1 Introduction

The transfer of electronic devices and tools into clean rooms, especially the temporary transfer, is critical in terms of microbial contamination risk. Devices like tablets or other electronics cannot be disinfected by heat. The common way is a wet wipe disinfection, which is done manually. This process is prone to error due to human error. Moreover, frequent exposure to chemical disinfectants can increase the risk of surface damage [1]. Another way to disinfect surfaces is the use of UVC radiation. UVC radiation is an electromagnetic wave with wavelength between 200–280 nm that can inactivate most microorganisms due to nucleic acid (DNA and RNA) damaging. Nucleic acids absorb the UVC radiation, resulting in the development of cross-links between nucleic acids. Due to this change in the genetic material, the pathogen is incapable of reproducing [2].

UVC radiation has been commonly used for decades in water disinfection. In 1985, over 1,500 UV water treatment systems were installed in Europe [3]. The first applications for UV water disinfection were reported in Europe in 1909 and in the US in 1916 [4]. Disinfection of air and surface with UVC has become increasingly important, especially with the emerging pandemic in 2020 [5]. The most commonly used UVC light source is the low-pressure mercury lamp, which emits at 254 nm. Nevertheless, UVC-LEDs catch up more and more to the low-pressure lamps because of some unique advantages. LEDs do not contain any heavy metals like mercury and can be manufactured in almost any wavelength within the UVC spectrum [6]. The challenge in using UVC-LEDs is the low energy efficiency compared to low-pressure lamps. The best UVC-LEDs on the market only provide an efficiency of 5–10%, compared to around 30–40% for low-pressure lamps [7]. The price of UVC-LEDs cannot compete to low-pressure lamps as well.

Clean rooms are a necessity in many scientific and industrial environments. In life science and pharma industry, bioburden needs to be under control or even a sterile environment is indispensable. To ensure product safety, it is necessary to minimize microbial contamination within a pharmaceutical clean room. The main sources, around 80–90%, of microbial contamination are typically affiliated to humans [8]. According to Wu et al. [9], over 80% of microorganisms found in clean rooms are gram positive Cocci, which are associated with human skin cells. In addition, non-spore forming rods (10–20%) and spore forming bacilli are regularly detected which could be transferred to the cleanroom by tools, which are brought from outside the clean area. Besides bacteria, mould and yeast are detected occasionally.

Plenty of UVC-devices for disinfection purposes have been described and investigated in the literature, mostly in healthcare environments. Most of them are using low pressure mercury lamps with an emission wavelength of 254 nm [10, 11]. The potential of UVC-light in disinfecting electronic devices such as phones is demonstrated with mercury lamps as well [12,13,14,15]. The usage of UVC-LEDs in a tabletop device for surface disinfection of electronic devices such like tablets and phones has not been described in literature properly yet. Surface disinfection with UVC-LEDs was investigated on a stethoscope [16]. Also, the disinfection of high-touch environmental surfaces with a UVC-LED light source in a healthcare setting was described [17].

The aim of this study is to decrease the risk of microbial contamination of clean rooms in pharmaceutical production with an automated process. Therefore, a UVC-LED device was developed to perform a surface disinfection on electronic devices, such as phones and tablets, which are frequently transferred in the clean room. In the next section we show the how the microbial experiments were done and what the technical parameters of the device are. Section 3 provide the results of the microbial experiments with the log reductions of different microorganisms. In Sect. 4 we discuss the results in context with recent literature. The last section provides a conclusion and shows the next steps.

2 Methods

2.1 The disinfection device

UVC radiation has a strong disinfecting effect, but only where the radiation reaches the surface. The inactivation rate depends on the specific sensitivity of the microorganism itself, the emission wavelength of the radiation source, and the irradiation dose impinging on the surface. The latter is the product of irradiation intensity per unit area and irradiation time in Ws/m2. To ensure safe and effective disinfection, it is necessary to achieve a minimum irradiation dose on each surface element of a device to be disinfected. To achieve this, a special structure of the irradiation room was developed (Fig. 1).

Fig. 1
figure 1

Disinfection device with a phone on the quartz glass plate, the white inner walls are coated with PTFE, which has a high diffuse reflectivity. On the bottom of the device the LEDs can be seen

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At the top and bottom of the irradiation room there are two flat UVC LED emitters, each equipped with 10 LEDs with an emission wavelength of 272 nm and an optical power of 100 mW per LED. This results in a total radiation power of 2 W. The LED modules are mounted on a heat sink and equipped with temperature sensors to monitor the LED temperature. The inner walls of the irradiation chamber are lined with films of sintered optical PTFE of type PMR10. This has a reflectance of 94 percent at 272 nm. In contrast to other frequently used UV reflectors, PMR10 also scatters the radiation, enabling very homogeneous irradiation of the object from all directions. This means that even interstitial spaces and hard-to-reach areas on the disinfection material can be easily reached. In the lower third of the irradiation area there is a quartz glass plate as a shelf for the material to be disinfected. Fused quartz glass was used because of its high UV transparency. In addition, this material is very scratch-resistant. In contrast to frequently used supports in the form of metal grids, no shadowing occurs. The homogeneity of the irradiation on a tablet surface was simulated using COMSOL Multiphysics (see Fig. 2). According to this simulation, the achievable intensity on a tablet surface is about 20 W/m2. In the rear wall of the irradiation room, a SiC UV photo sensor of the type sgLux SG-01 is integrated to monitor the real irradiation power. This sensor was calibrated to the irradiation intensity in the center of the irradiation room. The calibrated sensor together with the irradiation time, the dose can be calculated by the controller. It is possible to predefine a specific dose for the device, this feature was used in the experiments.

Fig. 2
figure 2

Raytracing simulation of a tablet into the disinfection device, the intensity of radiation on the surface is shown

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2.2 Material and method

To perform microbial tests, a variety of five different types of microorganisms have been chosen. As mentioned elsewhere [4], the majority of microorganisms found in clean rooms are gram positive cocci, therefore Staphylococcus aureus was tested. The second most organism found are gram negative rods, with Pseudomonas aeruginosa as a representative. Additionally, Aspergillus brasiliensis spores, as mould and two spore forming bacilli; Bacillus subtilis spores and Paenibacillus glucanolyticus spores were tested. The load of microorganisms in the test samples were much higher than it is expected in real world environment of a clean room (with e.g. an action limit of 5 cfu/cm2 (colony forming units/cm2) on floor-surfaces) to create inactivation curves and determine the limits of the system.

$$ \begin{gathered} {\text{Test}}\;{\text{surfaces}}: \quad \quad \quad \quad {\text{Stainless}}\;{\text{steel}},\;{\text{glass}},\;{\text{plastic}}\;\left( {{\text{acrylonitrile}}\;{\text{butadiene}}\;{\text{styrene}},\;{\text{ABS}}} \right) \hfill \\ {\text{Size}}\;{\text{of}}\;{\text{test}}\;{\text{surfaces}}: \quad {\text{approx}}. \, 5\;{\text{cm}} \times 5\;{\text{cm}} \hfill \\ {\text{Test}}\;{\text{temperature}}:\quad \quad \quad 18\;^\circ {\text{C}} - 25\;^\circ {\text{C}} \hfill \\ \end{gathered} $$

2.2.1 Preparation of microbial suspensions (vegetative bacteria, yeasts)

The test strains were taken from the agar plate with an inoculating loop or a pad and were diluted in NaCl-peptone. The amount of microbial material, which was needed to reach the required microbial count, was determined in preliminary tests. Vegetative bacteria and bacterial spores were incubated for a maximum of 3 days at 30 °C–35 °C. Yeasts and moulds were incubated for a maximum of 5 days at 20 °C–25 °C.

2.2.2 Preparation of spore suspensions

A. brasiliensis: the test strain was plated on malt-agar (Merck Chemicals GmbH, Darmstadt, Germany) and incubated for 3 days at 28 °C–31 °C. The test strain was then subcultivated on malt-agar (Merck Chemicals GmbH, Darmstadt, Germany) and incubated for 19 days at 28 °C–31 °C. The fungal spores were washed down with aqua ad injectabilia and filtered through sterile gauze. Afterwards the spore suspension was stored at 2 °C–8 °C.

B. subtilis: the test strain was plated on minimal medium agar (standard I medium under addition of manganese sulfate; Merck Chemicals GmbH, Darmstadt, Germany) and incubated for 24 h at 30 °C–35 °C. Afterwards the agar plates were incubated for 14 days at 20 °C–25 °C. The bacterial spores were washed down with aqua ad injectabilia and heated for 10 min at 80 °C in order to inactivate vegetative strains. The spore suspension was stored at 2 °C–8 °C.

Pae. glucanolyticus: the test strain was plated on minimal medium agar (standard I medium under addition of manganese sulfate; Merck Chemicals GmbH, Darmstadt, Germany) and incubated for 2 days at 30 °C–35 °C. Afterwards the agar plates were incubated for 14 days at 20 °C–25 °C. The bacterial spores were washed down with aqua ad injectabilia and heated for 10 min at 80 °C in order to inactivate vegetative strains. The spore suspension was stored at 2 °C–8 °C.

2.2.3 Evaluation of initial microbial count

The microbial suspensions were diluted 1:10 in NaCl-peptone and 1.0 ml of dilutions were plated on agar plates and incubated as follows: bacteria were plated on tryptic soy agar (bioMérieux Deutschland GmbH, Nürtingen, Germany; exception: Pae. glucanolyticus was plated on mannitol salt agar; bioMérieux Deutschland GmbH, Nürtingen, Germany) and incubated at 30 °C–35 °C for a maximum of 3 days (according to the requirements stated in the European Pharmacopeia). Yeasts and moulds were plated on sabouraud dextrose agar (bioMérieux Deutschland GmbH, Nürtingen, Germany) and incubated at 20 °C–25 °C for a maximum of 5 days (according to the requirements stated in the European Pharmacopeia). Afterwards colonies were counted and the initial microbial count was determined.

2.2.4 Treatment of test surfaces

Test surfaces were placed for 60 min in 5% Decon (detergent; Decon Laboratories Ltd., Hove, United Kingdom) and then washed with sterile water. The clean surfaces were placed for 15 min in 70% isopropanol (Laborhaus Scheller GmbH & Co.KG, Euerbach, Germany; isopropanol evaporates without leaving any residue on the surfaces and therefore has no influence on the subsequent UV-irradiation) and then dried under the laminar flow. The test surfaces were placed dust-free in sterile petri dishes to be protected against microbial contamination.

2.2.5 Contamination of test surfaces

0.05 ml of microbial suspension (initial microbial count: 1.0–9.9 × 109 cfu/ml for vegetative bacteria and yeasts, 1.0–9.9 × 108 cfu/ml for bacterial spores (B. subtilis), 1.0–9.9 × 107 cfu/ml for bacterial spores (Pae. glucanolyticus) and 1.0–9.9 × 106 cfu/ml for moulds) was applied drop by drop on the entire test surfaces. The application of microbial suspensions was performed based on the routine procedure for the evaluation of microbicidal efficacy for chemical disinfectants, where suspensions are applied with a defined volume on surfaces to ensure comparable conditions. Afterwards the contaminated test surfaces were kept for drying at 36 °C for a maximum of 60 min.

2.2.6 UV-irradiation of test surfaces – in triplicate (different testing days)

After drying of the microbial suspension, one contaminated test surface per irradiation dose was placed in the middle of the testing device. The UV-irradiation was started automatically. As soon as the irradiation dose was reached, the UV-irradiation was stopped automatically (irradiation dose see Table 1).

Table 1 Test strains and irradiation dose used
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2.2.7 Positive control – in triplicate (different testing days)

As control, test surfaces were treated in parallel as described above but without performing the UV-irradiation.

2.2.8 Quantitative reisolation of microorganisms (recovery of microorganisms)

At the end of the UV-irradiation each test surface was transferred (upside down) in a sterile petri dish containing 10 ml inactivation combination (3.0% Tween 80 + 3.0% saponin + 0.1% histidine + 0.1% cysteine in casein-soy-broth) and sterile glass beads. The petri dish was then shaken for a minimum of 1 min (= test neutralization mixture, TNM) to reisolate the remaining microorganisms from the surface. Afterwards from the TNM serial 1:10 dilutions (100–10–6 for vegetative bacteria and yeasts, 100–10–5 for bacterial spores (B. subtilis), 100–10–4 for bacterial spores (Pae. glucanolyticus) and 100–10–3 for moulds) were prepared and 2 × 1.0 ml (alternative 4 × 0.5 ml) of the TNM as well as the dilutions were plated on agar plates. From the positive control serial 1:10 dilutions were prepared (10–4–10–6 for vegetative bacteria and yeasts, 10–3–10–5 for bacterial spores (B. subtilis), 10–2–10–4 for bacterial spores (Pae. glucanolyticus) and 10–1–10–3 for moulds) and 2 × 1.0 ml (alternative 4 × 0.5 ml) of the dilutions were plated on agar plates. After incubation under conditions mentioned at point 2.2.1, colonies were counted manually, and the microbial count evaluated using MS Excel.

2.2.9 Calculation of reduction

The number of colony forming units (cfu) of each UV-irradiation sample and the corresponding positive control was counted. This was done for all dilutions. For the subsequent calculation of results plates were evaluated where the number of cfu ranged between 14 and 330 (for vegetative bacteria, bacterial spores and yeast) and 14–165 cfu (for moulds), if possible. The log10 of number of cfu per ml were then calculated according to the following formula.

$$N_{c} = \log_{10} \frac{{x + x^{\prime } }}{2}*\frac{10}{d}$$
(1)
$$N_{d} = \log_{10} \frac{{y + y^{\prime } }}{2}*\frac{10}{d}$$
(2)

with:

Nclog10 of cfu of 1.0 ml positive control.x, x’cfu of positive control

Ndlog10 of cfu of 1.0 ml irradiation sample.y, y’cfu of irradiationddilution factor

The reduction factor of UV-irradiation was then calculated following:

$${\text{Reduction}}\;{\text{factor}}\;\left( R \right):R = N_{c} - N_{d}$$
(3)

The different surfaces were investigated in detail, because the log10-reductions differ also depending on the surface type. Due to this fact, the roughness is shown in Table 3. Ra and Rz values are shown related to the different samples, where Ra is the average profile height deviation from the mean line and Rz the maximum peak to valley height of the profile. The roughness for stainless steel and plastic is measured using an optical method, whereas the data for glass is taken from literature [18, 19], because the optical method does not work with the transparent glass.

3 Results

The UV-inactivation of microorganisms was tested on different surfaces. The results are shown in Fig. 3a-e. A distinct inactivation can be seen for all pathogens, but the required dose strongly varies according to the type of pathogen and the surface. Additionally, the detailed results are shown in Table 2 with the corresponding confidence interval.

Fig. 3
figure 3

Inactivation graphs of 5 different microorganisms, irradiated with 272 nm radiation on 3 different surface samples (glass \(\cdots\), ABS \(-\) and stainless steel \(\cdots -\)). a S. aureus (b) P. aeruginosa (c) A. brasiliensis sp. (d) B. subtilis sp. (e) Pae. glucanolyticus sp.

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Table 2 Detailed results of the microbial inactivation with corresponding 95% CI
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S. aureus (Fig. 3a) reaches an inactivation of almost 8 log10 with a corresponding dose of 150 J/m2 on plastic and glass surface. The graphs of these surfaces are almost identically. On stainless steel, it only reaches 5.5-log10 inactivation with the same dose. All three inactivation curves show a two-stage decay.

P. aeruginosa (Fig. 3b) reaches more than 7 log10 steps on plastic and glass surfaces with a dose of 100 J/m2. On plastic, it reaches the 7- log10 reduction for 40 J/m2 and stays approximately constant for higher doses. The log10-reduction on glass reaches 7 log10 for doses higher 60 J/m2. The rise in log10-reduction for small doses below 60 J/m2 is slightly lower than on plastic surface. The steel surface shows an almost linear rise in log10-reduction. It reaches 4.5 log10 steps at 100 J/m2.

A. brasiliensis spores (Fig. 3c) shows low log10-reduction on all surfaces. It only achieves slightly above 2 log10 steps for both, plastic and glass. Steel is slightly below 2 log10 steps. The graphs are almost identically and show a clear two-stage decay. The dose to achieve these low log10-reductions is 3000 J/m2 and thus much higher than for the other pathogens.

B. subtilis spores (Fig. 3d) are treated with the same dose as A. brasiliensis spores (3000 J/m2). The results for plastic and glass surface are almost the same with approximately 7 log10 steps of reduction. On stainless steel, it achieves 4.5- log10 reduction. All three graphs show a two-stage decay.

Pae. glucanolyticus spores (Fig. 3e) is also treated with 3000 J/m2 and this leads to a 6- log10 reduction for plastic and glass surface. The reduction on steel surface is again much lower with 3.5 log10. Glass and steel surface show clearly a two-stage decay whereas plastic shows a linear behavior, except for the point corresponding 1200 J/m2.

The roughness of the different surface materials is shown in Table 3. Glass has a very low roughness compared to steel and ABS. Stainless steel has the highest roughness because it provides a shot blasted surface. Especially the Rz value is much higher than for plastic, which means, that there are deep cavities in the surface. Plastic lies somewhere in between glass and stainless steel.

Table 3 Surface roughness of the 3 different material samples stainless steel, ABS and glass, the standard deviation of the measured values is calculated from a sample size of n = 3. The values for glass are provided by literature [18, 19]
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In addition to the microbial test, a numerical simulation model was developed investigating the irradiance on the surface of an electronic device (Fig. 2). On top of the device, the irradiance is almost constant with an average intensity of 15.3 W/m2. On the bottom side of the device, the intensity is slightly higher because the distance to the LEDs is shorter than to the top.

The intensity on the sides of the device is lower than on the top and bottom surface. Because of the diffuse reflection of the walls, sufficient intensities can also be reached on the sides. The intensity on the two short sides is lower than on the long sides, because the device can barely fit into the box; as a result, the irradiance of the short sides is not ideal. The irradiance of smaller devices will be much better than the device shown in Fig. 2. The corresponding values are shown in Table 4 in addition with the calculated dose for a 60-s irradiation time.

Table 4 Simulation results of a tablet for a 60 s irradiation for all sides, COMSOL Multiphysics is used for the simulations
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4 Discussion

In general, the highest dose is required to inactivate fungal spores, followed by bacterial spores and vegetative bacteria, who have the highest susceptibility to ultraviolet radiation [2]. The presented results fit in this fundamental correlation.

Apart from the differences for the type of pathogens, also the surfaces which are to be disinfected are affecting the disinfection efficacy. The roughness of shot blasted steel is compared to [20, 21] and with Ra around 1.5 µm in good agreement with both references. The roughness of polymer materials can vary depending on the surface treatment and the material itself. The dose on glass and plastic surfaces are in similar magnitude, but to achieve the same inactivation rate a slightly higher dose for the plastic surface is needed. On the other hand, the spores of B. subtilis and A. brasiliensis have almost the same inactivation at the same dose on both plastic and glass. The remaining pathogens need slightly higher doses for the same inactivation, between 25% and 70% higher doses. In all cases, the log10-reduction on the steel surface is lower than on the other two surfaces.

Table 5 shows the mean size of the microorganisms in this study. S. aureus, A. brasiliensis spores and Pae. glucanolyticus spores have a spherical shape and P. aeruginosa and B. subtilis spores are rod-shaped [22,23,24,25,26]. The size is provided with the dose for a 3-log10 reduction of each organism on glass and stainless steel, with exception of A. brasiliensis spores. For this microorganism a 1-log10 reduction is shown in Table 5, because 3-log10 reduction was not reached. The reduction differs on the compared surfaces, and we assume, that the size of the microorganisms in conjunction with the surface roughness influences the dose requirements for said surfaces. This finding is supported by the studies of Kim et al. [19], which show that the inactivation of microorganisms on a glass surface is higher than on plastic and steel surfaces. Ratliff et al. show a similar correlation between the surface roughness and the inactivation efficacy [27]. Contrary to the studies of Kim and Ratliff, the inactivation on stainless steel surface was lower in our work compared to the plastic surface. In both referenced publications, the surface roughness of stainless steel is distinctly lower than in our work, because we used a shot blasted stainless steel. Smaller organisms which fit better in the cavities of the stainless steel surface, tend to require higher doses compared to the smooth glass surface. A. brasiliensis spores do not fit in the cavities and therefore the dose requirements for the steel and glass surface are almost identically. Only S. aureus seems not to fit in this assumption, even though the size of this organism is small compared to the surface roughness, but this organism tends to form clusters [28] Therefore, a large proportion of the organisms gets higher irradiation. This assumption is supported by the fact, that for higher log10 reductions the difference between steel and glass surface increase rapidly.

Table 5 Mean size and 3-log10 reduction of the investigated microorganisms on stainless steel and glass surface
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References for log10 reductions of the investigated microorganisms are not provided at this point, because there is no standardized method to measure the microbial inactivation on surfaces [29]. Duering et al. show that there are many influences on the outcome, that makes it difficult to compare data from other studies [30]. But we are the first one to publish data about the UV susceptibility of Pae. glucanolyticus spores.

The microbial inactivation study has some limitations, because the experiments are performed on flat samples with limited size, which are contaminated on the surface. The results for this samples cannot be transferred directly to devices with more complex geometries. Larger and mostly flat surfaces are expected to reach similar results as the small surfaces investigated in this study, because the illumination into the disinfection device is almost homogenous, due to the diffuse reflection of the inner walls. This minimizes shadowing on the surface of devices with more complex geometries, but it is not impeded for each geometry. Small gaps and cavities in the surface cannot be illuminated properly. And as mentioned before, there is no standardized method for inactivation studies on surfaces, which can lead to slightly different results, if there are only small changes in the methods for this study.

5 Conclusions

This study showed that UV-LEDs with 272 nm achieve sufficient inactivation rates on 5 different microorganisms. It was also shown that different surfaces influence the efficacy of the UV-LED device to inactivate microorganisms. We assume that these differences are due to the surface roughness of the materials. The provided UV-intensity inside the device is high enough to inactivate even spore-forming bacteria and mould, which are persistent to UV-radiation and to chemical disinfectants. The automated disinfection process is reproducible and doesn’t rely on consumables such as chemicals.

In general, the UV-LED device has the potential to fulfill the requirements for being used in a clean room environment and improve the process of transferring items into the clean room with minimal risk of contamination. Furthermore, for the application of UV irradiation in a pharmaceutical environment, shorter irradiation times are probably sufficient, because the microbiological load on the surfaces is expected to be lower than the initial microbial counts used in this study. Validation studies will follow to make sure the disinfection of specific devices fulfill the regulations and requirements in pharmaceutical clean rooms.