conditions, both treatments achieved superior disinfection in which E. coli was more sensitive than S. aureus with the increase in exposure time. E. coli, particularly at higher exposure durations, appeared to be more susceptible to maximum UV exposure than S. aureus (p<0.001). Building on recent research, the study by [29] demonstrated that disinfection efficacy is influenced by both distance and exposure duration, where the effect was primarily attributed to focal distance, as moving the UV source farther from the target area proportionally increased the irradiation diameter in both positive and negative directions. Moreover, using a similar distance [30], used a Krypton disinfection setup to examine its antibacterial efficiency. Even after extending the treatment period to 40 min in order to obtain an effective degree of suppression, the effects were less noticeable compared to this study where more effective suppression was achieved in less time and higher distances.

However, in case of both the treatments, 80 % PWM led to less effective disinfection than that achieved with maximum settings, however, still exhibited a pattern with increasing exposure durations resulting in enhanced suppression. This result suggests that S. aureus exhibited weaker suppression compared to E. coli but showed increasing suppression over the same duration. Results showed that closely packed dense colonies persisted after treatment for a minimum duration of 120-s test, indicating that the dosage imparted was not enough to inactivate the bacteria completely and the surviving cells were able to re-colonize. However, with increment in duration formation of clumped colonies degenerated into smaller colonies but viable bacteria thence persisted till 210-s of exposure for both the treatments. The corresponding UV doses varied between 12.24 and 21.42 mJ/cm², which was significantly lower than that at full intensity, explaining the decrease of the suppression effect.

Nonetheless, lower PWM setting (60 %) resulted in weaker suppression where S. aureus initially showed a weaker response compared to E. coli. However, with prolonged UV exposure up to 210 s, increased bacterial suppression was observed, suggesting that higher irradiation duration led to greater bacterial reduction. However, even the maximum duration of S. aureus treatment resulted in residual dense opaque bacterial growth in regions of UV irradiation that were insufficient to achieve the substantial suppression. This indicated that in addition to duration of treatment, efficiency in which the treatment is carried out was next in line on the scale of achieving enhanced anti-microbial efficacy.

PWM dependency on log reduction

The reduction values resulted for S. aureus and E. coli at different PWM settings (100 , 80 and 60 %) and exposure times (120 s, 150 s, 180 s and 210 s) showed promising disinfection when duration was extended to approximately 4 min (see Figure 5). The effective reduction was increased with higher PWM intensitives and longer exposure durations attributed to the effect of irradiance dose and PWM on bacterial reduction. At 100 % PWM intensity, reduction factor consistently increased from 0.83 log at 120 s to 1.54 log₁₀ at 210-s, corresponding to the doses from 29.28 mJ/cm² to 51.24 mJ/cm². The results demonstrated a clear dose-response relationship: as the dose increased, bacterial reduction became more significant [31]. Compared to the study by [32] which achieved a 1-log₁₀ reduction at an exposure dose of 113 mJ/cm², the inactivation attained with lower doses were more pronounced. The log reduction values at 80 % PWM were lower but followed the identical rising trend, where the reduction factor was evaluated to be 0.28-log₁₀ inactivation at 120 s to 1.11-log₁₀ at 210-s, corresponding to the doses which ranged between 12.24 mJ/cm² to 21.42 mJ/cm² in line with the lower irradiance intensity due to the PWM ratio. At 60 % PWM, the reduction was negligible, resulting at 0.19-log₁₀ inactivation at 120 s and 0.28-log₁₀ inactivation at 210-s, with corresponding doses of 8.64 mJ/cm² to 15.12 mJ/cm², exhibiting hardly an effect with low irradiance Intensity and extended exposure times.

Figure 5:

Log reduction of S. aureus and E. coli following UV irradiation at different pulse width modulation (PWM) settings (60 , 80, and 100 %) and exposure times (120 s, 150 s, 180 s, and 210 s).

Unlike S. aureus, the reduction values observed with E. coli was more pronounced. One study explores the effectiveness of UV-C irradiation as a microbial control strategy in indoor public spaces. At comparable PWM intensities and exposure durations, log reduction results indicated similar inactivation trends in E. coli as a function of dose and irradiance intensity. Accoridng to the findings, the reduction values at 100 % PWM varied from 1.02-log₁₀ at 120-s to 2.05-log₁₀ inactivation at 210-s, with corresponding doses ranging between 29.28 mJ/cm² and 51.24 mJ/cm², respectively, representing the maximum bacterial inactivation achieved in the various PWM levels. The present study aligns with previous research on E. coli O157:H7 inactivation using UV irradiation [33], where nearly higher dose was required to achieve the highest reduction 1.95-log₁₀ inactivation and 1.83-log₁₀ inactivation with Far UV-C. Hence, the present study showcased a linear dose-reduction relationship, suggesting that higher doses at peak PWM levels produce the enhanced inactivation of E. coli. With the decrease in PWM level, the bacterial reduction was comparatively lower at 80 %, valuing at 0.48-log₁₀ to 1.2-log₁₀ at 120-s and 210-s respectively, with the lower doses ranging from 12.24 mJ/cm² to 21.42 mJ/cm². At a PWM of 60 %, the reduction was even lesser for the first couple of exposure times, which later grew with the extension in treatment time. In that case, log reduction was found to be 0.12-log₁₀ at 120-s which rose to 1.35-log₁₀ inactivation at 210-s, while doses in the range of 8.64 mJ/cm² to 15.12 mJ/cm² were the indication that for lower irradiance intensity, longer exposure times were needed for an effective bacterial inactivation. These results wer more pronounced compared to the recent study which demonstrated the effectiveness of UVC irradiation against L. monocytogenes which required 21.6 mJ/cm² achieved a 1.0–1.6-log₁₀ reduction. Overall, the results indicated that E. coli was susceptible to the UV irradiance dose and exposure time, where with maximum exposure exposure and PWM level, highest reduction was achieved which was effective for the inactivation of microbes.

This heightened inactivation of E. coli could be due to its cellular structure and susceptibility to UV light. E. coli is Gram-negative and its cell wall is thinner and more susceptible to the UV damage than S. aureus’s thick peptidoglycan layer in its cell wall that provides for additional protection to its cells. As mentioned previously, the primary lesion of UV irradiation is the pyrimidine dimers in the DNA, while structural weaknesses of E. coli may be the cause of better absorption of the UV energy and more disruption of the cellular functions.

Overall, the results emphasized the importance of optimizing both dose and exposure time to achieve effective microbial inactivation. Higher PWM settings and longer exposure times consistently resulted in greater log reductions for both microorganisms. However, the difference in inactivation rates between E. coli and S. aureus highlighted the need to consider microbial species’ specific characteristics when designing disinfection protocols. The findings suggest that while E. coli is more easily inactivated, achieving comparable reduction levels for S. aureus requires higher doses or extended exposure durations.

Irradiance distribution and treatment coverage

As four UV LEDs were positioned at the center point facing downward, with one additional LED at each edge of the chamber tilted at cetain angle, the arrangement ensured optimal coverage and maximum average attainability of irradiance at the center where the sample is to be placed for effective disinfection. Figure 6 further illustrated the irradiance distribution across the entire disinfection chamber.

Figure 6:

Irradiance distribution profile over petri dishes at a 15 cm distance for (a) S. aureus and (b) E. coli.

The resulting irradiance values showed a gradient from 244 μW/cm² (PWM setting at 100 %) to 72 μW/cm² (PWM setting at 60 %) for vertical distances from the centre of the beam over 15 cm (Figure 7). The reported range of irradiance achieved was higher than that from studies done by Nunayon et al. [34], achieving 0.31 μW/cm². In contrary [35], showed similar efficiencies with treatment requiring 60 min of exposure. The horizontal measurements at the distance across comparable vertical distances showed a consistent trend, with the values decreasing from 244 μW/cm² to 45 μW/cm², 102 μW/cm² to 8 μW/cm² and 72 μW/cm² to 7 μW/cm², for 100 , 80 and 60 % PWM levels, respectively. Hence, at maximum PWM intensity, the optimal coverage for effective disinfection was reported to be 21 cm in diameter.

Figure 7:

Varying level of PWMs influencing irradiance attainability and distribution across targeted surface.

SEM observations

To validate bacterial cell disruption, SEM analysis was conducted on both treated and untreated samples. In the control samples, S. aureus exhibited a cocci shape, while E. coli displayed a rod-shaped morphology [36], [37]. Upon exposure to the irradiation at a distance of 15 cm for 210 s, major changes in morphology were observed (Figure 8c and d) when compared to untreated samples (Figure 8a and b). Figure 8d depicted the SEM images of S. aureus cells when subjected to 210 s of irradiation. Interestingly, the treated cells revealed the formation of micro-colonies distributed across the surface. The irradiated E. coli cells revealed the incidence of lysis and bleb protrusions on their surfaces (Figure 8c) [37], [38], [39], [40], highlighting the impact of the UV irradiaiton in damaging cellular structure. This observation suggested that a 275 nm UV-C LEDs irradiation could induce diverse morphological changes in E. coli and S. aureus both, potentially influencing its growth and physical integrity. Furthermore, the adverse effect of UV radiation resulted in leakage of extracellular matrix resulting in bacterial lysis and appearance of medium-sized amorphous structures (Figure 8c and d) shown by orange, red and cyan colored arrow, widespread along the surface of the agar which was consistent with [38]. The microgram validated enough evidences in damaging the cellular integrity of S. aureus and E. coli, disrupting the DNA at greater extent, supporting the evidence of cellular damage.

Figure 8:

SEM images depicting controlled and irradiated bacteria under the influence of a UV irradiation: (a) Control sample revealing unharmed S. aureus, (b) control sample revealing unharmed E. coli (c) irradiated samples revealing lysis and bleb protrusion of damaged S. aureus, (d) irradiated samples revealing bleb protrusion of damaged E. coli.