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. 2020 May 13;29(9):1281–1287. doi: 10.1007/s10068-020-00765-z

Aluminum based reflective nanolens arrays to improve the effectiveness of ultraviolet inactivation of Escherichia coli O157:H7 and Listeria monocytogenes in water and a sucrose solution

Junsoo Park 1, Changhoon Chai 1,✉
PMCID: PMC7406604  PMID: 32802567

Abstract

Aluminum based reflective nanolens arrays were developed via a series of aluminum electropolishing and anodization steps with subsequent selective dissolution of anodic aluminum oxide (AAO). The diameter of nanolenses (d) on arrays can be controlled by altering electrolytes and voltages used for aluminum anodization. The d values of arrays produced by anodization in 0.3 M oxalic acid at 40, 60, and 80 V, and in 1.0 M phosphoric acid at 100, 110, and 120 V were 71.94, 121.90, and 161.53 nm, and 220.16, 252.06, and 274.78 nm, respectively. The effectiveness of UV (254 nm) inactivation of Escherichia coli O157:H7 and Listeria monocytogenes at concentrations of 5–6 log CFU/mL in water and in a 10% (w/v) sucrose solution was improved using a nanolens array having a d value of 252.06 nm.

Electronic supplementary material

The online version of this article (10.1007/s10068-020-00765-z) contains supplementary material, which is available to authorized users.

Keywords: Nanolens array, Aluminum anodization, UV inactivation, E. coli O157:H7, L. monocytogenes

Introduction

Ultraviolet (UV) irradiation is frequently used to inactivate microorganisms, including viruses, bacteria, and fungi in water and beverages (Chatterley and Linden, 2010; Islam et al., 2016; Shachman, 2005). UV irradiation on microorganisms induces lesions in genomic DNA, including formation of pyrimidine dimers between adjacent pyrimidines on the same DNA strand (Oppezzo and Pizarro, 2001). RNA synthesis and DNA replication fail in microorganisms with such DNA lesions, thus inhibiting growth and proliferation (Chatterley and Linden, 2010). Inactivation of microorganisms by UV irradiation is dependent on the UV dose, which is linearly related with UV irradiation time (Kowalski, 2009). For example, 90%, 99%, 99.9%, and 99.99% reductions of E. coli in water by UV irradiation were reported to require irradiation with UV at near 260 nm in doses of 5, 9, 14, and 18 mJ cm−2, respectively (Hijnen et al., 2006).

Light, including UV, traveling in a straight line can be refracted by a lens and reflected by a reflector. Reflective metal based sheets are frequently used in lamp bases to improve the illumination effectiveness, and focusing light using lenses can be used to start a fire. The effectiveness of UV inactivation is improved by placing a reflective metal sheet behind a quartz cell containing microorganisms (Sommer et al., 1996). The effectiveness of UV inactivation can be improved by focusing UV radiation on microorganisms in water and beverages. However, it is necessary to consider the size and distribution of microorganisms to develop lenses capable of focusing UV, and to improve the effectiveness of UV inactivation. Microorganisms that contaminate water and beverages include viruses and bacteria, which are 20–1000 nm and 1–8 μm in size, respectively (Tortora et al., 2015), and show a random distribution in these liquids. It was postulated that UV can be focused on randomly distributed microorganisms using an array of lenses separated by distances of less than a micrometer, which would improve the effectiveness of UV inactivation. Furthermore, electricity consumed by UV irradiation for pathogen inactivation can be saved using reflective nanolens arrays. To the best of our knowledge, a reflective nanolens array for improving UV inactivation of pathogens in liquid foods has not been reported.

Aluminum is highly reflective and is, therefore, widely used as a reflector in automotive applications, ceiling lights, and wall lighting. The reflectivity of aluminum depends on the smoothness of the surface. As aluminum is electrochemically reactive, the surface can be polished electrochemically to a mirror finish (Hou et al., 2018). Aluminum can be fabricated as a nanoporous structure with hexagonally arranged nanopores by application of an electrical voltage under acidic conditions in a process known as anodization (Ling and Li, 2015). Nanopores hexagonally arranged on aluminum during anodization are formed by simultaneous electrochemical processes involved in the growth and partial dissolution of aluminum oxide (Fig. 1). The oxide layer consists of hexagonal cells (Fig. 1A). A cylindrical nanopore with a hemispherical bottom is formed in the center of the cell during anodization of aluminum in an acidic environment (Fig. 1B) (Keller et al., 1953). Cell and nanopore sizes on anodic aluminum oxide (AAO) are dependent on the electrolytes and voltage used for anodization (Lee, 2015). Anodization of aluminum in oxalic acid and in phosphoric acid produced nanoporous aluminum with nanopores of 20–100 nm and 20–500 nm in diameter, respectively (Cheng and Ngan, 2015; Ono and Masuko, 2003). Cell and nanopore sizes on AAO were positively related to the anodization voltage (Cheng and Ngan, 2015; Ono and Masuko, 2003). Hemispherically shaped nanolens arrays can be obtained by removing AAO using a chemical solution that is selectively reactive to aluminum oxide (Fig. 1C) (ASTM, 2014).

Fig. 1.

Fig. 1

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Schematic representations of (A) hexagonally arranged nanopores on anodic aluminum, (B) the anodization process of nanopore formation on an aluminum surface, and (C) fabrication of a nanolens array by chemical stripping of anodized aluminum

Nanolens arrays were developed via a series of aluminum electropolishing and anodization steps, with subsequent selective dissolution of AAO. The diameter of nanolenses on the array was controlled by altering the voltage of aluminum anodization in oxalic acid and in phosphoric acid. Fabrication of nanolenses on aluminum surfaces was examined using a scanning probe microscope (SPM). Nanolens arrays that can improve UV inactivation of pathogens in water and soft drinks is proposed. A sucrose solution was used as a model soft drink. Escherichia coli O17:H7 and Listeria monocytogenes, one of the most life-threatening Gram-negative and Gram-positive food poisoning bacteria associated with hemorrhagic colitis and listeriosis, respectively (Cohen and Giannella, 1992; Farber and Peterkin, 1991), were used as pathogens for inactivation by UV irradiation. Nanolens arrays with a proper geometry that could improve the effectiveness of UV inactivation of E. coli O157:H7 and L. monocytogenes were chosen based on an investigation of UV inactivation in water where different reflective nanolens arrays were applied. It was demonstrated that use of a reflective nanolens array improved the effectiveness of UV inactivation of E. coli O157:H7 and L. monocytogenes in a sucrose solution.

Materials and methods

Preparation of nanolens arrays

Aluminum sheets (alloy 1050; Dongbang Metal Co., Siheung-si, Gyeonggi-do, Korea) were cut into a rectangular shape (15 mm × 80 mm). Aluminum strips were degreased in acetone using a sonication bath (Branson 3800; Branson, Danbury, CT, USA), washed with sterilized distilled water (DW), dried under a stream of HEPA-filtered air, and stored under sterilized conditions. For aluminum electropolishing, an aluminum strip was placed in an electrochemical reactor consisting of a regulated DC power supply (HYP-12005D; Han Young Electronic, Incheon, Korea), an anode clamp, a platinum cathode (size: 30 mm × 50 mm; Made Lab Co., Hanam-si, Gyeonggi-do, Korea), a jacketed beaker, and a circulating constant temperature bath (CCA-112A; Eyela, Tokyo, Japan). The aluminum strip connected to the anode clamp and a cathode were placed in a jacketed beaker filled with an electropolishing solution composed of ethyl alcohol, ethylene glycol, perchloric acid, and water (71:10:7:12). Electropolishing was carried out by applying 42 V to the aluminum strip for 40 s at 5 °C. Electropolished aluminum strips were rinsed with sterilized DW, dried under a stream of filtered air, and stored under sterilized conditions.

An electropolished aluminum strip was connected to an anode clamp to anodize the surface. An aluminum strip and a cathode were placed in a jacketed beaker containing 0.3 M oxalic acid and 1.0 M phosphoric acid. The aluminum strip was anodized at 40, 60, and 80 V in 0.3 M oxalic acid for 30 min at 5 °C, and at 100, 110, and 120 V in 1.0 M phosphoric acid for 30 min at 5 °C. To remove AAO from the anodized aluminum and produce a nanolens array, the anodized aluminum strip was kept in a solution composed of 0.15 M chromic acid and 0.6 M phosphoric acid overnight at 65 °C, rinsed with sterilized DW, dried under a stream of filtered air, and stored under aseptic conditions.

UV inactivation of E. coli O157:H7 and L. monocytogenes using nanolens arrays

E. coli O157:H7 ATCC 35150 and L. monocytogenes ATCC 7644 obtained from the American Type Culture Collection (Rockville, MD, USA) were cultured in Luria–Bertani broth (LB broth; BD Difco, Sparks, MD, USA) and Brain Heart Infusion broth (BHI broth; BD Difco), respectively. Cultures were centrifuged at 5000 × g for 20 min and the supernatant was discarded. Pellets were resuspended in sterilized DW, then diluted to prepare a 50 mL DW and a 10% (w/v) sucrose (CJ Cheiljedang Corp., Seoul, Korea) solution containing the microorganisms at 5–6 log CFU/mL and added in a 250 mL quartz beaker. Five strips of bare aluminum, electropolished aluminum, and nanolens array were placed in parallel on the center of the top of magnetic stirrer, then a 250 mL quartz beaker containing a 50 mL of one of the bacterial solutions on five aluminum strips. A UV lamp with emission primarily at 254 nm (8 W, type G8T5; Sankyo Denki, Hiratsuka, Kanagawa, Japan) was placed above the beakers at a distance from the UV lamp to the water surface of approximately 270 mm. The UV inactivation system, including UV lamp, quartz beaker, and magnetic stirrer, was enclosed in a black acrylic box. Aliquots of a solution of 1 mL or 0.1 mL were taken from the quartz beaker every 5 s for 35 s with stirring and irradiation. Aliquots were then diluted and spread on LB agar plates for E. coli O157:H7 (BD Difco) and BHI agar plates for L. monocytogenes (BD Difco). Numbers of viable E. coli O157:H7 and L. monocytogenes were enumerated after incubation of plates at 37 °C for 24 h. Analysis of microorganism inactivation using nanolens arrays was performed in triplicate. Log reductions of viable microorganism populations at 15 s were examined in each group using a one-way analysis of variance (AVONA) with SPSS software (Ver. 24, Statistical Package for the Social Sciences Inc., Chicago, USA). After ANOVA, Duncan’s multiple range test was performed to identify nanolens arrays that significantly improved the effectiveness of UV inactivation (p < 0.05).

UV transmittance of 50 mL sucrose solution was measured in triplicate as a function of the sucrose concentration using a radiometer (VLX-3X; Vilber Lourmat, Torcy, France) equipped with a UV (254 nm) sensor (CX-254; Vilber Lourmat). The height of the 50 mL sucrose solution in the 250 mL quartz beaker was approximately 13 mm.

Analysis of the nanolens array surface geometry

An SPM (Easyscan 2; Nanosurf AG, Liestal, Switzerland) was used to analyze the surface geometry of nanolens arrays. Surface geometric parameters were obtained in contact mode using a silicon cantilever with a nominal spring constant of 0.14 N m−1 (ShoconA; Applied Nanostructures, Inc., Mountain View, CA, USA). Surface geometric parameters were processed using SPIP software (Ver. 6.7.3; Image Metrology, Lyngby, Denmark) to produce images of surface geometries, which were also processed using an option in the SPIP software for analysis of the surface roughness of bare aluminum and electropolished aluminum and the nanolens diameter of (d) of the array. Surface roughness of bare and electropolished aluminum, and the d value of the array were analyzed in triplicate.

Results and discussion

Development of aluminum based reflective nanolens arrays

Alloy 1050, used in this study, is industrial grade aluminum commonly used in electrical, chemical, and food industries. It is generally finished mechanically and the surface is rough. Nanolens arrays based on alloy 1050 were required to contain orderly structured nanolenses on the surface to improve the effectiveness of UV inactivation. Protruding ridges of bare aluminum can be corroded electrochemically by electropolishing, which makes the surface smooth and allows production of a mirror-like surface. Aluminum strips were electropolished to smoothen the surface (Fig. 2), then subjected to anodization and subsequent selective AAO dissolution. Bare aluminum exhibited a surface roughness (arithmetic mean height) of 21.74 ± 3.14 nm. The roughness of the aluminum surface polished to a mirror-like finish by electropolishing showed a reduction in roughness to 1.33 ± 0.28 nm. Despite use of an industrial grade aluminum (alloy 1050), orderly structured nanolens arrays were achieved (Fig. 3). However, nanolens arrays produced by anodization in oxalic acid at voltages > 80 V and in phosphoric acid at voltages > 120 V were not included in further experiment due to surface burn defects. Since a higher voltage could be applied to aluminum in phosphoric acid than oxalic acid, the d value of arrays produced by anodization in phosphoric acid was larger than for production in oxalic acid (Fig. 3). The d value was increased with an increased anodization voltage. The average d values of arrays produced by anodization in oxalic acid at 40 V and in phosphoric acid at 100 V were 71.94 and 220.16 nm, respectively, and increased to 161.53 and 274.78 nm with an increase of anodization voltage to 80 V in oxalic acid and 120 V in phosphoric acid, respectively (Fig. 3A, C, D, and E). However, the standard deviation of the d value ranged from approximately 11 to 35 nm for both arrays. Large standard deviations of d values were probably due to impurities included in the industrial grade aluminum that was used (alloy 1050).

Fig. 2.

Fig. 2

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Scanning probe microscope (SPM) images of the surface of (A) bare and (B) electropolished aluminum

Fig. 3.

Fig. 3

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SPM images of nanolens arrays that were developed by selective chemical dissolution of electropolished aluminum strips anodized in 0.3 M oxalic acid at (A) 40, (B) 60, and (C) 80 V, and in 1.0 M phosphoric acid at (D) 100, (E) 110, and (F) 120 V

Choice of nanolens arrays to improve the effectiveness of UV inactivation

Microorganisms subjected to the UV inactivation system would be affected by both direct UV radiation from the UV lamp and radiation reflected by nanolens arrays. Thus, E. coli O157:H7 and L. monocytogenes in water with arrays were inactivated more rapidly than without use of arrays (Fig. 4A and B). It took 30 s to inactivate 5–6 log CFU/mL E. coli O157:H7 and L. monocytogenes to 0 log CFU/mL with only direct UV. Concentrations of both organisms were reduced to 0 log CFU/mL within 15 s with use of arrays produced by anodization in phosphoric acid at 100, 110, 120 V, and 110 V (Fig. 4A and B). Reductions of viable E. coli O157:H7 and L. monocytogenes populations at 10 and 15 s using nanolens arrays produced by anodization in phosphoric acid at 110 V and 110 and 120 V were significantly greater than under other test conditions for each organism (Fig. 4C and D). Thus, nanolens arrays produced by anodization in phosphoric acid at 110 V were used for application of UV inactivation of E. coli O157:H7 and L. monocytogenes in a 10% sucrose solution.

Fig. 4.

Fig. 4

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UV inactivation of (A) E. coli O157:H7 and (B) L. monocytogenes in water using aluminum based nanolens arrays. Log reductions of viable (C) E. coli O157:H7 at 10 s and (D) L. monocytogenes at 15 s with UV irradiation using nanolens arrays. Different superscripts indicate significant differences (p < 0.05)

The sum of UV energy reflected by electropolished aluminum strips should be equivalent to the sum reflected by nanolens arrays, as UV inactivation was tested under the same conditions and the electropolished aluminum strips and nanolens arrays were identical in size (15 mm × 80 mm). Accordingly, the difference in UV inactivation between electropolished aluminum strips and nanolens arrays was probably due to nanolenses.

Patterns of light reflection from reflective lens arrays are governed by the wavelength of incident light and the lens diameter (Tsao et al., 2013). Light reflection from a lens array tends to exhibit characteristics of total reflection if the diameter of lenses in the array is not similar to the wavelength of incident light. The degree of light scattering is increased as the lens diameter becomes close to the light wavelength. The d value of nanolens arrays produced by anodization in phosphoric acid at 110 V was 252.06 nm (Fig. 3E). A UV lamp emitting primarily at 254 nm was used to inactivate E. coli O157:H7 and L. monocytogenes. More incident 254 nm UV radiation was probably focused near E. coli O157:H7 and L. monocytogenes by reflection from nanolens arrays having a d value of 252.06 nm than by arrays having a d value that was not close to 254 nm in wavelength of UV. This probably improved UV inactivation (Fig. 4).

Use of nanolens arrays to improve the effectiveness of UV inactivation of E. coli O157:H7 and L. monocytogenes in 10% sucrose solutions

Water transmits UV, but the presence of suspended or dissolved food ingredients curtails UV transmittance. Although sucrose transmits UV comparably well in comparison with other food ingredients, such as proteins and lipids, UV transmittance was decreased with an increase in the sucrose concentration (Fig. 5). The total sugar concentration of the majority of soft drink products is approximately 10% (Ventura et al., 2011). Approximately 84% of UV at 254 nm is transmitted through a 10% sucrose solution with a height of 13 mm (Fig. 5). E. coli O157:H7 and L. monocytogenes at concentrations of 5–6 log CFU/mL in both water and a 10% sucrose solution were inactivated by UV irradiation with populations reduced to 0 log CFU/mL within 30 s and 35 s, respectively, with no nanolens array (Fig. 4A and B and Fig. 6). More UV irradiation time was required to inactivate these populations to 0 log CFU/mL in a 10% sucrose solution than in water as sucrose molecules in the solution adsorb UV. When nanolens arrays having a d value of 252.06 nm were used for UV inactivation of E. coli O157:H7 and L. monocytogenes in a 10% sucrose solution, UV transmitted through the solution was reflected by the arrays and focused near the organisms, resulting in inactivation to a concentration of 0 log CFU/mL within 25 s (Fig. 3E and Fig. 6). L. monocytogenes, a Gram positive bacterium, has a thicker peptidoglycan layer than E. coli O157:H7, a Gram negative bacterium. Despite the difference in the cell wall structure between the two, the effectiveness of UV inactivation of E. coli O157:H7 was not much different from the effect on L. monocytogenes, even in a 10% sucrose solution (Fig. 6). The inactivation effectiveness was improved by use of nanolens arrays having a d value of 252.06 nm (Fig. 6). Therefore, use of a nanolens array having a d value of 252.06 nm can save energy and time for UV inactivation in the food industry.

Fig. 5.

Fig. 5

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Transmittance (%) of UV (at 254 nm) as a function of the concentration of sucrose in solution (% w/v)

Fig. 6.

Fig. 6

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UV inactivation of (A) E. coli O157:H7 and (B) L. monocytogenes in a 10% (w/v) sucrose solution using aluminum based nanolens arrays produced by anodization in 1.0 M phosphoric acid at 110 V (the average of d = 252.06 nm)

Electronic supplementary material

Below is the link to the electronic supplementary material.

10068_2020_765_MOESM1_ESM.tif (2.2MB, tif)

Fig. S1. Experimental setups for ultraviolet (UV) inactivation.(TIFF 2221 kb)

Acknowledgments

This study was supported by a grant from the National Research Foundation of Korea (NRF-2016R1A2B1012571) funded by the Korean government.

Compliancee with ethical standards

Conflict of interest

The nanolens array manufactured and used for this report is protected by Korea patent filing serial number ‘10-2018-0106899’, “Ultraviolet sterilizing apparatus using a lens array,” filed Sep 7, 2018. This does not alter author adherence to all Food Science and Biotechnology policies regarding sharing data and materials.

Footnotes

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Supplementary Materials

10068_2020_765_MOESM1_ESM.tif (2.2MB, tif)

Fig. S1. Experimental setups for ultraviolet (UV) inactivation.(TIFF 2221 kb)

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