Ultrafast light induced inactivation of microbes based on BIO-affinity ligands

Abstract

Constructs and methods for microbial inactivation that effectively combines the affinity ligands with photochemicals at spatially distinct locations on a substrate to provide effective binding of the microbial targets while preventing any quenching of the reactive oxygen species (ROS) by the targeting ligands. The binding assisted photoinactivation is a key mechanism for enhancing the rate of inactivation of bacteria and viruses over photochemical applications alone.

Description

ULTRAFAST LIGHT INDUCED INACTIVATION OF MICROBES BASED ON BIO-AFFINITY LIGANDSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. provisional patent application serial number 63 / 619,450 filed on January 10, 2024, incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under grant number 2022-67017-36308, awarded by The U.S. Department of Agriculture (USDA- NIFA). The Government has certain rights in the invention.NOTICE OF MATERIAL SUBJECT TO COPYRIGHT PROTECTION

[0003] A portion of the material in this patent document may be subject to copyright protection under the copyright laws of the United States and of other countries. The owner of the copyright rights has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the United States Patent and Trademark Office publicly available file or records, but otherwise reserves all copyright rights whatsoever. The copyright owner does not hereby waive any of its rights to have this patent document maintained in secrecy, including without limitation its rights pursuant to 37 C.F.R. § 1.14.BACKGROUND

[0004] 1. Technical Field

[0005] This technology pertains generally to devices and techniques for bacteria and virus neutralization and more particularly to photo-active matrices modified with bio-affinity ligands to capture target microbes to the surface of the matrix for rapid inactivation of the microbes. The constructs can be used for rapid inactivation of bacteria and viruses in areas, such asclinical treatment, air filtration, water purification and food safety.

[0006] 2. Background

[0007] Due to the rapid spread of pathogenic microorganisms, there is an urgent and pressing need to develop alternative antimicrobial approaches for applications that need rapid inactivation of bacteria and viruses, such as clinical treatment environments, air filtration, water purification and food safety. Since the abuse of chemical bactericides can bring pollution, toxicity and drug-resistant bacteria, it is highly desired to develop new and environmentally friendly ways to kill bacteria and other microbes.

[0008] Photoactive chemicals can produce reactive oxygen species (ROS), including hydroxyl radicals (HO’), superoxide radical anions (O2— ), hydrogen peroxide (H2O2), and singlet oxygen (1O2), under irradiation of light. However, the key limitations are the short half-life of many of these ROS species and limited rate of inactivation of bacteria due to various constraints. Many of the current photoactive chemicals require extended treatment time (including extended exposure to light sources). The treatment time of current approaches can range from a few minutes to hours to generate adequate inactivation of bacteria. This limits the application of these materials in biomedical, environmental hygiene and food industries. In addition, many of the photo-active agents in use are not food grade compounds or food approved chemicals and thus have very restrictive applications.

[0009] In addition, many of the applications with photoactive chemicals require direct addition of these photochemical agents to the target system in a liquid suspension and thus have limitations such as possible concerns with the residues of the chemicals in the target system after the treatment. Furthermore, many existing photochemical systems have limited efficacy for aerosol treatments. Accordingly, there is a need for innovative photosensitizer materials and systems that are capable of rapid inactivation of pathogenic bacteria and viruses that overcome these challenges.BRIEF SUMMARY

[0010] Unlike most antimicrobials, that have significant toxicity and environmental impact, food and human safe compositions are provided thatenable easy adaptations of the technology to food, environmental and biomedical applications. Many existing antimicrobial materials require close contact between microbes and surface associated antimicrobial compositions. Due to surface properties of both microbes and contact surfaces, however, this contact can be limited and thus greatly reduces both the efficacy and kinetics for the inactivation of the antimicrobial compositions. This is vital for applications targeted for reducing cross-contamination through surfaces, air and water as rate of inactivation significantly influences the risk of crosscontamination.

[0011] The provided compositions have a substrate with affinity ligands configured to capture and couple to a target microbe and a photosensitizer adjacent to the affinity ligands that preferably produces a reactive oxygen species (ROS) such as hydroxyl radicals (HO ), hydrogen peroxide (H2O2), and singlet oxygen (1O2) that neutralize the captured target microbe. The photo-active matrices are modified with one or more types of bio-affinity ligands to capture the microbes to the surface of the matrix in proximity to photosensitizer molecules for rapid inactivation of the microbes. With exposure to light, the photosensitizer is induced to generate ROS which inactivates the microbe target. It is believed that enhanced absorption of microbes improves interaction between the generated reactive oxygen species (ROS) and target microbes enhances the antimicrobial effectiveness of short-lived ROS species.

[0012] Specific designs aimed at a faster microbial inactivation rate have proven to be quite successful with Gram-negative and Gram-positive bacterial models as well as viral models. The novel prepared matrices exhibited between about 15 to about 360-fold increase in the rate of inactivation against tested bacterial and viral models compared to the results of unmodified matrices under the induction of different accessible light sources.

[0013] To address these challenges, innovative formulations of photochemicals have been developed that: (a) significantly enhance the rate of inactivation of the bacteria and viruses (in some compositions over 360 fold enhancement in the rate of inactivation) in both liquid and aerosolized droplets using a unique combination of affinity ligands and photochemicals; (b)develop solid photosensitizer (including fibers) compositions that effectively combines the affinity ligands with photochemicals at spatially distinct locations to provide effective binding of the microbial targets while preventing any quenching of the ROS by the targeting ligands. The binding assisted photoinactivation is one key mechanism for enhancing the rate of inactivation of bacteria and viruses; (c) the development of these novel compositions eliminates the requirement to add these photochemicals directly to the system and thus enables applications for wound dressings, air filters, water purification without directly contaminating the treated system; (d) Inactivation could be achieved using daylight (indoor visible light), cool LED light irradiation, mobile phone flashlight irradiation, sunlight and UV-A, thus enabling applications in diverse environments and light power levels in the range of 1 ,050- 90,000 Lux.

[0014] The inactivation performance of the developed matrices, designed to enhance the interaction between the generated ROS and the microbe cell via capture the microbes’ cells by bio-affinity ligand. By comparison, current photo-induced approaches provide this rapid inactivation of more than 7 log of the microbes’ population in short time between 10 seconds to about 5 minutes. Moreover, there is not any system that uses bio-affinity ligands to accelerate the inactivation rate.

[0015] In one embodiment, a construct is provided with multiple affinity ligands on a small-scale support that binds the target in proximity to inactivating photochemicals that produce a zone of inactivation. The construct strategically combines the affinity ligands with selected photochemicals at spatially distinct locations to provide effective binding of the microbial targets while preventing any quenching of the ROS by the targeting ligands and enhancing the rate of photoinactivation. Preferred substrates include Zein films, curcum in-based nanofibrous membranes and a Rose Bengal-modified nanofibrous membranes. The substrate may also include synthetic polymers including nylon, PVA, PVA-co-PE and various forms of these polymers.

[0016] Accordingly, the technology provides a microbe neutralizing particle platform with neutralizing means such as one or more types of photosensitizer molecules bound to the surfaces or inside the substrate, ligand or linker thatare initiated by an exposure to light. The photosensitizers are preferably one or both of two classes of photosensitizer molecules: 1 ) Small molecules, such as curcumin or rose Bengal. These molecules can be adsorbed or conjugated to the substrate or can in some cases, may be encapsulated. 2) In some cases, the substrate itself can have photosensitizer / neutralization properties, such as zein films, that have photosensitization capabilities. In another case, a photosensitizer is a macromolecule (could be a protein such as zein). In one embodiment, the means for neutralizing is a photochemical that produces a reactive oxygen species (ROS) coupled to the substrate adjacent to an affinity ligand. Preferred means for neutralizing is a photochemical that produces a reactive oxygen species (ROS) selected from the group of hydroxyl radicals (HO’), superoxide radical anion (O2_) hydrogen peroxide (H2O2), and singlet oxygen (1O2) upon exposure to light.

[0017] The rate of inactivation can also be enhanced by using an affinity ligand with reduced MW / size as small ligands may bring the microbe closer to the ROS penetration / migration area.

[0018] Further aspects of the technology described herein will be brought out in the following portions of the specification, wherein the detailed description is for the purpose of fully disclosing preferred embodiments of the technology without placing limitations thereon.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The technology described herein will be more fully understood by reference to the following drawings which are for illustrative purposes only:

[0020] FIG. 1 is a side-view diagram of a construct for neutralizing microbes shown schematically according to an embodiment of the technology.

[0021] FIG. 2 is a flowchart of one embodiment of the methods of the technology.

[0022] FIG. 3 is a graph of antimicrobial activity of yeast cell wall particles (YCW) under the daylight irradiation against L. innocua.

[0023] FIG. 4A is a graph showing binding affinity of stained YCW to L innocua biofilm.

[0024] FIG. 4B is a graph showing binding affinity of stained innocua to different zein matrices.

[0025] FIG. 5A is a graph of antibacterial activity of Zein film under daylight irradiation against: E. coli.

[0026] FIG. 5B is a graph of antibacterial activity of Zein film under daylight irradiation against L. innocua.

[0027] FIG. 6 is a graph of antiviral activity of zein film against phage T7 under daylight irradiation.

[0028] FIG. 7A is a graph of daylight-induced antibacterial activity of zein- YCW matrix against E. coli.

[0029] FIG. 7B is a graph of daylight-induced antibacterial activity of zein- YCW matrix against L. innocua.

[0030] FIG. 8 is a graph of daylight-induced antiviral performance of zein- YCW matrix against phage T7.

[0031] FIG. 9A is a graph of antibacterial activity of CrNFM matrix under daylight irradiation against L. innocua.

[0032] FIG. 9B is a graph of antibacterial activity of CrNFM matrix under daylight irradiation against E. coli.

[0033] FIG. 10 is a graph of antiviral activity of CrNFM matrix under daylight irradiation against bacteriophage T7.

[0034] FIG. 11 A is a graph of daylight-induced antibacterial activity of CrNFM- YCW matrix against E. coli.

[0035] FIG. 11 B is a graph of daylight-induced antibacterial activity of CrNFM- YCW matrix against L innocua.

[0036] FIG. 12 is a graph of daylight-induced antiviral activity of: CrNFM-YCW matrix against bacteriophage T7 under daylight irradiation.

[0037] FIG. 13 is a graph of antibacterial activity of Free Mn (100 mg / mL) against E. coli under daylight irradiation.

[0038] FIG. 14 is a graph of daylight-induced antibacterial activity of RB-NFM matrix against E. coli.

[0039] FIG. 15 is a graph of daylight-induced antibacterial activity of RBMn- NFM matrix against E. coli.

[0040] FIG. 16 is a graph of LED-induced antibacterial activity of RBMn-NFM matrix against E. coli.

[0041] FIG. 17 is a graph of mobile phone flashlight-induced antibacterial activity of RBMn-NFM matrix against E. coli.

[0042] FIG. 18 is a graph of the survival of E. coli O157:H7 on coupons coated with OPE and gelatine (“OPE coating”) or gelatine alone (“Gelatin only”) after 15-min incubation with or without UVA light treatment.DETAILED DESCRIPTION

[0043] Referring more specifically to the drawings, for illustrative purposes, constructs and methods for the rapid inactivation of microbes are generally shown. Several embodiments of the technology are described generally in FIG. 1 to FIG. 18 to illustrate the characteristics and functionality of the apparatus and methods. It will be appreciated that the methods may vary as to the specific steps and sequence and the systems and apparatus may vary as to structural details without departing from the basic concepts as disclosed herein. The method steps are merely exemplary of the order that these steps may occur. The steps may occur in any order that is desired, such that it still performs the goals of the claimed technology.

[0044] Turning now to FIG. 1 , the basic structure of the microbe inactivation constructs and methods are generally illustrated schematically. The constructs 10 are efficient light-induced antimicrobial matrices against a wide range of the microbes under activating conditions such as daylight, cool LED light irradiation and mobile phone flashlight illuminations. Generally, the matrix constructs 10 have a substrate 12 with outer surfaces or porous surfaces 14. Although a planar substrate is shown in the embodiment of FIG. 1 , the substrate 12 may have a particulate or spherical structure. The outer surfaces 16 of the substrate 1 are modified with one or more types of affinity ligands 16 that are capable of coupling with target microbes 18. Also coupled to the substrate surface 14 are photosensitizer molecules 20 that are capable of releasing neutralizing molecules 24 upon exposure to light. In another embodiment, the substrate is capable of releasing or producing neutralizing molecules 24. Photosensitizers 20 and affinity ligands 16 may be chemicallyattached or adsorbed to the surface of the substrate.

[0045] One embodiment of an associated method 100 for inactivation of bacterial or virus targets using a photo activated construct is shown in FIG. 2. At block 110 of FIG. 2, a target bacteria or virus for neutralization is identified and evaluated. The characteristics of the target identified at block 110 will influence the later selection of the affinity ligands and photosensitizer molecules. In some instances, the target may be a genus or other grouping of microbes with common characteristics that have affinity with a ligand or a sensitivity to a particular or ROS or photochemical.

[0046] After the identification of the target at block 110, the substrate, affinity ligand and photosensitizer molecules are selected at block 120 of FIG. 2. More than one substrate, affinity ligand or photosensitizer can be selected at block 120. For example, affinity ligands with different affinities can be selected at block 120. Similarly, different photosensitizers can be selected that are effective with the selected target or work synergistically can be selected.

[0047] Once the selections have been made, the substrate, affinity ligand and photosensitizer materials are obtained, and a construct is assembled at block 130 of FIG. 2. It can be seen that the density of the selected affinity ligands and photo applied to the surfaces of the substrate can be selected and optimized for specific target microbes and applications. The ratio of the number of selected affinity ligands to the number of selected photosensitizer molecules applied to the surfaces of the substrate can also be adjusted and optimized.

[0048] The constructs are preferably assembled at block 130 so that the affinity ligands and photosensitizer molecules on the surfaces of the substrate are separated by distances in a range from about 10 nm to about 100 microns. The preferred surface density or concentrations on the substrate has the adjustable concentration range of photosensitizer molecules from the micromolar and millimolar range per unit area and the corresponding affinity ligands to be within nanomolar to micromolar range of concentration.

[0049] In one embodiment the construct is a film, particle, fiber, coated fiber or coated particle with affinity ligands mounted on the outer surfaces of the filmor fiber. In another embodiment, more than one type of affinity ligand is coupled to the surface of the film or fiber. The selection, concentration and spatial distribution of the affinity ligands and the selection of the one or more photochemicals that are applied to the construct can be controlled. The type, form and dimensions of the construct can also be controlled and adapted to specific inactivation settings. For example, the concentration of inactivation molecules experienced by the captured microbe can be regulated by the selected density of photosensitizers mounted to the substrate.

[0050] The substrate may also be developed using cellulose or other carbohydrate-based polymers in the form of micro and nanofiber membranes, films, aerogels and particles. These carbohydrate structures are further modified with photosensitizer molecules. With exposure to light, the photosensitizer is induced to generate a ROS which inactivates the microbe target. These photosensitizer molecules may be on the surface of these structures and these ligands bring the microbes within the range of ROS penetration zone and enhance the inactivation. Thus, smaller ligands may bring the microbes closer to the surface than micron scale yeast cell wall particles. This results in an enhanced rate of inactivation of the microbes with smaller ligands.

[0051] In one embodiment, the substrate is selected from a range of naturally occurring plant and animal derived proteins or glycoproteins including zein, gelatin, collagen, keratin and their subsequent treatment with acidic (2 pH to 6.5 pH) and / or basic (8 pH to 2 pH) solutions to induce structural transformations enhancing ROS generation upon exposure to light.

[0052] Preferred substrates or coatings that produce a reactive oxygen species upon exposure to light. Preferred coatings include yeast cell wall particles (YCW), D-mannose, mucins, oligosaccharides, lactoferrin, chitosan, isolated RBC cell membranes, platelets, and antimicrobial peptides such as Lipid A, Lipid II, Lipopolysaccharide transport (LptD) and Peptidoglycan binding peptides.

[0053] In one embodiment, the photo-active matrices are modified with conjugated yeast cell wall particles (YCW) or D-mannose to improve the antimicrobial performance through capture of diverse pathogens to closeproximity to the solid matrix consequently efficiently utilize the generated ROS. The experimental validation of YCW / D-mannose role in enhancing the antimicrobial performance and rate of the different solid light-active naturalbased matrices (Zein film and curcum in-based nanofibrous membranes) and synthetic photosensitizer-based matrices (Rose Bengal-modified nanofibrous membranes) are shown in the examples.

[0054] In addition to YCW particles and D-mannose, the other compositions that may provide similar enhancement in the photo-activity of the material for the inactivation of bacteria and viruses include: mucins; oligosaccharides lactoferrin; chitosan as well as host of bio-based molecules such as isolated cell membranes from RBCs and platelets and antimicrobial peptides such as Lipid A, Lipid II, Lipopolysaccharide transport (LptD) and Peptidoglycan binding peptides.

[0055] The final constructs prepared at block 130 may be deployed in a variety of different contexts to allow the affinity ligands to bind with targets at block 140 of FIG. 2. For example, the constructs can be prepared and used in medical and food storage and service contexts as well as with air filtration and water purification applications. The targets will be captured by the affinity ligands over time. The time of capture of targets can also be optimized through the selection of the density of affinity ligands and photosensitizers.

[0056] Finally, at block 150 of FIG. 2, the constructs are exposed to light to activate the photosensitizer molecules or the photosensitive substrate to release the neutralizing molecules or chemicals for rapid inactivation of the captured microbes. Inactivation can be achieved using daylight (indoor visible light), cool LED light irradiation, mobile phone flashlight irradiation, sunlight and UV-A, thus enabling applications in diverse environments and light power levels in the range of 1 ,050 to 90,000 Lux.

[0057] The technology described herein may be better understood with reference to the accompanying examples, which are intended for purposes of illustration only and should not be construed as in any sense limiting the scope of the technology described herein as defined in the claims appended hereto.

[0058] Example 1

[0059] To demonstrate the functionality of the constructs and neutralization methods, a construct with yeast cell wall affinity ligands was fabricated. Initially, the antimicrobial activity of YCW particles against L innocua under the light irradiation condition was evaluated. The graph of FIG. 3 shows that the tested YCW particles alone did not demonstrate any antibacterial activity toward the bacteria.

[0060] The efficiency of YCW as an affinity ligand to bind with bacteria and bacterial biofilms was then assessed. To evaluate the relative efficiency of YCW to bind bacterial biofilm, the fluorescence-labeled YCW was incubated with biofilm at room temperature for 1 hour. After incubation, the attachment of YCW to L innocua biofilm was quantified based on the measurement of fluorescence intensity. This measurement represented the relative concentration or density of the bound YCW to the biofilm substrate. As shown in FIG. 4A, 1 hour incubation of fluorescently labeled YCW resulted in a significant binding of the particles to the selected model biofilm. The binding efficiency of the fluorescently labeled YCW was approximately 15% for the L. innocua biofilm.

[0061] To investigate the binding affinity of bacteria to YCW-modified matrices, the fluorescence-labeled L. innocua was incubated with pristine matrices and YCW-modified matrices. The immobilization of YCW onto the zein film improved the binding of L. innocua cells by more than 40% as seen in FIG. 4B.

[0062] Example 2

[0063] To further demonstrate the functionality of the constructs with different substrates, a construct with a zein film substrate was modified with YCW affinity ligands. Initially, a zein film (10% w / v) was prepared in ethanol after totally dissolving at 50 °C, the solution was cast and left to dry overnight at 50 °C. Thereafter, YCW at a concentration of 50 mg / mL was immobilized onto the zein film surfaces using carbodiimide conjugation reaction.

[0064] The light-induced antimicrobial activity of zein-based matrices was then evaluated. After immersing the prepared pristine zein films and YCW- modified zein films in acidic buffer for 5 minutes and drying, the antimicrobialactivity was investigated against two bacterial strains which are Listeria innocua as a Gram-positive model and Escherichia coli a Gram-negative bacteria model. Also, the antimicrobial activity was tested against bacteriophage T7 as viral surrogate.

[0065] As shown in FIG. 5, the zein film provided more than 6 log reduction (>99.9999%) against E. coli after 60 minutes of the daylight irradiation in comparison with the control sample exposed to the same light conditions. The entire reduction (> 6 log) of on L. innocua the prepared zein films was required to 120-m inute exposure to daylight irradiation. Similar levels of inactivation were achieved against bacteriophage T7 in 60 minutes as shown in FIG. 6. The control zein films (in the dark) generated no significant inactivation of the selected microbes.

[0066] After modification of the zein films with YCW, the modified zein films illustrated 30-fold and 24-fold increase in the rate of inactivation of E. coli and L. innocua cells respectively compared to the results of unmodified zein films. The reduction of 7 log (99.99999%) of L. innocua required 5 minutes of exposure time to daylight with inactivation of more than 3 log after only 2 minutes. In the case of E. coli, the 7-log reduction was achieved within only 2 minutes under daylight irradiation as illustrated in FIG. 7.

[0067] In addition to bacteria, similar rates of rapid inactivation were also observed in the case of viral particles using T7 bacteriophage as a model system (FIG. 8). The T7 bacteriophage required about 5 minutes of daylight irradiation on the surface of YCW-modified zein films to achieve reduction of more than 6 log.

[0068] Example 3

[0069] Fabrication of light-active natural-based matrices was further evaluated with non-fibrous membrane (NFM) constructs. Curcum in-based nanofibrous membranes (CrNFMs) were fabricated. The PVA-co-PE nanofibrous membranes were fabricated using electrospinning. The prepared NFMs were modified with curcumin at a concentration of 2 mg / mL using glutaraldehyde (0.5% v / v) in an acidic medium. The PVA-co-PE NFM constructs were modified with YCW ligands at concentration of 50 mg / mL and curcumin at concertation of 2 mg / mL using glutaraldehyde (0.5% v / v) in acidic medium.

[0070] Light-induced antimicrobial activity of curcum in-based nanofibrous membranes was also evaluated. The antimicrobial function of the prepared CrNFM under daylight exposure was investigated against L innocua, E. coli and T7 bacteriophage targets.

[0071] The CrNFM exhibited a desired antimicrobial function against all the tested microorganisms. CrNFMs under the daylight induction provided 6 log reduction (>99.9999%) after 75 minutes against L innocua as shown in FIG. 9A. However, no significant reductions were achieved either in the control nanofibrous membranes under the light exposure or the CrNFMs in the dark conditions. Only the combination of daylight irradiation, curcumin, and polymer could demonstrate a significant antibacterial effect. The CrNFMs needed a shorter time to achieve a complete inactivation against E. coli than that of L innocua, whereas the same log reduction was achieved within only 30 minutes of exposure (FIG. 9B).

[0072] As shown in FIG. 10, Phage T7 was required to the daylight exposure for 30 min on the surface of CrNFM for achieving the more than 6 log reduction (>99.9999%), providing more than 1 log reduction after only 5 minutes of the daylight irradiation.

[0073] After immobilization of YCW, the prepared matrix (CrNFM-YCW) exhibited rapid bacterial inactivation, achieving more than 7 log inactivation in 2 min and 5 min of both model of bacteria (E. coli and L. innocua, respectively) under the daylight irradiation as seen in FIG. 11. Similarly, the modification of the photosensitizer-based material with YCW significantly accelerates the inactivation rate of viral particles. CrNFM-YCW reduced more than 2 log (99%) of phage T7 population in only 1 minute of the daylight exposure and the 6-log reduction acquired in 5 minutes under the daylight induction as shown in FIG. 12.

[0074] Example 4

[0075] Light-induced antimicrobial activity of Rose Bengal and Mannosebased nanofibrous membranes was evaluated to further demonstrate the functionality of the constructs. For evaluation of different photosensitizer agents and further enhancement of the antimicrobial performance of the lightbased matrix, D-mannose (Mn) was used as one of the affinity ligands andone of the microbial binding receptors on the surface of the YCW particles and Rose Bengal (RB) was employed as photosensitizer agent.

[0076] The antimicrobial activity of Mn against E. cali under the light irradiation condition was evaluated at the concentration of 100 mg / mL. The plot of FIG.13 confirmed that Mn does not have any antibacterial activity toward the bacteria.

[0077] The prepared NFMs were modified with RB (250 mg / L) using carbodiimide crosslinking chemistry followed by the attachment of Mn at concertation of 5 mg / mL using glutaraldehyde.

[0078] The antimicrobial function of the prepared RBMn-NFM was investigated against E. coli under daylight, LED light and mobile phone flashlight exposure to prove the superior activity of the developed matrix under broad and different light sources.

[0079] RB-NFM without Mn modification exhibited a desired antimicrobial function against E. coli under the daylight induction providing 6 log reduction (>99.9999%) after 60 minutes (FIG. 14).

[0080] After the modification with of Mn, the prepared matrix (RBMn-NFM) exhibited ultrafast bacterial inactivation, provided more than 1 log reduction (>90%) within 5 seconds and more than 6 log inactivation (>99.9999%) was achieved in only 10 seconds under the daylight irradiation with more than 360- folds faster than the RB-NFM without Mn modification (FIG. 15). While no significant reductions were achieved either in the control nanofibrous membranes under the light exposure or the RBMn-NFMs in the dark conditions. Only the combination of daylight irradiation, with the novel matrix could demonstrate a significant antibacterial effect.

[0081] Under the visible light (cool LED light) exposure, the developed matrix (RBMn-NFM) depicted a similar ultrafast antimicrobial performance with more than 6 log inactivation (>99.9999%) within only 10 seconds while no antimicrobial activity was achieved with RB-NFM without Mn modification under LED exposure for a longer time (1 min) (FIG. 16).

[0082] Under the mobile phone flashlight induction, E. coli was required to the flashlight exposure for 30 seconds on the surface of the developed matrix (RBMn-NFM) for achieving the more than 6 log reduction (>99.9999%),providing more than 2 log reduction after only 10 seconds of the phone flashlight irradiation (FIG. 17).

[0083] Example 5

[0084] Light-assisted antimicrobial properties of the plant derived extract coating against bacteria was evaluated to show the breadth of possible constructs. The survival of E. coli O157:H7 on coupons coated with OPE and gelatine (“OPE coating”) or gelatine alone (“Gelatin only”) after 15-min incubation with or without UVA light treatment is shown in FIG. 18.

[0085] To improve the coating efficiency of Olive Pomace Extract (OPE) on polypyrene (PP) and stainless steel (SS), gelatine was added as a crosslinking agent. Briefly, sixty microliter of 20% gelatine solution and OPE (5 mg GAE / mL) were sequentially deposited onto the surface of PP or SS coupons. Coated coupons were rinsed with DI water and air dried for 24 hours. Coupons coated with 20% gelatine alone (without OPE) were included as negative controls. Coated coupons were then inoculated with stationeryphase culture of rifampicin-resistant E. coli O157:H7 (ATCC 700728) and subsequently incubated with or without UVA light for 15 min. Bacteria on coupons before or after the treatment were recovered with maximum recovery diluent and enumerated by plating on tryptic soy agar with 50 ppm rifampicin. As shown on FIG. 18, E. coli O157:H7 population on OPE-coated coupons was reduced by 2.5-3 log after 15-min UVA treatment, where no significant reduction was observed on coupons coated with gelatine only. Bacterial inactivation on coated coupons without UVA light treatment was less than 1 log. The results suggest that the OPE coating and the UVA light treatment can induce synergistic antimicrobial effect within 15 minutes on both PP and SS coupon surface.

[0086] This result demonstrates the application of plant derived extracts that may contain complex mixture of plant bioactives including phenolics, phenolic acids, and other bioactives including peptides. These results also illustrate that it is possible to develop coatings with a crude plant extract with photoactive properties to inactivate target bacteria. In contrast to pure compounds such as curcumin, rose Bengal and their linkages to biopolymers for coating the surface, this set of results is for a coating approach based onphysisorption of complex plant extract on biopolymers and using this coating system to develop photoactive antimicrobial surfaces. Thus, extending both the range of compositions of photoactive materials and methods to develop affinity based photoactive coating.

[0087] From the description herein, it will be appreciated that the present disclosure encompasses multiple implementations of the technology which include, but are not limited to, the following:

[0088] A construct for neutralizing microbes, comprising: (a) a substrate with an outer surface; (b) a plurality of affinity ligands configured to bind to at least one moiety of a target bacteria, fungi or virus with high affinity, the ligands physically adsorbed or chemically bound to the outer surfaces of the substrate; and (c) means for neutralizing a target placed on the outer surface of the substrate so that exposure to light neutralizes a captured target.

[0089] The construct of any previous or following implementation, wherein the substrate comprises a substrate selected from the group of an aerogel, a film, a particle, a film modified with particles, a fiber, a film coated fiber and microfiber and nanofiber membranes.

[0090] The construct of any previous or following implementation, wherein the substrate comprises a synthetic polymer selected from the group of nylon, PVA and PVA-co-PE.

[0091] The construct of any previous or following implementation, wherein the substrate comprises a natural polymer selected from the group of a cellulose- based polymer and a carbohydrate-based polymer.

[0092] The construct of any previous or following implementation, wherein the ligand comprises a bacteriophage, bacteriophage derived tail fiber proteins, antibodies, nanobodies, fungal hyphae and algal cell walls.

[0093] The construct of any previous or following implementation, wherein the substrate comprises a substrate selected from the group of a Zein film, a curcum in-based nanofibrous membrane and a photosensitizer-modified nanofibrous membrane.

[0094] The construct of any previous or following implementation, further comprising a substrate composition or surface additives that enhance the dispersion of the fluid on the substrate to reduce the overall contact angle.

[0095] The construct of any previous or following implementation, wherein the means for neutralizing is a photochemical that produces a reactive oxygen species (ROS) coupled to the substrate adjacent to the affinity ligands.

[0096] The construct of any previous or following implementation, wherein the means for neutralizing is a photochemical that produces a reactive oxygen species (ROS) selected from the group of hydroxyl radicals (HO’), superoxide radical anion (O2_), hydrogen peroxide (H2O2), and singlet oxygen (1O2).

[0097] The construct of any previous or following implementation, wherein the means for neutralizing is a photosensitizer configured to produce a reactive oxygen species (ROS) photochemical upon exposure to light.

[0098] The construct of any previous or following implementation, further comprising a linker coupled to each affinity ligand configured to mount the affinity ligands to the surface of the substrate.

[0099] The construct of any previous or following implementation, wherein the substrate is modified with an affinity ligand from the group of yeast cell wall particles (YCW) and D-mannose.

[0100] The construct of any previous or following implementation, wherein the substrate is modified with at least one affinity ligand material selected from the group of mucins, oligosaccharides, lactoferrin, chitosan, isolated RBC cell membranes, platelets, and antimicrobial peptides such as Lipid A, Lipid II, Lipopolysaccharide transport (LptD) and Peptidoglycan binding peptides.

[0101] The construct of any previous or following implementation, wherein the substrate surface is modified with both a plurality of affinity ligands and a plurality of photosensitizers configured to produce a reactive oxygen species (ROS) adjacent to each other.

[0102] The construct of any previous or following implementation, further comprising a linker coupled to each photosensitizer configured to mount each the photosensitizer to the surface of the substrate.

[0103] The construct of any previous or following implementation, wherein the light is sunlight, UV-A light, LED visible light or a mobile phone flashlight.

[0104] The construct of any previous or following implementation, wherein the exposure to light is for a duration of between approximately 5 seconds to approximately 120 minutes.

[0105] The construct of any previous or following implementation, wherein the light exposure has a light power level in the range of 1 ,050 Lux to 15,000 Lux.

[0106] The construct of any previous or following implementation, wherein the light exposure has a light power level in the range of 1 ,050 Lux to 90,000 Lux.

[0107] A method of inactivation of bacteria and viruses, the method comprising: (a) providing construct of a substrate with an outer surface, a plurality of affinity ligands configured to bind to at least one moiety of a target bacteria, fungi or virus with high affinity, the ligands mounted on the outer surfaces of the substrate, and a means for neutralizing a target upon light exposure placed on the outer surface of the substrate in proximity to one or more affinity ligand; (b) binding targets to the affinity ligands of the substrate; and (c) exposing the bound constructs to a light of an intensity and duration to activate the means for neutralizing the target.

[0108] The method of any previous or following implementation, wherein the means for neutralizing of target is a photochemical that produces a reactive oxygen species (ROS) coupled to the substrate adjacent to the affinity ligands; and wherein the photosensitizer is induced to generate ROS upon exposure to light which inactivates the target.

[0109] The method of any previous or following implementation, wherein the means for neutralizing is a photochemical that produces a reactive oxygen species (ROS) selected from the group of hydroxyl radicals (HO ), superoxide radical anion (O2— ), hydrogen peroxide (H2O2), and singlet oxygen O2).

[0110] The method of any previous or following implementation, wherein the substrate comprises a substrate selected from the group of an aerogel, a film, a particle, a film coated particle, a fiber, a film coated fiber and microfiber and nanofiber membranes.

[0111] The method of any previous or following implementation, wherein the substrate comprises a synthetic polymer selected from the group of nylon, PVA and PVA-co-PE; a natural polymer selected from the group of a cellulose-based polymer or a carbohydrate-based polymer.

[0112] The method of any previous or following implementation, wherein the substrate comprises an affinity ligand from the group of a bacteriophage, bacteriophage derived tail fiber proteins, antibodies, nanobodies, fungalhyphae and algal cell walls.

[0113] The method of any previous or following implementation, wherein the substrate comprises a substrate selected from the group of a Zein film, a curcum in-based nanofibrous membrane and a photosensitizer-modified nanofibrous membrane.

[0114] The method of any previous or following implementation, wherein the substrate is modified with yeast cell wall particles (YCW) or D-mannose.

[0115] The method of any previous or following implementation, wherein the substrate is modified with at least one material selected from the group of mucins, oligosaccharides, lactoferrin, chitosan, isolated RBC cell membranes, platelets, and antimicrobial peptides such as Lipid A, Lipid II, Lipopolysaccharide transport (LptD) and Peptidoglycan binding peptides.

[0116] The method of any previous or following implementation, wherein the substrate comprises a substrate selected from the group of naturally occurring plant and animal derived proteins or glycoproteins including zein, gelatin, collagen, keratin and their subsequent treatment with acidic (2-6.5 pH) and basic (8-12 pH) conditions to induce structural transformations enhancing ROS generation upon exposure to light.

[0117] The method of any previous or following implementation, the construct further comprises coupling a linker to each affinity ligand; and mounting the linker and affinity ligands to the surface of the substrate.

[0118] The method of any previous or following implementation, the construct further comprises coupling a linker to each photosensitizer; and mounting the linker and affinity ligands to the surface of the substrate.

[0119] The method of any previous or following implementation, wherein the substrate is modified with an affinity ligand from the group of yeast cell wall particles (YCW) and D-mannose.

[0120] The method of any previous or following implementation, wherein the substrate is modified with at least one affinity ligand material selected from the group of mucins, oligosaccharides, lactoferrin, chitosan, isolated RBC cell membranes, platelets, and antimicrobial peptides such as Lipid A, Lipid II, Lipopolysaccharide transport (LptD) and Peptidoglycan binding peptides.

[0121] The method of any previous or following implementation, wherein thelight is sunlight, UV-A light or LED visible light, with a light power level in the range of 1 ,050 Lux to 15,000 Lux and a duration of approximately 5 seconds to approximately 120 minutes.

[0122] A construct for neutralizing microbes, comprising: (a) a substrate with an outer surface; (b) a plurality of affinity ligands physically adsorbed or chemically bound to the outer surfaces of the substrate, the ligands configured to bind to at least one moiety of a target microbe with high affinity thereby actively capturing the microbe; and (c) a plurality of photosensitizer molecules coupled to the surface or pores of the substrate in proximity to the ligands; (d) wherein the target microbes are neutralized by the photosensitizer molecules upon exposure to light.

[0123] A construct for neutralizing microbes, comprising: (a) a substrate with an outer surface, said substrate configured to neutralize target microbes upon exposure to light; and (b) a plurality of affinity ligands physically adsorbed or chemically bound to the outer surfaces of the substrate, the ligands configured to bind to at least one moiety of a target microbe with high affinity thereby actively capturing the microbe; (c) wherein the target microbes are neutralized by the photosensitizer substrate upon exposure to light.

[0124] As used herein, the term "implementation" is intended to include, without limitation, embodiments, examples, or other forms of practicing the technology described herein.

[0125] As used herein, the singular terms "a," "an," and "the" may include plural referents unless the context clearly dictates otherwise. Reference to an object in the singular is not intended to mean "one and only one" unless explicitly so stated, but rather "one or more."

[0126] Phrasing constructs, such as “A, B and / or C,” within the present disclosure describe where either A, B, or C can be present, or any combination of items A, B and C. Phrasing constructs indicating, such as “at least one of” followed by listing a group of elements, indicates that at least one of these groups of elements is present, which includes any possible combination of the listed elements as applicable.

[0127] References in this disclosure referring to “an embodiment”, “at least one embodiment” or similar embodiment wording indicates that a particularfeature, structure, or characteristic described in connection with a described embodiment is included in at least one embodiment of the present disclosure. Thus, these various embodiment phrases are not necessarily all referring to the same embodiment, or to a specific embodiment which differs from all the other embodiments being described. The embodiment phrasing should be construed to mean that the particular features, structures, or characteristics of a given embodiment may be combined in any suitable manner in one or more embodiments of the disclosed apparatus, system, or method.

[0128] As used herein, the term "set" refers to a collection of one or more objects. Thus, for example, a set of objects can include a single object or multiple objects.

[0129] Relational terms such as first and second, top and bottom, upper and lower, left and right, and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0130] The terms "comprises," "comprising," "has", "having," "includes", "including," "contains", "containing" or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, apparatus, or system, that comprises, has, includes, or contains a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, apparatus, or system. An element proceeded by "comprises . . . a", "has . . . a", "includes . . . a", "contains . . . a" does not, without more constraints, preclude the existence of additional identical elements in the process, method, article, apparatus, or system, that comprises, has, includes, contains the element.

[0131] As used herein, the terms "approximately", "approximate", "substantially", "substantial", "essentially", and "about", or any other version thereof, are used to describe and account for small variations. When used in conjunction with an event or circumstance, the terms can refer to instances in which the event or circumstance occurs precisely as well as instances in which the event or circumstance occurs to a close approximation. When used in conjunction with a numerical value, the terms can refer to a range ofvariation of less than or equal to ± 10% of that numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1 %, less than or equal to ±0.5%, less than or equal to ±0.1 %, or less than or equal to ±0.05%. For example, "substantially" aligned can refer to a range of angular variation of less than or equal to ±10°, such as less than or equal to ±5°, less than or equal to ±4°, less than or equal to ±3°, less than or equal to ±2°, less than or equal to ±1 °, less than or equal to ±0.5°, less than or equal to ±0.1 °, or less than or equal to ±0.05°.

[0132] Additionally, amounts, ratios, and other numerical values may sometimes be presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[0133] The term "coupled" as used herein is defined as connected, although not necessarily directly and not necessarily mechanically. A device or structure that is "configured" in a certain way is configured in at least that way but may also be configured in ways that are not listed.

[0134] Benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or element of the technology described herein or any or all the claims.

[0135] In addition, in the foregoing disclosure various features may be grouped together in various embodiments for the purpose of streamlining the disclosure. This method of disclosure is not to be interpreted as reflecting an intention that the claimed embodiments require more features than are expressly recited in each claim. Inventive subject matter can lie in less than allfeatures of a single disclosed embodiment.

[0136] The abstract of the disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims.

[0137] It will be appreciated that the practice of some jurisdictions may require deletion of one or more portions of the disclosure after the application is filed. Accordingly, the reader should consult the application as filed for the original content of the disclosure. Any deletion of content of the disclosure should not be construed as a disclaimer, forfeiture, or dedication to the public of any subject matter of the application as originally filed.

[0138] All text in a drawing figure is hereby incorporated into the disclosure and is to be treated as part of the written description of the drawing figure.

[0139] The following claims are hereby incorporated into the disclosure, with each claim standing on its own as a separately claimed subject matter.

[0140] Although the description herein contains many details, these should not be construed as limiting the scope of the disclosure, but as merely providing illustrations of some of the presently preferred embodiments. Therefore, it will be appreciated that the scope of the disclosure fully encompasses other embodiments which may become obvious to those skilled in the art.

[0141] All structural and functional equivalents to the elements of the disclosed embodiments that are known to those of ordinary skill in the art are expressly incorporated herein by reference and are intended to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. No claim element herein is to be construed as a "means plus function" element unless the element is expressly recited using the phrase "means for". No claim element herein is to be construed as a "step plus function" element unless the element is expressly recited using the phrase "step for".

Claims

CLAIMSWhat is claimed is:1 . A construct for neutralizing microbes, comprising:(a) a substrate with an outer surface;(b) a plurality of affinity ligands configured to bind to at least one moiety of a target bacteria, fungi or virus with high affinity, said ligands physically adsorbed or chemically bound to the outer surfaces of the substrate; and(c) means for neutralizing a target placed on the outer surface of the substrate so that exposure to light neutralizes a captured target.

2. The construct of claim 1 , wherein said substrate comprises a substrate selected from the group of an aerogel, a film, a particle, a film modified with particles, a fiber, a film coated fiber and microfiber and nanofiber membranes.

3. The construct of claim 1 , wherein said substrate comprises a synthetic polymer selected from the group of nylon, PVA and PVA-co-PE.

4. The construct of claim 1 , wherein said substrate comprises a natural polymer selected from the group of a cellulose-based polymer and a carbohydrate- based polymer.

5. The construct of claim 1 , wherein said ligand comprises a bacteriophage, bacteriophage derived tail fiber proteins, antibodies, nanobodies, fungal hyphae and algal cell walls.

6. The construct of claim 1 , wherein said substrate comprises a substrate selected from the group of a Zein film, a curcum in-based nanofibrous membrane and a photosensitizer-modified nanofibrous membrane.

7. The construct of claim 1 , further comprising: a substrate composition or surface additives that enhance the dispersion of the fluid on the substrate to reduce the overall contact angle.

8. The construct of claim 1 , wherein said means for neutralizing is a photochemical that produces a reactive oxygen species (ROS) coupled to said substrate adjacent to said affinity ligands.

9. The construct of claim 1 , wherein said means for neutralizing is a photochemical that produces a reactive oxygen species (ROS) selected from the group of hydroxyl radicals (HO’), superoxide radical anion (O? -), hydrogen peroxide (H2O2), and singlet oxygen (1O2).

10. The construct of claim 1 , wherein said means for neutralizing is a photosensitizer configured to produce a reactive oxygen species (ROS) photochemical upon exposure to light.11 . The construct of claim 1 , further comprising: a linker coupled to each affinity ligand configured to mount said affinity ligands to the surface of the substrate.

12. The construct of claim 1 , wherein said substrate is modified with an affinity ligand from the group of yeast cell wall particles (YCW) and D-mannose.

13. The construct of claim 1 , wherein said substrate is modified with at least one affinity ligand material selected from the group of mucins, oligosaccharides, lactoferrin, chitosan, isolated RBC cell membranes, platelets, and antimicrobial peptides such as Lipid A, Lipid II, Lipopolysaccharide transport (LptD) and Peptidoglycan binding peptides.

14. The construct of claim 1 , wherein said substrate surface is modified with both a plurality of affinity ligands and a plurality of photosensitizers configured to produce a reactive oxygen species (ROS) adjacent to each other.

15. The construct of claim 1 , further comprising: a linker coupled to each photosensitizer configured to mount each said photosensitizer to the surface of the substrate.

16. The construct of claim 1 , wherein said light is sunlight, IIV-A light, LED visible light or a mobile phone flashlight.

17. The construct of claim 1 , wherein said exposure to light is for a duration of between approximately 5 seconds to approximately 120 minutes.

18. The construct of claim 1 , wherein said light exposure has a light power level in the range of 1 ,050 Lux to 15,000 Lux.

19. The construct of claim 1 , wherein said light exposure has a light power level in the range of 1 ,050 Lux to 90,000 Lux.

20. A method of inactivation of bacteria and viruses, the method comprising:(a) providing construct of a substrate with an outer surface, a plurality of affinity ligands configured to bind to at least one moiety of a target bacteria, fungi or virus with high affinity, said ligands mounted on the outer surfaces of the substrate, and a means for neutralizing a target upon light exposure placed on the outer surface of the substrate in proximity to one or more affinity ligand;(b) binding targets to said affinity ligands of said substrate; and(c) exposing said bound constructs to a light of an intensity and duration to activate said means for neutralizing the target.21 . The method of claim 20, wherein said means for neutralizing of target is a photochemical that produces a reactive oxygen species (ROS) coupled to said substrate adjacent to said affinity ligands; and wherein said photosensitizer is induced to generate ROS upon exposure to light which inactivates the target.

22. The method of claim 20, wherein said means for neutralizing is a photochemical that produces a reactive oxygen species (ROS) selected from the group of hydroxyl radicals (HO’), superoxide radical anion (O2— ), hydrogen peroxide (H2O2), and singlet oxygen (1O2).

23. The method of claim 20, wherein said substrate comprises a substrate selected from the group of an aerogel, a film, a particle, a film coated particle, a fiber, a film coated fiber and microfiber and nanofiber membranes.

24. The method of claim 20, wherein said substrate comprises a synthetic polymer selected from the group of nylon, PVA and PVA-co-PE; a natural polymer selected from the group of a cellulose-based polymer or a carbohydrate-based polymer.

25. The method of claim 20, wherein said substrate comprises an affinity ligand from the group of a bacteriophage, bacteriophage derived tail fiber proteins, antibodies, nanobodies, fungal hyphae and algal cell walls.

26. The method of claim 20, wherein said substrate comprises a substrate selected from the group of a Zein film, a curcum in-based nanofibrous membrane and a photosensitizer-modified nanofibrous membrane.

27. The method of claim 20, wherein said substrate is modified with yeast cell wall particles (YCW) or D-mannose.

28. The method of claim 20, wherein said substrate is modified with at least one material selected from the group of mucins, oligosaccharides, lactoferrin, chitosan, isolated RBC cell membranes, platelets, and antimicrobial peptides such as Lipid A, Lipid II, Lipopolysaccharide transport (LptD) and Peptidoglycan binding peptides.

29. The method of claim 20, wherein said substrate comprises a substrate selected from the group of naturally occurring plant and animal derived proteins or glycoproteins including zein, gelatin, collagen, keratin and their subsequenttreatment with acidic (2-6.5 pH) and basic (8-12 pH) conditions to induce structural transformations enhancing ROS generation upon exposure to light.

30. The method of claim 20, said construct further comprising: coupling a linker to each affinity ligand; and mounting said linker and affinity ligands to the surface of the substrate.31 . The method of claim 20, said construct further comprising: coupling a linker to each photosensitizer; and mounting said linker and affinity ligands to the surface of the substrate.

32. The method of claim 20, wherein said substrate is modified with an affinity ligand from the group of yeast cell wall particles (YCW) and D-mannose.

33. The method of claim 20, wherein said substrate is modified with at least one affinity ligand material selected from the group of mucins, oligosaccharides, lactoferrin, chitosan, isolated RBC cell membranes, platelets, and antimicrobial peptides such as Lipid A, Lipid II, Lipopolysaccharide transport (LptD) and Peptidoglycan binding peptides.

34. The method of claim 20, wherein said light is sunlight, UV-A light or LED visible light, with a light power level in the range of 1 ,050 Lux to 15,000 Lux and a duration of approximately 5 seconds to approximately 120 minutes.

35. A construct for neutralizing microbes, comprising:(a) a substrate with an outer surface;(b) a plurality of affinity ligands physically adsorbed or chemically bound to the outer surfaces of the substrate, said ligands configured to bind to at least one moiety of a target microbe with high affinity thereby actively capturing said microbe; and(c) a plurality of photosensitizer molecules coupled to the surface or pores of the substrate in proximity to said ligands;(d) wherein said target microbes are neutralized by said photosensitizer molecules upon exposure to light.

36. A construct for neutralizing microbes, comprising:(a) a substrate with an outer surface, said substrate configured to neutralize target microbes upon exposure to light; and(b) a plurality of affinity ligands physically adsorbed or chemically bound to the outer surfaces of the substrate, said ligands configured to bind to at least one moiety of a target microbe with high affinity thereby actively capturing said microbe; (c) wherein said target microbes are neutralized by said photosensitizer substrate upon exposure to light.

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