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Title:
PHOTONIC INACTIVATION OF PATHOGENS
Document Type and Number:
WIPO Patent Application WO/2018/222856
Kind Code:
A2
Abstract:
Devices, systems and methods for the inactivation of pathogens such as viruses in a liquid medium containing products such as pharmaceutical compounds are presented. These are useful, for example, for the inactivation of viruses in liquids containing antibody pharmaceuticals, and make use of one or more of flow-through inactivation, plasmonic virus inactivation enhancement, mode matching between hydrodynamic flow profile and laser intensity profile, and nonlinear flow geometries. The methods allow flow-through pathogen inactivation while avoiding collateral damage to products to be decontaminated.

Inventors:
ASHER DAMON (US)
ERRAMILLI SHYAMSUNDER (US)
GAGNE GEORGE (US)
GILLESPIE CHRISTOPHER (US)
GUMMULURU SURYARAM (US)
HONG MI (US)
MIURA AYAKO (US)
NAZARI MINA (US)
REINHARD BJOERN (US)
SOUZA KATHLEEN (US)
XI MIN (US)
Application Number:
PCT/US2018/035369
Publication Date:
December 06, 2018
Filing Date:
May 31, 2018
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
EMD MILLIPORE CORP (US)
UNIV BOSTON (US)
International Classes:
A23L3/26; A61L2/00; A61L2/08; A61M1/36; C02F1/30
Other References:
XU L. ET AL.: "Role of Risk Assessments in Viral Safety: An FDA Perspective PDA", J. PHARM. SCI. TECHNOL., vol. 68, 2014, pages 6 - 10
DEPALMA A.: "Viral Safety Methods for Manufacturing", GENETIC ENGINEERING & BIOTECHNOLOGY NEWS, vol. 30, no. 8, 2010, pages 38 - 9
DYKEMAN, E. C.; SANKEY, O. F.: "Vibrational energy funneling in viruses-simulations of impulsive stimulated Raman scattering in M13 bacteriophages", J.PHYS.:CONDENS.MATTER, vol. 21, 2009, pages 505102, XP020168296
VIGDERMAN, L.; ZUBAREV, E. R.: "High-Yield Synthesis of Gold Nanorods with Longitudinal SPR Peak Greater than 1200 nm Using Hydroquinone as a Reducing Agent", CHEM. MATER., vol. 25, 2013, pages 1450 - 1457
KOLB, H. C.; FINN, M. G.; SHARPLESS, K. B.: "Click Chemistry: Diverse Chemical Function from a Few Good Reactions", ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 40, 2001, pages 2004 - 2021, XP002554101
PURYEAR, W. B. ET AL.: "Interferon-Inducible Mechanism of Dendritic Cell-Mediated HIV-1 Dissemination is Dependent on the Siglec, CD 169", PLOS PATHOGENS, vol. 9, 2013, pages el003291
HATCH, S. C.; ARCHER, J.; GUMMULURU, S.: "Glycosphingolipid composition of human immunodeficiency virus type 1 (HIV-1) particle is a crucial determinant for dendritic cell-mediated HIV01 trans-infection", JOURNAL OF VIROLOGY, vol. 83, 2009, pages 3496 - 3506
MURPHY, C. J. ET AL.: "Anisotropic metal nanoparticles: Synthesis, assembly, and optical applications", JOURNAL OF PHYSICAL CHEMISTRY B, vol. 109, 2005, pages 13857 - 13870
NIKOOBAKHT, B.; EL-SAYED, M. A.: "Preparation and Growth Mechanism of Gold Nanorods (NRs) Using Seed-Mediated Growth Method", CHEM. MATER., vol. 15, 2003, pages 1957 - 1962, XP002487039, DOI: doi:10.1021/cm020732l
JOHNSON, C. J.; DUJARDIN, E.; DAVIS, S. A.; MURPHY, J. C.; MANN, S.: "Growth and form of gold nanorods prepared by seed-mediated, surfactant-directed synthesis", JOURNAL OF MATERIALS CHEMISTRY, vol. 12, 2002, pages 1765 - 1770, XP055125944, DOI: doi:10.1039/b200953f
GAO, L. ET AL.: "Plasmon-Mediated Generation of Reactive Oxygen Species from Near-Infrared Light Excited Gold Nanocages for Photodynamic Therapy in vitro", ACS NANO, vol. 8, 2014, pages 7260 - 7271
Attorney, Agent or Firm:
CARROLL, Alice, O. et al. (US)
Download PDF:
Claims:
CLAIMS

What is claimed is:

1. A method for inactivating a pathogen in a liquid medium, comprising (i) providing the liquid medium containing the pathogen and (ii) exposing the liquid medium and a plasmonic material to an excitation pulse, the liquid medium being in contact with the plasmonic material, and the plasmonic material having a localized surface plasmon resonance that overlaps with the excitation pulse.

2. The method of claim 1, wherein the liquid medium containing the pathogen is

exposed to the excitation pulse while flowing through a flow channel.

3. The method of claim 2, wherein at least part of the liquid medium has a laminar flow.

4. The method of claim 2 or 3, wherein the liquid medium has a flow in a flow direction and characterized by a velocity profile and the excitation pulse has an intensity profile corresponding to the velocity profile.

5. The method of claim 4, wherein the excitation pulse is applied in a direction

substantially parallel to the flow direction.

6. The method of claim 4, wherein the excitation pulse is applied in a direction

substantially perpendicular to the flow direction.

7. The method of claim 1, 2, 3, 4, or 6, wherein the liquid medium flows through a

nonlinear channel while being exposed.

8. The method of claim 7, wherein the nonlinear channel comprises a plurality of bends and/or turns in the channel.

9. The method of any one of the preceding claims, wherein the plasmonic material

comprises plasmonic nanoparticles.

10. The method of claim 9, wherein the plasmonic nanoparticles are selected from

plasmonic nanorods, nanospheres, nanocubes, nanoprisms, bipyramids, hollow nanoparticles, nanoshells comprising a metallic shell around a non-metallic core, nanoellipsoids, and nanoantenna, made of, at least in part, a metal, an alloy, a doped dielectric or semiconductor materials.

11. The method of claim 10, wherein the plasmonic nanoparticles are nanorods made of gold, titanium oxide, silicon oxide, silicon, germanium, transition metal nitrides, arsenides, phosphides, transparent conducting oxides, silicides, germanides, or sulfides.

12. The method of any one of the preceding claims, wherein the liquid medium is

characterized by a non-turbulent flow.

13. The method of any one of the preceding claims, wherein the excitation pulse is a laser excitation pulse.

14. The method of any one of claims 1 to 12, wherein the pathogen is a virus or a

bacterium.

15. The method of any one of claims 1 to 12, wherein the pathogen is a bacterium from the genus Mycoplasma, a strain of Escherichia coli, porcine circovirus (PCV), a human adenovirus, a reovirus, an orbivirus, a minute mouse virus (MMV), Cache Valley virus (CVV), an epizootic hemorrhagic disease virus (EHDV), a murine minute virus, a vesivirus, or a murine leukemia virus (MLV).

16. The method of any one of claims 1 to 15, wherein the pathogen has a size in the range from 18 nm to 100 nm.

17. The method of any one of the preceding claims, wherein the excitation pulse is a femtosecond pulse of a wavelength in the range from 600 nm to 1000 nm.

18. The method of any one of the preceding claims, wherein the excitation pulse is one out of a continuous train of 10 to 100 femtosecond, picosecond or nanosecond pulses, at a repetition rate of 0.1 kHz to 5 kHz, centered at 600 nm to 1000 nm, with energies between 1 to 20 mJ/cm2.

19. The method of any one of the preceding claims, wherein the excitation pulse is one out of a continuous train of 35 femtosecond pulses at a repetition rate of 1 kHz, centered at about 805 nm, with energies up to 7.5 mJ/cm2, and transform-limited spectral widths of about 25 nm.

20. The method of any one of the preceding claims, wherein the liquid medium is

exposed to excitation pulses for 1 second to 2 minutes, while within an illumination zone.

21. The method of any one of the preceding claims, wherein the liquid medium is flown through the illumination zone within 1 second to 2 minutes.

22. A method for inactivating a pathogen in a liquid medium, comprising (i) flowing the liquid medium containing the pathogen and (ii) exposing the liquid medium, while flowing, to an excitation pulse centered at a wavelength between 350 nm and 450 nm.

23. The method of claim 22, wherein the liquid medium is aqueous.

24. The method of claim 22 or 23, wherein the liquid medium has a laminar flow.

25. The method of claim 22, 23 or 24, wherein the liquid medium has a flow

characterized by a velocity profile and the excitation pulse has an intensity profile matched to the velocity profile.

26. The method of claim 25, wherein the excitation pulse is applied in a direction

substantially parallel to the flow direction.

27. The method of claim 25, wherein the excitation pulse is applied in a direction

substantially perpendicular to the flow direction.

28. The method of claim 22, 23, 24, 25 or 27, wherein the liquid medium is being flown through a nonlinear channel within an illumination zone.

29. The method of claim 28, wherein the nonlinear channel comprises a plurality of bends and/or turns in the channel.

30. The method of anyone of claims 22 to 29, wherein the liquid medium being flown is characterized by a non-turbulent flow.

31. The method of claims 22 to 30, wherein the pathogen is a bacterium from the genus Mycoplasma, a strain of Escherichia coli, an enveloped virus, and non-enveloped virus, or a bacteriophage.

32. The method of claims 22 to 30, wherein the pathogen is a virus selected from dsDNA virus, ssDNA virus, dsRNA virus, (+)ssRNA virus, (-)ssRNA virus, ssRNA-RT virus, and dsDNA-RT virus.

33. The method of claims 22 to 30, wherein the pathogen is a porcine circovirus (PCV), a human adenovirus, a reovirus, an orbivirus, a minute mouse virus (MMV), Cache Valley virus (CVV), an epizootic hemorrhagic disease virus (EHDV), a murine minute virus, a vesivirus, or a murine leukemia virus (MLV).

34. The method of any one of claims 22 to 30, wherein the pathogen has a size in the range from 18 nm to 100 nm.

35. The method of any one of claims 22 to 34, wherein the excitation pulse is one out of a continuous train of 10 to 100 femtosecond, picosecond or nanosecond pulses, at a repetition rate 0.1 kHz to 5 kHz, centered at 600 nm to 1000 nm, with energies between 1 to 20 mJ/cm2.

36. The method of any one of claims 22 to 34, wherein the excitation pulse is one out of a continuous train of 35 femtosecond pulses at a repetition rate 1 kHz, centered at about 402 nm, with energies up to 7.5 mJ/cm2, and transform-limited spectral widths of about 25 nm.

37. The method of any one of claims 22 to 36, wherein the liquid medium is exposed to excitation pulses for 1 second to 5 minutes, while within an illumination zone.

38. The method of any one claim 22 to 37, wherein the liquid medium is flown through the illumination zone within 1 second to 5 minutes.

39. A flow channel cell comprising an enclosure having (i) a first optical window; (ii) an inlet; (iii) an outlet, the first optical window being adapted and positioned to allow light from a laser or light emitting diode to enter into the enclosure to expose a volume within the enclosure to the light thereby forming an illumination zone within the enclosure, the inlet and outlet being positioned such that a liquid medium to be exposed to the light flows from the inlet through the illumination zone and out of the outlet; and (iv) a plasmonic material within the illumination zone.

40. The flow channel cell of claim 39, wherein the flow channel cell has a second optical window, the second optical window being adapted and positioned to allow additional light from a laser or light emitting diode to enter into the enclosure to expose a volume within the enclosure to the additional light thereby forming a second illumination zone within the enclosure.

41. The flow channel cell of claim 40, wherein the first and second illumination zones overlap.

42. The flow channel cell of claim 40 or 41, wherein the first and second optical windows are parallel and at opposing sides of the enclosure.

43. The flow channel cell of any one of claims 39-42, further comprising a nonlinear channel connected to the inlet and outlet and positioned within at least one illumination zone.

44. The flow channel cell of claim 43, wherein the nonlinear channel comprises a

plurality of bends and/or turns in the channel.

45. The flow channel cell of any one of claims 39-44, wherein the plasmonic material comprises plasmonic nanoparticles.

46. The flow channel cell of claim 45, wherein the plasmonic nanoparticles are selected from plasmonic nanorods, nanospheres, nanocubes, nanoprisms, bipyramids, hollow nanoparticles, nanoshells comprising a metallic shell around a non-metallic core, nanoellipsoids, and nanoantenna, made of, at least in part, a metal, an alloy, a doped dielectric or semiconductor materials.

47. The flow channel cell of claim 45 or 46, wherein the plasmonic nanoparticles are nanorods made of gold, titanium oxide, silicon oxide, silicon, germanium, transition metal nitrides, arsenides, phosphides, transparent conducting oxides, silicides, germanides, or sulfides.

48. The method of any one of claims 1-21, wherein the plasmonic material is embedded in a polymer matrix, the polymer matrix being optically transparent to the excitation pulse, and biochemical product passes through the polymer matrix.

49. The method of any one of claims 1-21 wherein the plasmonic material is suspended within the liquid medium and confined to an illumination zone by use of an enclosure having filters that allow the liquid medium to pass through but not the plasmonic material.

50. The method of any one of claims 1-21, wherein the plasmonic material is embedded within a polymer matrix, the polymer matrix being optically transparent to allow plasmonic material to be exposed to the laser excitation pulse.

51. The method of any one of claims 48-50, wherein the polymer matrix is a hydrogel.

52. The method of claim 51, wherein the hydrogel is in the form of a membrane matrix.

53. The method of claim 51, wherein the hydrogel is in particle form, and the hydrogel is dispersed in the liquid medium.

54. The method of any one of claims 51-53, wherein the hydrogel is agarose.

55. A pathogen inactivation system comprising (a) a laser or light emitting diode and (b) a flow channel cell having an enclosure having (i) a first optical window; (ii) an inlet; (iii) an outlet, the first optical window being adapted and positioned to allow light from the laser or light emitting diode to enter into the enclosure to expose a volume within the enclosure to light thereby forming an illumination zone within the enclosure, the inlet and outlet being positioned such that a liquid medium to be exposed to the light flows from the inlet through the illumination zone and out of the outlet; and (iv) a plasmonic material within the illumination zone.

56. A pathogen inactivation system comprising (a) a laser or light emitting diode and (b) a flow channel cell of anyone of claims 39-47.

57. A plasmonic material for inactivating a pathogen, wherein the plasmonic material has a localized surface plasmon resonance that overlaps with an excitation pulse.

58. The plasmonic material of claim 57, wherein the localized surface plasmon resonance and the excitation pulse are adapted for inactivation of a pathogen in a liquid medium containing the plasmonic material.

59. The plasmonic material of claim 57 or 58, wherein the plasmonic material is

geometrically tuned for inactivation of the pathogen.

60. The plasmonic material of any one of claims 57-59, wherein the plasmonic material comprises plasmonic nanoparticles.

61. The plasmonic material of any one of claims 57-60, wherein the plasmonic material comprises plasmonic nanoparticles selected from plasmonic nanorods, nanospheres, nanocubes, nanoprisms, bipyramids, hollow nanoparticles, nanoshells comprising a metallic shell around a non-metallic core, nanoellipsoids, and nanoantenna, made of, at least in part, a metal, an alloy, a doped dielectric or semiconductor materials.

62. The plasmonic material of any one of claims 57-60, wherein the plasmonic material comprises (1) nanorods made of gold, titanium oxide, silicon oxide, silicon, germanium, transition metal nitrides, arsenides, phosphides, transparent conducting oxides, silicides, germanides, or sulfides; or (2) bipyramidal gold nanoparticles.

63. The plasmonic material of claim 57, wherein the plasmonic material is (1) gold

nanorods; or (2) bipyramidal gold nanoparticles. A material for inactivation of a pathogen, the material comprising a plasmonic material of any one of claims 57-63, the plasmonic material being embedded in polymer matrix.

The material of claim 64, wherein the polymer matrix is optically transparent to the excitation pulse, and allows liquid medium and the pathogen contained therein to pass through the polymer matrix.

66. The method of any one of claims 64-65, wherein the polymer matrix is a hydrogel.

The method of claim 66, wherein the hydrogel is in the form of a membrane matrix.

The method of claim 66, wherein the hydrogel is in particle form.

The method of any one of claims 66-68, wherein the hydrogel is agarose.

Description:
PHOTONIC INACTIVATION OF PATHOGENS RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No.

62/514,530, filed on June 2, 2017, the entire teachings of which application are incorporated herein by reference.

BACKGROUND

[0002] Pathogen inactivation, and selective inactivation of pathogens in the presence of other biomolecules such as antibodies, or even living cells in food, feed stock in

pharmaceutical bioreactors, therapeutic compounds and other sensitive areas, is of great importance for human and animal health. For many of these applications, harsh chemical or ionizing radiation techniques are not appropriate as they lack sufficient selectivity.

[0003] Viral contamination is an integral risk common to all biopharmaceuticals derived from cell lines, for example, antibody based pharmaceuticals. See Xu L. et al., Role of Risk Assessments in Viral Safety: An FDA Perspective PDA, J. Pharm. Sci. Technol. (2014); 68:6-10. doi: doi: 10.573 l/pdajpst.2014.00959; and DePalma A., Viral Safety Methods for Manufacturing, GENETIC ENGINEERING & BIOTECHNOLOGY NEWS

(2010);30(8):38-9.

[0004] A pathogen contamination event can lead to service system shutdown, leading to drug scarcity and interrupted treatments for patients, and thereby seriously compromise the public health system.

[0005] The best clearance method in current practice is passive filtration that exploits the difference in the hydrodynamic radius between the contaminant and the pharmaceutical. Difficult to handle are un-enveloped viruses, with parvoviruses being perhaps the most difficult, because of the small size ranging from 18-20 nm, which is only slightly larger than antibodies. The small size difference makes it difficult to discriminate with conventional nanofiltration methods, relying on sieving, or on diffusion. While it is possible to inactivate pathogens such as viruses using heat or pH or detergents many of these methods can adversely affect the pharmaceutical. [0006] Generally, current methods for the prevention of pathogen contamination have several disadvantages including high cost, batch processing, low pathogen inactivation rates, long irradiation times, low decontamination selectivity and/or significant collateral damage rates. There is a need for new devices, systems and methods for the inactivation of pathogens in liquid medium that improve upon these methods.

SUMMARY

[0007] Devices, systems and methods for the inactivation of pathogens in a liquid medium are presented which provide lower cost, flow-through processing, higher pathogen inactivation rates, shorter irradiation times, higher decontamination selectivity and/or lower collateral damage rates.

[0008] The device, system and method embodiments are useful for the inactivation of pathogens, such as viruses, in liquids containing pharmaceutical products, such as antibody pharmaceuticals. The device, system and method embodiments allow pathogen inactivation while avoiding collateral damage to antibody pharmaceuticals, and they allow both batch and flow-through processing. Flow-through processing provides scalability for large scale inline processing.

[0009] One embodiment is a method for inactivating a pathogen in a liquid medium. The method includes (i) providing the liquid medium containing the pathogen and (ii) exposing the liquid medium and a plasmonic material to an excitation pulse, the liquid medium being in contact with the plasmonic material, and the plasmonic material having a localized surface plasmon resonance that overlaps with the excitation pulse.

[0010] A further embodiment is a method for inactivating a pathogen in a liquid medium. The method includes (i) flowing the liquid medium containing the pathogen and (ii) exposing the liquid medium, while flowing, to an excitation pulse centered at a wavelength between about 350 nm and about 450 nm within an illumination zone.

[0011] Yet a further embodiment is a flow channel cell, which can be used in methods described herein. The flow channel cell includes an enclosure having (i) a first optical window; (ii) an inlet; (iii) an outlet, the first optical window being adapted and positioned to allow light from a laser or light emitting diode to enter into the enclosure to expose a volume within the enclosure to the light, thereby forming an illumination zone within the enclosure, the inlet and outlet being positioned such that a liquid medium to be exposed to the light flows from the inlet through the illumination zone and out of the outlet; and (iv) a plasmonic material within the illumination zone.

[0012] Another embodiment is a pathogen inactivation system. The system includes (a) a laser or light emitting diode and (b) a flow channel cell having an enclosure having (i) a first optical window; (ii) an inlet; (iii) an outlet, the first optical window being adapted and positioned to allow light from the laser or light emitting diode to enter into the enclosure to expose a volume within the enclosure to light thereby forming an illumination zone within the enclosure, the inlet and outlet being positioned such that a liquid medium to be exposed to the light flows from the inlet through the illumination zone and out of the outlet; and (iv) a plasmonic material within the illumination zone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale; emphasis instead being placed upon illustrating embodiments.

[0014] FIG. 1 illustrates streamlines in a cylindrical flow channel (left), and a

corresponding mode matched laser intensity profile (right).

[0015] FIG. 2 provides a schematic of a test bench for flow channel evaluation.

[0016] FIG. 3 shows the Log Reduction Value (LRV) results for five separate flow- through PhiX-174 bacteriophage inactivation runs.

[0017] FIG. 4 provides a schematic of a flow channel cell design with transparent Quartz end windows for inline laser exposure (left) and a 3D printed model of the schematic illustrating the window sealing method and inserts for the incorporation of designed plasmonic filter systems in inline flow.

[0018] FIG. 5 provides a schematic of a nonlinear flow channel geometry, specifically, a spiral channel configuration.

[0019] FIG. 6 provides a schematic of a nonlinear flow channel geometry, specifically, a serpentine channel configuration.

[0020] FIG. 7 provides a graph showing peak resonance wavelengths and spectral widths, quantified by the Full-Width-at-Half-Maximum (FWFDVI) of the longitudinal nanorod plasmon mode as function of the average power of the laser irradiation. [0021] FIG. 8 provides the UV-Vis absorption spectra of nanorods recorded after 10s of fs laser irradiation with different average powers (Control refers to rods without irradiation; Inset: TEM image of nanorod; Size bar = 5 nm).

[0022] FIG. 9A provides virus log-reduction- value (LRV) obtained for virus (V) alone, virus + PEGylated nanorods ( R), virus + annexinV functionalized nanorod ( R+A5) and virus + spherical nanoparticles (NP) obtained after irradiation to a pulsed (35 fs) 805 nm laser with an average power of 3 W for 10 s exposure time.

[0023] FIG. 9B provides binding curves for antibody irradiated with the pulsed 805 nm laser for 10 s at an average power of 3 W in the presence and absence of nanorods as determined by ELISA.

[0024] FIG. 9C provides LRV measured for virus and virus + PEGylated nanorods (both in the presence of IgG antibody) as function of average laser power. The irradiation time was held constant at t = 10 s. Inset: TEM image of laser irradiated virus + pegylated nanorod. Size bar = 20 nm.

[0025] FIG. 9D provides binding curves for the IgG antibody for the different experimental conditions noted above for FIG. 9C as determined by ELISA. Control = no laser irradiation.

[0026] FIG. 9E provides LRV obtained under similar conditions as in FIG. 9C but with two-fold increased virus concentration. The increase in LRV with virus concentration indicates that the LRV values are limited by the sensitivity of the infection assay.

[0027] FIG. 9F provides percentage of viable cells for no-treatment control (mock), cells after incubation with a nanorod solution that was exposed to the laser, and cells treated with a nanorod solution that was not exposed to the laser. Laser irradiation conditions were identical to the ones described for FIG. 9B.

[0028] FIG. 10 provides the UV-Vis spectra of bipyramidal nanoparticles recorded after 10s of fs laser irradiation with different average powers.

[0029] FIG. 11 provides a graph showing resonance peak wavelengths and associated spectral widths of the longitudinal bipyramidal nanoparticle plasmon modes as function of the average power of the laser irradiation.

[0030] FIG. 12 provides an image of agarose gel electrophoresis runs for gold

bipyramidal nanoparticles exposed to 10 s of fs laser irradiation with the indicated average powers; "control" indicating the absence of laser irradiation. [0031] FIG. 13 provides an image of agarose gel electrophoresis runs for gold nanorods exposed to 10 s of fs laser irradiation with the indicated average powers; "control" indicating the absence of laser irradiation.

[0032] FIG. 14 shows the Log Reduction Value (LRV) results for flow-through PhiX-174 bacteriophage inactivation runs for three different samples: sample "V" containing PhiX-174 but no plasm onic material, sample "V+ R" containing PhiX-174 and gold nanorods, and sample "V+BP" containing PhiX-174 and gold bipyramidal nanoparticles.

[0033] FIG. 15 shows the Log Reduction Value (LRV) results for laidlawii (cultured in GHB) photonic inactivation, including those in which the photonic inactivation was plasmonically enhanced with gold nanorods ("Red+ R").

[0034] FIG. 16 shows the Log Reduction Value (LRV) results for laidlawii (in buffer) photonic inactivation, including those in which the photonic inactivation was plasmonically enhanced with gold nanorods functionalized with Concanavalin A ("Red+Conc A NR").

[0035] FIG. 17 shows the Log Reduction Value (LRV) results for E. coli photonic inactivation with plasmonic enhancement using gold bipyramidal nanoparticles.

[0036] FIG. 18 shows the Log Reduction Value (LRV) results for PhiX-174 photonic inactivation with plasmonic enhancement using agarose bead embedded gold nanorods.

DETAILED DESCRIPTION

[0037] A description of example embodiments follows.

Methods for Inactivating a Pathogen in a Liquid Medium

[0038] One embodiment is a method for inactivating a pathogen in a liquid medium, comprising (i) providing the liquid medium containing the pathogen and (ii) exposing the liquid medium to a laser excitation pulse within an illumination zone and in the presence of a plasmonic material, which is within the illumination zone and in contact with the liquid medium and whose localized surface plasmon resonance overlaps with the excitation pulse.

[0039] As used herein, "inactivating a pathogen" generally refers to plasmonically trapping and/or rendering the pathogen less harmful, for example, by reduction or loss of infectivity. In specific embodiments of the methods in which viruses are inactivated, the viruses are made less harmful by inactivation of viral functions responsible for early stages of the infection, including host cell binding and fusion. [0040] As used herein, "plasmonically trapping" a pathogen refers to confining the pathogen in space due to optical gradient forces experienced by the pathogen in a locally enhanced electric field generated by the plasmonic material.

[0041] Log reduction values (LRV) are used to quantify pathogen inactivation. The ratio of the intensity of the luminescence to a calibration standard is measured in RLU

(relative light units), and is a linear function of the reporter concentration. If RLU C and RLU S are the measured RLU values of the control and sample respectively, the Log-Reduction Value (LRV) defined as LRV = log 10 (RLU c / RLU S ) gives a quantitative measure of the extent of virus infection in the culture. Percent reduction P is calculated from the LRV as follows: P = {\-\0 'LRV ) x 100. For example, 1 log reduction corresponds to 90% reduction, 2 log reduction corresponds to 99% reduction, and 3 log reduction corresponds to 99.9% reduction.

[0042] Pathogens that can be inactivated include, but are not limited to, viruses. The virus can be selected from dsDNA virus, ssDNA virus, dsRNA virus, (+)ssRNA virus, (-)ssRNA virus, ssRNA-RT virus, and dsDNA-RT virus.

[0043] Pathogens include, but are not limited to, a porcine circovirus (PCV), a human adenovirus, a reovirus, an orbivirus, a minute mouse virus (MMV), Cache Valley virus (CVV), an epizootic hemorrhagic disease virus (EHDV), a murine minute virus, a vesivirus, or a murine leukemia virus (MLV).

[0044] The pathogen can have a size, for example, in the range from 18 nm to 200 nm, in the range from 18 to 100 nm, or in the range from 35 nm to 100 nm.

[0045] Liquid mediums for decontamination using the methods, devices and systems described herein can be aqueous. Typically, the liquid mediums are produced in

pharmaceutical processes, and comprise pathogens as well as a desired pharmaceutical product. Such pharmaceutical products include, but are not limited to, small molecule pharmaceuticals and biopharmaceuticals, such as bio-reactive proteins and antibodies.

[0046] The methods can be performed in flow channel cells which are adapted to permit both liquid medium flow and access to optical radiation, for example, from lasers or high power LEDs.

[0047] Suitable lasers, include, but are not limited to, continuous wave lasers and pulsed lasers. For example, the laser is a pulsed laser, such as a femtosecond laser, a picosecond laser, or lasers with longer pulses in the nanosecond range. Robust picosecond laser diodes are available as turnkey devices from a broad range of vendors (e.g., Hamamatsu). [0048] The excitation pulse(s) can be provided, for example, by a laser or a light emitting diode.

[0049] In certain embodiments, the laser or light emitting diode provide light at a wavelength in the range from 400 nm to 1000 nm. In other embodiments, the lasers provide light at a wavelength in the range from 600 nm to 1000 nm. In yet other embodiments, the lasers provide light at a wavelength in the range from 600 nm to 900 nm. In yet other embodiments, the lasers provide light at a wavelength in the range from 750 nm to 950 nm.

[0050] In some embodiments, the laser excitation pulse can be, for example, one out of a continuous train of 10 to 100 pulses at a repetition rate of 0.1 kHz to 5 kHz, centered at 600 nm to 1000 nm, with energies between 1 to 20 mJ. In other embodiments, the following operation parameters are suitable: (i) Laser pulse width: 65 -100 femtoseconds; (ii) Laser repetition rate: 1 kHz - 10 kHz; and (iii) energies per pulse: 1 mJ-3mJ. Spectral width and laser pulse width are inversely related and not independent. Δλ~0Α4 λ 2 0 /εΔί for a Gaussian pulse.

[0051] As referred to herein, "illumination zone" refers to a region in which the liquid medium with pathogens to be inactivated is exposable to an excitation pulse. Typically, the illumination zone is within an enclosure for the liquid medium to be decontaminated, for example, within the enclosure of a flow channel cell.

[0052] The ability of excitation pulses from a laser or light emission diode to inactivate viruses can be further enhanced by the incorporation of plasmonic materials, reducing the intensity requirements and lowering collateral damage, for example, to biopharmaceutical products, such as antibodies.

[0053] Plasmonic materials are materials in which a part of their associated electrons can behave like a plasma, a new state of matter in which the ions are assumed to be nearly fixed in space, and mobile electrons that can propagate throughout the material, either throughout the volume or along the surface. Under appropriate coupling to an incident electromagnetic wave, all the electrons lose their individual identities and behave like a single collective quantum quasiparticle called the plasmon. The collective nature of the electron response endows plasmonic materials with extraordinary properties, an ability to interact with, enhance and scatter electromagnetic radiation with wavelengths far larger than the length-scales of the plasmonic material particles. The properties of the plasmon can be altered by controlling and modifying the quantum states of the plasmonic material. [0054] Suitable plasmonic materials include plasmonic nanoparticles with geometric shapes including, but not limited to, plasmonic nanorods, nanospheres, nanocubes, nanoprisms, bipyramids, hollow nanoparticles, nanoshells comprising a metallic shell around a non-metallic core, nanoellipsoids as well as nanoantenna structures of more complex geometry, the plasmonic nanoparticles made of, at least in part, a metal, alloy, or doped dielectric and semiconductor materials (including but not limited to, titanium oxide, silicon oxide, silicon, germanium, transition metal nitrides, arsenides, phosphides, transparent conducting oxides, silicides, germanides, and sulfides).

[0055] Suitable liquid media include, but are not limited to, water, aqueous solvents including salt solutions, glycerol-water mixtures, ethanol-water mixtures, polyethylene glycol mixtures, and organic solvents.

[0056] In certain embodiments the plasmonic material is gold nanorods.

[0057] The size and shape of the plasmonic materials are engineered for operation at selected electromagnetic radiation frequencies and their associated wavelengths. For example, plasmonic nanorods can be synthesized to have a longitudinal resonance of about 800 nm (see Example 4), as well as a transverse resonance at another wavelength such as 530 nm by adjustment of the aspect ratio. Suitable aspect ratios for plasmonic nanorods range from smaller than 0.1 to greater than 10, allowing for operation over a range of wavelengths. Shapes of nanoparticles can also be tailored for multiple resonance frequencies, greater than just the two fundamental resonances associated with longitudinal or transverse modes. By modifying the size, shape and composition of the plasmonic material, the quantum states can be engineered for selective inactivation of the pathogen. Geometry tuning of the plasmonic quantum material properties enhances the efficiency of pathogen inactivation and robustness of the plasmonic materials. For example, in embodiments, the length of biyramidal gold nanoparticles can be about 80nm. Also, the width can be about 25 nm. Further, the aspect ratio can be about 3.

[0058] Plasmonic nanorods can also be functionalized using methods known in the art. For example, plasmonic nanorods can be PEGylated nanorods or AnnexinV-functionalized nanorods (see also Example 4 below).

[0059] Generally, the plasmonic materials are in contact with the liquid medium that contains the pathogen to be inactivated. Typically, this means that the plasmonic material can be free floating within the liquid medium. For example, plasmonic nanorods can be suspended throughout the liquid medium or they can be attached to or incorporated into a matrix such as a polymer matrix that is optically transparent to allow laser excitation pulses to pass through to the incorporated plasmonic nanorods. For example, plasmonic nanorods can be incorporated into a hydrogel polymer matrix. For example, plasmonic nanorods can be loaded in agarose gels (for example, 2% agarose gels). Other suitable polymer matrices for incorporation of plasmonic nanorods include, but are not limited to, polyacrylamide gels such as PNIPAM (poly(N-isopropylacrylamide)), PMMA (polymethylmethacrylate), PES

(polyethylene sulfones), and cellulose. In an exemplification of this, plasmonic nanorods are loaded in agarose gels (for example 2% agarose gels). The hydrogels can be formulated in the form of a membrane matrix, or in the form of particles dispersed in the liquid medium and used for photonic inactivation.

[0060] When plasmonic materials (e.g., bipyramidal nanoparticles) are embedded in a polymer matrix such as a hydrogel, the polymer matrix is adapted to allow the liquid medium containing the pathogen interact with the plasmonic material.

[0061] As used herein in regard to polymer matrices with embedded plasmonic material, "optically transparent to an excitation pulse" refers to allowing a sufficient percentage of the photons of the excitation pulse to pass through the polymer matrix to the embedded plasmonic material to allow plasmonic enhancement. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 90%, or at least 95%, of the photons of the excitation pulse pass through the polymer matrix to the embedded plasmonic material.

[0062] In some embodiments, the liquid medium, while within the illumination zone (either contained without flow or flowing through the illumination zone), is exposed to excitation pulses for 1 second to 1 hour. In other embodiments, the liquid medium is exposed to excitation pulses for 1 second to 10 minutes. In other embodiments, the liquid medium is exposed to excitation pulses for 1 second to 5 minutes. In other embodiments, the liquid medium is exposed to excitation pulses for 1 second to 2 minutes. In other embodiments, the liquid medium is exposed to excitation pulses for 1 second to 60 seconds. In other embodiments, the liquid medium is exposed to excitation pulses for 1 second to 30 seconds.

[0063] In some embodiments, the plasmonic material is a plasmonic nanorod. In an aspect of these embodiments, the illumination zone has a plasmonic nanorod to pathogen (e.g., vims) ratio in the range from 1 : 1 to 20,000: 1, in the range from 1 : 1 to 10,000: 1, in the range from 1 : 1 to 5,000, or in the range from 1 : 1 to 1000: 1. In another aspect of these embodiments, the illumination zone has a plasm onic nanorod to pathogen (e.g., virus) ratio in the range from 10: 1 to 20,000: 1, in the range from 10: 1 to 10,000: 1, in the range from 10: 1 to 5,000, or in the range from 10: 1 to 1000: 1. In another aspect of these embodiments, the illumination zone has a plasmonic nanorod to pathogen (e.g., virus) ratio in the range from 50: 1 to 20,000: 1, in the range from 50: 1 to 10,000: 1, in the range from 50: 1 to 5,000, or in the range from 50: 1 to 1000: 1. In another aspect of these embodiments, the illumination zone has a plasmonic nanorod to pathogen (e.g., virus) ratio in the range from 100: 1 to 20,000: 1, in the range from 100: 1 to 10,000: 1, in the range from 100: 1 to 5,000, or in the range from 100: 1 to 1000: 1. In another aspect of these embodiments, the illumination zone has a plasmonic nanorod to pathogen (e.g., virus) ratio in the range from 100: 1 to 1,000: 1. In another aspect of this embodiment, the illumination zone has a plasmonic nanorod to pathogen (e.g., virus) ratio of 10,000: 1.

[0064] To plasmonically inactivate pathogens in a liquid medium while minimizing the risk of collateral damage to the product contained in the liquid medium, the liquid medium can be exposed to excitation pulses in such a manner that the pathogens are exposed to excitation pulses within the illumination zone such that they receive about the same number of photons, that is, a uniform dose. Laser pulse profile shaping methods can be used to match the laser intensity profile to the streamline flow in the liquid medium flowing through the illumination zone.

[0065] Additionally, conventionally in fluid flows, uniformity of mixing is attempted by forcing turbulent flow. While the methods can be used with liquid medium that is

characterized by turbulent flow in the illumination zone, non-turbulent flow such as laminar flow is desirable because turbulent flow can result in increased light scatter, decreasing the uniformity of laser exposure. Further, shear forces in turbulent flow can cause damage to the desired products such as biopharmaceuticals, reducing yield of the biopharmaceuticals.

[0066] In some embodiments, uniformity of dosage is enabled without requiring turbulent flow: (i) in laminar flow, the intensity profile of the light source (e.g, laser or light emitting diode) is matched to the velocity field in the streamline in such a manner as to expose particles in each streamline to receive substantially the same total dosage of photons; (ii) nonlinear channel designs of the flow channel ensure increased exposure time without requiring the expansion of the light beam profile; (iii) the incorporation of plasmonic materials enhances photonic viral inactivation using longer wavelength lasers, reducing the need for ultra-high intensity sources, and allowing to avoid the use of short wavelength sources (e.g., UV laser sources) that can cause additional collateral damage.

[0067] In some embodiments of the methods, the liquid medium flow is laminar while the liquid is exposed to laser light. In flow channels with "no-slip" boundary conditions, particles traveling along different streamlines in the flow velocity profile will traverse the laser illumination zone with different speed.

[0068] In some embodiments of the methods, the laser mode is shaped to match the velocity profile of the streamlines, ensuring uniform laser treatment of the entire flow channel. This is accomplished by matching the laser intensity profile with the flow stream in such a manner as to ensure that pathogen particles (e.g., virus particles), independent of the streamline, receive the identical laser dosage.

[0069] For inactivation methods requiring a particular minimum threshold of laser photon dose, nonlinear channels can be used. Such nonlinear channels can include a plurality of bends and/or turns in the channel.

[0070] The nonlinear channel can, for example, meander in two or three dimensions. For example, nonlinear channel geometries include, but are not limited to, a labyrinthine channel, a serpentine channel, a spiral channel, or combinations thereof. In another example, the nonlinear channel geometries include, but are not limited to a planar labyrinthine channel, a planar serpentine channel, a planar spiral channel, or combinations thereof.

[0071] FIG. 1 illustrates mode matching the laser beam intensity to the streamline flow. As an example, for a cylindrical flow channel of circular cross-section, in laminar flow, particles at the center of the channel are traveling the fastest, and dwell the shortest time in the flow channel. Concomitantly, particles close to the channel walls travel the slowest. For Poiseulle flow, the velocity profile in laminar flow is quadratic. The use of a laser beam with a flat intensity profile in a direction perpendicular to the flow channel therefore fails to provide uniform dosage. As illustrated in FIG. 1 (right side), the laser beam intensity is maximum at the center, and decreases away from the center. The laser intensity profile can be chosen to mode-match the hydrodynamic flow, thereby providing the particles in

substantially all the streamlines with the substantially the same dose of laser photons. For perfect matching, a quadratic laser intensity profile is desired, and attainable using conventional laser pulse shaping methods. In practice, in a Gaussian mode, illustrated in FIG. 1 (right), the central region can approximately match the velocity field. The mode matching is effective both in longitudinal flow mode as well as in transverse flow mode in which the laser beam is perpendicular to the flow.

[0072] In flow-through mode, the flow rate, flow channel geometry, dimensions of the illumination zone, laser light source parameters such as wavelength, spectral width, repetition rate, pulse duration, and pulse energies, are selected to inactivate a given pathogen (such as a virus).

[0073] A first embodiment is a method for inactivating a pathogen in a liquid medium, comprising (i) providing the liquid medium containing the pathogen and (ii) exposing the liquid medium and a plasmonic material to an excitation pulse, the liquid medium being in contact (and/or interacting) with the plasmonic material, and the plasmonic material having a localized surface plasmon resonance that overlaps with the excitation pulse. An alternative first embodiment is a method for inactivating a pathogen in a liquid medium, comprising exposing a liquid medium that contains the pathogen and a plasmonic material to an excitation pulse, the liquid medium being in contact (and/or interacting) with the plasmonic material, and the plasmonic material having a localized surface plasmon resonance that overlaps with the excitation pulse.

[0074] In an aspect of this first embodiment or alternative first embodiment, the liquid medium containing the pathogen is exposed to the excitation pulse while flowing through a flow channel. In another aspect of this embodiment, at least part of the liquid medium has a laminar flow. In another aspect of this embodiment or the foregoing aspect, the liquid medium has a flow in a flow direction and characterized by a velocity profile and the excitation pulse has an intensity profile corresponding to the velocity profile. In an aspect of the foregoing aspect, the excitation pulse is applied in a direction substantially parallel to the flow direction. In an alternative aspect, the excitation pulse is applied in a direction substantially perpendicular to the flow direction. In an aspect of this embodiment or any of the foregoing aspects, the liquid medium flows through a nonlinear channel while being exposed. In an aspect of the foregoing aspect, the nonlinear channel comprises a plurality of bends and/or turns in the channel. In an aspect of this embodiment or any of the foregoing aspects, the plasmonic material comprises plasmonic nanoparticles. In an aspect of the foregoing aspect, the plasmonic nanoparticles are selected from plasmonic nanorods, nanospheres, nanocubes, nanoprisms, bipyramids, hollow nanoparticles, nanoshells comprising a metallic shell around a non-metallic core, nanoellipsoids, and nanoantenna, made of, at least in part, a metal, an alloy, a doped dielectric or semiconductor materials. In an aspect of the foregoing aspect, the plasmonic nanoparticles are nanorods made of gold, titanium oxide, silicon oxide, silicon, germanium, transition metal nitrides, arsenides, phosphides, transparent conducting oxides, silicides, germanides, or sulfides. In an aspect of this embodiment or any of the foregoing aspects, the liquid medium is characterized by a non-turbulent flow. In an aspect of this embodiment or any of the foregoing aspects, the excitation pulse is a laser excitation pulse. In an aspect of this embodiment or any of the foregoing aspects, the pathogen is a virus. In an aspect of this embodiment or any of the foregoing aspects, the pathogen is a bacterium. In an aspect of this embodiment or any of the foregoing aspects, the pathogen is a bacterium from the genus Mycoplasma, a strain of Escherichia coli, a porcine circovirus (PCV), a human adenovirus, a reovirus, an orbivirus, a minute mouse virus (MMV), Cache Valley virus (CVV), an epizootic hemorrhagic disease virus (EHDV), a murine minute virus, a vesivirus, or a murine leukemia virus (MLV). In an aspect of this embodiment or any of the foregoing aspects, the pathogen has a size in the range from 18 nm to 100 nm. In an aspect of this embodiment or any of the foregoing aspects, the excitation pulse is a femtosecond pulse of a wavelength in the range from 600 nm to 1000 nm. In an aspect of this embodiment or any of the foregoing aspects, the excitation pulse is one out of a continuous train of 10 to 100 femtosecond, picosecond or nanosecond pulses, at a repetition rate of 0.1 kHz to 5 kHz, centered at 600 nm to 1000 nm, with energies between 1 to 20 mJ/cm 2 . In an aspect of this embodiment or any of the foregoing aspects, the excitation pulse is one out of a continuous train of 35 femtosecond pulses at a repetition rate of 1 kHz, centered at about 805 nm, with energies up to 7.5 mJ/cm 2 , and transform-limited spectral widths of about 25 nm. In an aspect of this embodiment or any of the foregoing aspects, the liquid medium is exposed to excitation pulses for 1 second to 2 minutes, while within an illumination zone. In an aspect of this embodiment or any of the foregoing aspects, the liquid medium is flown through the illumination zone within 1 second to 2 minutes. In an aspect of this embodiment or any of the foregoing aspects, the plasmonic material is embedded in a polymer matrix, the polymer matrix being optically transparent to the excitation pulse, and biochemical product passes through the polymer matrix. In an aspect of this embodiment or any of the foregoing aspects, the plasmonic material is embedded in a polymer matrix, the polymer matrix being optically transparent to the excitation pulse, and biochemical product passes through the polymer matrix. In an aspect of this embodiment or any of the foregoing aspects, the plasmonic material is embedded in a polymer matrix, the plasmonic material is suspended within the liquid medium and confined to an illumination zone by use of an enclosure having filters that allow the liquid medium to pass through but not the plasmonic material. In an aspect of this embodiment or any of the foregoing aspects, the plasmonic material is embedded within a polymer matrix, the polymer matrix being optically transparent to allow plasmonic material to be exposed to the laser excitation pulse.

[0075] A second embodiment is a method for inactivating a pathogen in a liquid medium, comprising (i) flowing the liquid medium containing the pathogen and (ii) exposing the liquid medium, while flowing, to an excitation pulse centered at a wavelength between 350 nm and 450 nm.

[0076] In an aspect of the second embodiment, the liquid medium is aqueous. In an aspect of the second embodiment or the foregoing aspect, the liquid medium has a laminar flow. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the liquid medium has a flow characterized by a velocity profile and the excitation pulse has an intensity profile matched to the velocity profile. In an aspect of the foregoing aspect, the excitation pulse is applied in a direction substantially parallel to the flow direction. In an alternative aspect of the foregoing aspect, the excitation pulse is applied in a direction substantially perpendicular to the flow direction. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the liquid medium is being flown through a nonlinear channel within an illumination zone. In an aspect of the foregoing aspect, the nonlinear channel comprises a plurality of bends and/or turns in the channel. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the liquid medium being flown is characterized by a non-turbulent flow. In an aspect of this embodiment or any of the foregoing aspects, the pathogen is a bacterium or a virus. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the pathogen is a bacterium from the genus Mycoplasma, a strain of Escherichia coli, an enveloped virus, and non-enveloped virus, or a bacteriophage. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the pathogen is a virus selected from dsDNA virus, ssDNA virus, dsRNA virus, (+)ssRNA virus, (-)ssRNA virus, ssRNA-RT virus, and dsDNA-RT virus. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the pathogen is a porcine circovirus (PCV), a human adenovirus, a reovirus, an orbivirus, a minute mouse virus (MMV), Cache Valley virus (CVV), an epizootic hemorrhagic disease virus (EHDV), a murine minute virus, a vesivirus, or a murine leukemia virus (MLV). In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the pathogen has a size in the range from 18 nm to 100 nm. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the excitation pulse is one out of a continuous train of 10 to 100 femtosecond, picosecond or nanosecond pulses, at a repetition rate 0.1 kHz to 5 kHz, centered at 600 nm to 1000 nm, with energies between 1 to 20 mJ/cm 2 . In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the excitation pulse is one out of a continuous train of 35 femtosecond pulses at a repetition rate 1 kHz, centered at about 402 nm, with energies up to 7.5 mJ/cm 2 , and transform-limited spectral widths of about 25 nm. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the liquid medium is exposed to excitation pulses for 1 second to 5 minutes, while within an illumination zone. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the liquid medium is flown through the illumination zone within 1 second to 5 minutes.

Flow Channel Cells and Systems

[0077] The methods for inactivating pathogens in a liquid medium can be performed in flow channel cells.

[0078] A third embodiment is a flow channel cell. The flow channel cell comprises an enclosure having (i) a first optical window; (ii) an inlet; (iii) an outlet, the first optical window being adapted and positioned to allow light from a laser or light emitting diode to enter into the enclosure to expose a volume within the enclosure to the light thereby forming an illumination zone within the enclosure, the inlet and outlet being positioned such that a liquid medium to be exposed to the light flows from the inlet through the illumination zone and out of the outlet; and (iv) a plasmonic material within the illumination zone.

[0079] In an aspect of the third embodiment, the flow channel cell has a second optical window, the second optical window being adapted and positioned to allow additional light from a laser or light emitting diode to enter into the enclosure to expose a volume within the enclosure to the additional light thereby forming a second illumination zone within the enclosure. In an aspect of the foregoing aspect, the first and second illumination zones overlap. In an aspect of any of the foregoing aspects, the first and second optical windows are parallel (i.e., substantially parallel) and at opposing sides of the enclosure. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the flow channel cell further comprises a nonlinear channel connected to the inlet and outlet and positioned within at least one illumination zone. In an aspect of the foregoing aspect, the nonlinear channel comprises a plurality of bends and/or turns in the channel. In an aspect of the second embodiment or any of the foregoing aspects of the second embodiment, the plasmonic material comprises plasmonic nanoparticles. In an aspect of the foregoing aspect, the plasmonic nanoparticles are selected from plasmonic nanorods, nanospheres, nanocubes, nanoprisms, bipyramids, hollow nanoparticles, nanoshells comprising a metallic shell around a non-metallic core, nanoellipsoids, and nanoantenna, made of, at least in part, a metal, an alloy, a doped dielectric or semiconductor materials. In an alternative aspect or further aspect of the foregoing aspect, the plasmonic nanoparticles are nanorods made of gold, titanium oxide, silicon oxide, silicon, germanium, transition metal nitrides, arsenides, phosphides, transparent conducting oxides, silicides, germanides, or sulfides.

[0080] Another embodiment is pathogen inactivation system. The system includes (a) a laser or light emitting diode and (b) a flow channel cell of the embodiments and aspects described above, for example, (b) a flow channel cell having an enclosure having (i) a first optical window; (ii) an inlet; (iii) an outlet, the first optical window being adapted and positioned to allow light from the laser or light emitting diode to enter into the enclosure to expose a volume within the enclosure to light thereby forming an illumination zone within the enclosure, the inlet and outlet being positioned such that a liquid medium to be exposed to the light flows from the inlet through the illumination zone and out of the outlet; and (iv) a plasmonic material within the illumination zone.

[0081] Another embodiment is a pathogen inactivation system comprising (a) a laser or light emitting diode and (b) a flow channel cell of the third embodiment or any of the foregoing aspects of the third embodiment.

EXAMPLES

Example 1 - Flow Through Inactivation [0082] Experiments were performed to demonstrate virus inactivation by lasers in flow- through mode. FIG. 2 shows the schematic of a test bench system 200 that was used for flow channel inactivation, comprising a height adjustable platform 210 with the components: Syringe pump assembly 220 for pumping of sample containing pathogen to be inactivated; tubing 230 for flow of the sample from the syringe 240 to and into a cell 250; tubing 260 for flow of exposed sample from the cell 250 (e.g., machined or quartz cell; here, the back face of the cell is visible) to and into a vial 270 for collection of the exposed sample; an optical component assembly 280 for coupling electromagnetic radiation into the cell 250; and a holder 290 for the cell bolted to translation stages 295 for alignment and focus adjustment. Mirror mounts with mirrors (297), for additionally routing laser light that is transmitted, back into the cell as needed to increase fluence, are shown. These mirror mounts with mirrors (297) were not needed or used in any of the runs shown in Figure 3.

[0083] This system incorporated a syringe pump method and high power coupling optics to evaluate and demonstrate flow through inactivation of the bacteriophage Phi-X 174. "Run" samples with titer ranging from 10 6 pfu/ml to 10 9 pfU/ml were loaded into the syringe pump. Fluid streams in quartz flow channels exposed the virus samples and controls to a

femtosecond laser illumination zone. A set of programmable protocols was developed.

Computer controlled operation was performed in both infuse and withdraw cycles under varying flow rates. Aliquots of the primary stock served to provide two sets of control samples - a "Feed" sample from the original stock; and a "Hold" sample that was subjected to the identical protocols of fluid flow, but without any exposure to the laser beam. Evaluation of the log reduction value using a standard plaque assay was made using the "Run", "Feed" and "Hold" samples.

[0084] FIG. 3 shows the LRV results in flow through photonic inactivation. Shown are the measured LRVs for a series of independent experiments on separate sample aliquots. The Run number identifies the sample and volume used in the particular measurement. RUN1 is the first experiment using a sample volume of 2 ml in the flow through photo-disruption experiment. RUN2 and RUN3 are measurements on two other aliquots of 2 ml each. RUN4 and RUN5 are measurements using larger sample volumes of 5 ml each. Control samples did not show virus inactivation, with an upper limit on the LRV that is at least 4.6 orders of magnitude smaller than with the laser on. Inactivation was demonstrated under two different set of laser parameter and flow conditions: (i) Using femtosecond laser pulses in the 800 nm wavelength range, laser inactivation of viruses was demonstrated in a system that contained designed plasmonic nanoparticles. The nanoparticles were designed for peak response tailored to the long wavelength radiation at 800 nm. (ii) Using frequency doubled femtosecond laser pulses in the wavelength range centered near 405 nm, virus inactivation was demonstrated without the use of nanoparticles for enhancement in flow-through mode.

Example 2 - Flow Channel Cell

[0085] FIG. 4 provides both a schematic illustration (left) as well as a photo of a 3D printed flow channel cell that was tested. The flow channel cell incorporates both fluid inlet and outlets, as well as optical windows capable of withstanding the high fluence of femtosecond laser systems. A central insert defines a region containing nanoparticles to enhance photonic inactivation. Inlet and outlet flow channels are visible at the top in the 3D printed model for illustrating the concept of incorporating fluid flow and photon flow pathways. The end inserts are designed to accept quartz, sapphire, calcium fluoride or other durable transparent high optical-quality windows. Illumination can be unidirectional, or bidirectional, using the two collinear end windows in operation. In laminar flow, the streamlines and photon flow paths are parallel.

Example 3 - Nonlinear Flow Channel Configurations

[0086] For a range of applications for process control, it may be sufficient to ensure a minimum threshold dose for laser inactivation of viruses. For such cases, non-linear flow channel configurations as shown in FIG. 5 and FIG. 6 can be used. FIG. 5 shows a spiral channel flow cell 500 providing a spiral channel 510. The optical window 520 is parallel to the plane of the spiral channel and is adapted to allow laser light to illuminate at least part of the spiral channel. The cell body includes a front part 530 and rear part 540, which jointly with the optical window 520 provide an enclosure for the spiral channel 510. The inlet and outlet ports (not shown) are on the rear part 530 of the cell, with one port at center and the other at the periphery.

[0087] Inlets and outlets in fluid communication with the spiral flow channel are preferably positioned outside the area expected to be exposed to laser light. For example, flow can enter the spiral channel from an inlet at the side of the enclosure and exit from an outlet in fluid communication with the spiral channel at the center of the spiral, or flow can enter the spiral channel from an inlet in fluid communication with the spiral channel at the center of the spiral and exit through an outlet at the side of the enclosure.

[0088] FIG. 6 shows a sinusoidal (i.e., example of a serpentine) flow channel cell 600, providing a sinusoidal channel 610. Flow into the sinusoidal flow channel is provided through inlet and outlet ports 620 and 630. As shown here, a sinusoidal channel can be arranged such that the general direction of the liquid flow within the channel is orthogonal to the direction in which the channel is adapted for exposure with laser light.

[0089] Inlets and outlets in fluid communication with the serpentine flow channel can be positioned outside the area expected to be exposed to laser light. Nonlinear flow channels are especially effective in methods of inactivation that make use of transverse illumination, where the laser beam is orthogonal to the liquid medium flow. Liquid medium is made to flow in nonlinear channels. The channels are fabricated in a transparent and durable substrate, either by lithography, machining, or printing. An optical window capable of operation with high power laser systems allows the flow channel to be illuminated. The nonlinear channel flow increases the time spent by pathogens in the in the illumination zone, ensuring that all the pathogens are exposed at least to a minimum required threshold for photonic inactivation. Additional efficiency can be gained in a double-sided geometry, with laser illumination from both sides using transparent windows on both faces of the cell shown in FIG. 5 or FIG. 6.

Example 4 - Plasmonic Enhancement of Selective Photonic Virus Inactivation

[0090] Enhancement of the photonic inactivation of Murine Leukemia Virus (MLV) via 805 nm femtosecond pulses through gold nanorods whose localized surface plasmon resonance overlaps with the excitation laser was found. Virus inactivation was found to be plasmonically enhanced, with greater than 3.7-log reduction measured by virus infectivity assays. Reliable virus inactivation was obtained for 10 s laser exposure with incident laser powers > 0.3 W. The fs-pulse induced inactivation was selective to the virus and did not induce any measurable damage to co-incubated antibodies. The loss in viral infection was associated with reduced viral fusion, linking the loss in infectivity with a perturbation of the viral envelope. It was observed that physical contact between and nanorods and virus particles was not required for viral inactivation and that reactive oxygen species (ROS) did not participate in the detected viral inactivation. [0091] Since noble metal nanoparticles convert incident electromagnetic waves into localized charge density oscillations, so called localized surface plasmon resonances (LSPRs), they generate high local E-field enhancements in electromagnetic hot-spots. (See Dykeman, E. C. & Sankey, O. F., Vibrational energy funneling in viruses-simulations of impulsive stimulated Raman scattering in M13 bacteriophages, J.Phys.:Condens. Matter 21, 505102 (2009)). It is believed that the strong E-field generated by the plasmonic

nanoparticles facilitated new virus inactivation processes. It was demonstrated that resonant nanoparticles whose LSPR overlapped with the excitation pulse enhanced the virus inactivation but that non-resonant nanoparticles had no effect. The effect of resonant plasmonic nanoparticles on virus inactivation as function of laser power when irradiated for a short exposure time of 10 s was characterized in detail. Since both efficiency and selectivity are important figures of merit for photonic virus inactivation, the selectivity towards virus particles was monitored by measuring the functionality of IgG antibodies co-incubated with the virus particles during laser exposure. The data indicated that the plasmonic enhancement effect is highly selective towards virus particles and generates no detectable collateral damage to the antibodies.

[0092] Methods and Materials

[0093] Laser Set-Up. The ultrashort pulsed (USP) excitation source used is a

femtosecond laser based upon a Legend Elite Duo (Coherent Inc.) Ti-sapphire regenerative amplifier. The laser produced a continuous train of 35 fs pulses at a repetition rate of 1 kHz centered at 805 nm with energies up to 7.5 mJ and spectral width of about 25 nm. The laser beam was incident on a quartz cuvette containing 250 μΙ_, of virus sample. Different laser powers on the sample were obtained using beam splitters and directing only a portion of the total laser beam to the sample area. The USP laser spot size was approximately 1 cm 2 and the typical exposure time of the sample to the laser irradiation was 10 sec. All laser irradiation studies were carried out at 22 °C. After irradiation, the samples were immediately stored at 4°C.

[0094] Photoacoustic Measurements. The acoustic signal generated by laser irradiated nanorods was collected in a homebuilt ultrasound detector. The nanorod sample was placed in a 1 mm quartz cuvette, which was located in a custom cell filled with water for ultrasound coupling. The cuvette was oriented at an angle of 45° relative to the 0.3 W fs laser beam. The photoacoustic signal (ultrasonic wave) was collected using a 2.25 MHz focused water immersion transducer.

[0095] Nanorod Synthesis and Functionalization. The seed-mediated growth technique described by Vigderman and Zubarev was used to synthesize gold nanorods. (See

Vigderman, L. & Zubarev, E. R., High- Yield Synthesis of Gold Nanorods with Longitudinal SPR Peak Greater than 1200 nm Using Hydroquinone as a Reducing Agent, Chem. Mater. 25, 1450-1457 (2013)). 460 μΐ. of a freshly prepared solution of 0.01 M sodium borohydride dissolved in 0.01 M sodium hydroxide was rapidly injected into 10 mL of 0.5 mM HAuCl 4 solution containing 0.1 M CTAB (cetyl trimethyl ammonium bromide) under extensive stirring. A change of color from greenish to light brown indicated the successful formation of gold nanoparticle seeds. To synthesize gold nanorods with a longitudinal resonance at ~ 800 nm, 11.5 μΐ. of 0.1 M silver nitrate solution was added to 10 mL of 0.5 mM HAuCl 4 solution containing 0.1 M CTAB. Subsequently, 500 μΕ of 0.1 M aqueous hydroquinone solution was added, and the resulting mixture was hand-stirred until it became clear. Then, 160 of gold seed solution was added under thorough stirring. The resulting mix was allowed to age overnight. To obtain PEGylated nanorods, 2.5 μΤ of 10 mM PEG2 (HS-CH 2 CH 2 - (C 2 H 4 0)77-N 3 ) were added together with 2.5 xL of 10 mM HS-(CH 2 )n-(CH 2 CH 2 0) 6 OCH 2 - COOH to lmL of gold nanorods in the presence of 3% v/v of Tween 20. The samples were incubated overnight and washed by centrifugation and subsequent resuspension. Alkyne- functionalized annexinV was cross-linked to the azide functionalized nanorods in the presence of 500 μΜ ascorbic acid and 100 μΜ CuS0 via Cu 1 catalyzed click-reaction. (See Kolb, H. C, Finn, M. G. & Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angewandte Chemie International Edition 40, 2004-2021 (2001)) The nanorods were cleaned again through repeated centrifugation and resuspension in DDI water or buffer.

[0096] Virus Production. Luciferase expressing, single cycle of replication competent MLV particles (MLV/luc) were derived via co-transfection of HEK293T cells with LNC-luc (luciferase expressing retroviral expression vector) and pCL-Eco (MLV packaging vector that expresses MLV Gag, Pol and ecotropic Env glycoproteins; Imgenex) plasmids, as previously described. (See Puryear, W. B. et al., Interferon-Inducible Mechanism of Dendritic Cell- Mediated HIV-1 Dissemination is Dependent on the Siglec, CD 169. PloS Pathogens 9, el003291 (2013)). For the virus fusion assay described below, HEK293T cells were co- transfected with LNC-luc, pCL-Eco and S15-Blam plasmids. To generate the S15-Blam expression plasmid that expresses a Src-eptiope tagged β-lactamase fusion protein upon transfection, the N-terminal 15 amino acid sequence of c-Src (SI 5) was cloned in frame upstream of β-lactamase or in pcDNA3.1/zeo+ (Invitrogen) eukaryotic expression plasmid. S15-Blam is incorporated into MLV particles upon virus particle budding from the plasma membrane of virus-producing cells. Virus-containing supernatants were harvested 2 days post transfection, and passed through 0.45 μιη filters. Virus was concentrated by

ultracentnfugation on a 20% sucrose cushion [28,000 rpm at 4°C for 2 hours with a SW32 Ti rotor (Beckman Coulter)]. Virus pellets were resuspended in PBS, aliquoted, and stored at - 80 °C until further use. Preparation of Nanorod- Virus Samples. PEGylated nanorods or nanorods loaded with annexin V were centrifuged at 5000 rpm for 10 min. The supernatant was discarded and 17 μΐ ^ of the pellet was added to 50 μΐ ^ of MLV/luc, followed by overnight incubation at 4°C. Then, 500 μΐ ^ of IgG antibody (anti-p24gag monoclonal antibody; Clone 183-H12-5C; NTH AIDS Research and Reference Reagent Program, contributed by Dr. Bruce Chesebro) in RPMI medium was added to the mixture and the mixture was diluted by adding lx Tris (pH 7) or PBS (pH 7.4) buffer to final volume of 1 ml. The nanorod to virus ratio was more than 10000: 1. The concentration of the nanorods was quantified using a UV-vis spectrometer (Cary 5000 spectrophotometer).

[0097] ROS Scavengers. A solution containing three different ROS scavengers were used. Sodium azide (NaN 3 ) and Manitol were dissolved in 20 mM Tris (pH 7.0). Then Manganese (III) Tetrakis(4-benzoic acid) Porphyrin (MnTBAP ) dissolved in 0.1 M NaOH was added. This solution was subsequently added to virus samples containing antibodies from a hybridoma preparation or to mixtures containing virus, PEGylated nanorods, and antibodies. All samples were diluted with Tris buffer. The final scavenger concentrations were 10 mM Manitol, 10 mM Sodium azide and 0.02 mM MnTBAP. The pH of the final solution was 7.

[0098] ELISA. To test the efficacy of the fs laser inactivation protocol on functionality (epitope recognition) of a monoclonal antibody, an anti-p24 §a§ monoclonal antibody (Clone 183-H12-5C) was exposed in the presence or absence of MLV/luc particles to fs laser, as described above. A sandwich ELISA was performed to test the ability of the anti-p24 a monoclonal antibody to quantify standard amounts of HIV-1 p24 ga s antigen, as described previously. (See Hatch, S. C, Archer, J. & Gummuluru, S. Glycosphingolipid composition of human immunodeficiency virus type 1 (HIV-1) particle is a crucial determinant for dendritic cell-mediated HIV01 trans-infection. Journal of Virology 83, 3496-3506 (2009)). Briefly, 2- fold dilutions of recombinant p24 §a§ antigen (ABI, Inc) was bound to HIV-Ig (from NABI and National Heart Lung and Blood Institute) coated wells and detected with unexposed control or laser exposed anti-p24 §a§ monoclonal antibody, and HRP-conjugated goat anti- mouse secondary antibody (Sigma). ELISA was developed with a peroxidase substrate (KPL, Inc) and the standard curves were generated with each of the control (untreated) or fs laser- exposed anti-p24 §a§ monoclonal antibodies.

[0099] Virus Fusion Assay. To investigate MLV/luc fusion to Rat2 target cells, a FACS based assay was utilized. Briefly, untreated or laser-exposed MLV/luc particles containing S15-BlaM fusion protein was used to infect target cells. After incubating 2 hours at 37°C, cells were washed with C0 2 -independent media (Invitrogen) and incubated in CCF2

(fluorogenic substrate of P-lactamase)-containing media at 18°C overnight, washed, fixed with 4% PFA. The number of β-lactamase positive cells was determined by FACS analysis using a LSRII flow cytometer (BD).

[00100] Quantification of Virus Inactivation . Virus inactivation was determined by infecting cells (mouse fibroblast cell line) with treated (laser-inactivated) or untreated viruses. Cells were then incubated for 48 h prior to lysis. The lysates were then used for measurement of luciferase activity using a chemiluminescent reporter.

[00101] Gel Electrophoresis of Virus - Nanorod Mix. Virus-Nanorod and Nanorod-only control samples were run on 2% agarose gels made from 0.5x TBE. The same buffer was used as running buffer. The samples were loaded with ficol and were run with a current of 200 mA, voltage of 140 V for 30 min.

[00102] Results and Discussion

[00103] Murine Leukemia Virus (MLV) was chosen as target for the viral inactivation studies of this example due to its similarity with endogenous mouse retroviruses which are known contaminants in the biopharmaceutical industry. All experiments were performed with a recombinant virus that expresses the luciferase reporter gene upon establishment of productive infection in target cells, providing a robust strategy for the quantification of virus infectivity. A first set of experiments established that nanorods whose LSPR overlaps with the incident fs laser pulse enhances the efficacy of light-induced laser activation. To that end, the viral infectivity obtained with virus particles exposed to fs laser irradiation with constant pulse duration but different average powers was compared. The exposure time of the individual samples was kept constant at 10 s, which is 3 orders of magnitude shorter than the exposure time in all previous photonic virus inactivation studies.

[00104] The LSPRs of gold nanoparticles are strongly morphology dependent, and the aspect ratio of nanorods represents a rational control parameter to tune the plasmon resonance across the visible range of the electromagnetic spectrum to the Near-Infrared (NIR) and beyond. (See Murphy, C. J. et al. Anisotropic metal nanoparticles: Synthesis, assembly, and optical applications. Journal of Physical Chemistry B 109, 13857-13870 (2005); and

Nikoobakht, B. & El-Sayed, M. A. Preparation and Growth Mechanism of Gold Nanorods (NRs) Using Seed-Mediated Growth Method. Chem. Mater. 15, 1957-1962 (2003)). Gold nanorods with an aspect ratio of 3.6 (length of long axis = 55±8 nm) sustain a longitudinal plasmon mode that peaks close to the wavelength of the fs laser emission (805 nm) as is shown in the UV-Vis spectra of the nanorod "control" (no laser exposure) in FIG. 7. A TEM image of a representative nanorod is provided as inset in FIG. 7 in the top right corner. The gold nanorods were obtained through a surfactant-directed synthesis (see Johnson, C. J., Dujardin, E., Davis, S. A., Murphy, J. C. & Mann, S. Growth and form of gold nanorods prepared by seed-mediated, surfactant-directed synthesis. Journal of Materials Chemistry 12, 1765-1770 (2002) using cetrimethylammonium bromide (CTAB) as ligand, which favors growth along the cry stall ographic [110] axis. Due to the functionalization of the nanorod surface with CTAB, the nanorods have a positive zeta-potential of approximately ζ = +23 mV. As CTAB is cytotoxic, it was replaced with polyethyleneglycol (PEG) ligands. In a first set of experiments, the stability of the nanorods was tested when irradiated with 805 nm laser pulses with 35 fs duration for 10 s. The laser average power was varied between 0.3 W, 1.2 W and 3 W and UV-Vis spectra were recorded after laser irradiation. These spectra and the spectrum of the non-irradiated control sample are summarized in FIG. 8. The UV-Vis spectra contain two characteristic peaks. One is centered at 520 nm and is assigned to the transverse plasmon mode of the nanorods, whereas the second peak at 800 nm is assigned to the longitudinal plasmon mode. (See Murphy, C. J. et al. Anisotropic metal nanoparticles:

Synthesis, assembly, and optical applications. Journal of Physical Chemistry B 109, 13857- 13870 (2005)). The peak wavelength of the longitudinal mode intensities and the associated full width at half maximum (FWHM), are plotted in FIG. 7. Interestingly, a blue shift and broadening of the longitudinal mode was observed. These effects are accompanied by a decrease in the spectral intensity of the longitudinal mode and an increase in the intensity of the transverse mode (FIG. 8).

[00105] Although the nanorods were found to show some morphological restructuring and agglomeration, both UV-vis data and the gel studies confirm that the sample still contained a substantial fraction of resonant nanorods under the chosen irradiation conditions. In a typical virus inactivation experiment, PEGylated nanorods were incubated with MLV particles in a ratio of more than 10000: 1 in a total volume of 67 μΐ ^ at 4°C for up to 12 h. The samples were then diluted by adding 933 μΐ ^ of PBS, pH 7.4 to a total volume of 1 mL. The final virus concentration was such that infections of Rat2 reporter cells with the luciferase encoding recombinant viruses resulted in approximately lxlO 6 relative light units (RLU). The inactivation studies were performed with PEGylated nanorods as well as with rods functionalized with annexin V that binds to phosphatidyl serine (PS) of the enveloped MLV. For the latter, Ca 2+ was added to the binding buffer when incubating annexin V- functionalized nanorods with virus particles to achieve a final Ca 2+ concentration of 2 mM.

[00106] In many applications it is necessary to inactivate the virus in a complex biological matrix. For instance, virus inactivation plays a critical role in the pharmaceutical industry for the fabrication of monoclonal antibodies (mAbs). Realistic process conditions were emulated by performing the virus inactivation in an IgG antibody (total protein concentration: 160 μΜ) containing Tris solution as biological matrix. The proteins were added after overnight co- incubation of nanorod and virus.

[00107] In a first set of inactivation experiment, the maximum average laser power of 3W was used and viral inactivation measured in the presence of three different nanoparticles: virus + PEGylated nanorods; virus + annexinV-functionalized nanorods; virus + spherical nanoparticles. Unlike for the nanorods, the LSPR of the 40 nm diameter gold nanoparticles (530 nm) did not overlap with the exciting laser pulse. The nanorod and nanoparticle to virus ratios was higher than 10000: 1. Additional controls included laser irradiation of viruses without nanoparticles. After laser irradiation for 10 s, virus infectivity was quantified (see FIG. 9A). The 805 nm fs laser irradiation had no effect on virus infectivity in the absence of R, or upon incubation of virus with spherical nanoparticles whose resonance does not overlap with the incident laser pulse. In contrast, addition of PEGylated nanorods whose longitudinal mode lies close to 800 nm led to a strong reduction in viral infectivity

(LRV-2.7). Interestingly, the LRV value obtained with PEGylated nanorods functionalized with annexinV, which can bind to phosphatidylserine (PS) in the viral membrane, was not higher than that for the PEGylated nanorods {vide infra). Since the nanorods used in the experiments did not show any systematic cytotoxicity before or after laser radiation (see FIG. 9F), it is believed that the decrease in infectivity observed for virus samples irradiated with the pulsed laser in the presence of resonant gold nanorods results from an enhancement of photonic virus inactivation through the plasmonic nanorods.

[00108] To assess the potential collateral damage that laser irradiation may have on IgG antibodies, the binding of an IgG antibody to its epitope before and after laser irradiation in the presence and absence of nanorods was compared using ELISA. These experiments were performed for an average laser power of 3 W and an irradiation time of 10 s. The same conditions resulted in a distinct drop in viral infectivity (see FIG. 9A) if nanorods were present. The ELISA studies provided essentially identical binding curves for the antibody before and after laser irradiation and in the presence or absence of the nanorods (see FIG. 9b). The antibody binding affinity was not significantly affected by the NIR fs laser irradiation even in the presence of resonant nanoparticles.

[00109] In a second independent study, the effect of the average laser power on virus inactivation in the presence and absence of resonant PEGylated nanorods was quantified. The laser irradiation time was 10 s. Based on the observation in FIG. 9A that annexinV- functionalized nanorods give rise to a less effective plasmonic enhancement of photonic virus inactivation, only PEGylated nanorods were included in the variable power studies. Again, all samples contained IgG antibodies. FIG. 9C shows the measured LRV values for average powers of 0 W (control), 0.06 W, 0.3 W, and 3 W. While in the absence of nanorods none of these average powers resulted in a measurable LRV, after addition of nanorods average powers > 0.3 W were sufficient to generate a measurable LRV. A LRV = 1.8 was obtained for an average power of 0.3 W and the viral inactivation further enhanced to LRV = 2.58 for an average power of 3 W. The striking difference in virus inactivation between samples with and without nanorods further corroborates a strong enhancement of photonic inactivation achieved through addition of nanorods. FIG. 9D shows the binding curves of the IgG antibody to its epitope after exposure to the different average powers of the pulsed lasers in the presence and absence of nanorods as well as for control without any laser irradiation. All conditions show essentially the same binding affinities; the antibody is not affected by the chosen experimental conditions. [00110] One finding from FIGS. 9A-F is that annexinV functionalization of the nanorods does not yield a stronger reduction of infectivity when compared with the PEGylated nanorods. In fact, the PEGylated nanorods without any virus binding functionality achieved higher LRV values in FIG. 9A. To check for non-specific binding of PEGylated nanorods to MLV particles the mix of virus and PEGylated nanorods was imaged in the TEM. No significant spatial colocalization between virus particles and PEGylated nanorods was detected, indicating that the non-specific binding of the PEGylated nanorods to the virus particles is low. A magnified TEM image of a virus particle is included as inset in FIG. 9C.

[00111] The fact that annexinV fails to enhance virus inactivation and the absence of a significant non-specific binding to MLV in the case of the PEGylated nanorods suggests that a direct binding between nanorods and virus particles is not required for the nanorod- enhanced photonic virus inactivation.

[00112] The LRVs measured in FIGS. 9A and 9C are limited by a relatively low virus titer, which was close to the detection limit of the luciferase activity assay used for the quantification of virus infection. Higher LRVs can be achieved by increasing the amount of initial virus input in the system. Indeed, when the virus concentration was increased by a factor of 2 and twice as much sample volume for virus infection was used as before, a LRV = 3.76 (FIG. 9E) was measured. Because the analysis revealed that no binding between nanorod and virus is required, overnight pre-incubation of virus and PEGylated nanorod was omitted for this measurement.

[00113] Without being bound by theory, it is believed that the fact that the enhancement does not require a physical contact between the nanorod and the virus particle argues against field-enhanced ISRS or multiphoton absorption effects as underlying mechanisms. Both of these effects are strongly E-field dependent and the E-field intensity decays rapidly with separation from the nanorod surface. At a distance of 100 nm, the E-field has already decayed to the value of the incident light field. Considering the nanorod concentration used in this example of -l x lO 11 particles/mL, which corresponds to one rod in a cube of solvent with side length of approximately 2 μπι, it is believed that it can be excluded that virus particles and nanorods co-localize close enough to generate a sufficient E-field enhancement to impact ISRS or multiphoton absorption of the virus. For similar reasons, it is unlikely that thermal effects are the underlying cause of the observed virus inactivation. Although the resonant fs laser excitation induces a temperature jump in the nanorod and its immediate environment, this effect is local and a subsequent thermalization with the heat bath of the solvent rapidly abrogates any temperature gradients. The measured temperature fluctuations during the course of the experiments were < 3°C.

[00114] One alternative mode of virus inactivation that does not require direct contact between nanorods and virus is the light induced generation of reactive oxygen species (ROS). It has been demonstrated in previous studies that plasmon excitation in gold nanocubes can generate singlet oxygen ( l 0 2 ), superoxide anion (0 2 " ), or hydroxyl radical (ΌΗ). (See Gao, L. et al. Plasmon-Mediated Generation of Reactive Oxygen Species from Near-Infrared Light Excited Gold Nanocages for Photodynamic Therapy in vitro. ACS Nano 8, 7260-7271 (2014)). To test whether ROS were involved in the virus inactivation, the fs laser irradiation of a mix of MLV and resonant nanorods was repeated in the presence of ROS scavengers. Sodium azide (scavenger for x 0 2 ), MnTBAP (scavenger for 0 2 " ), and mannitol (scavenger for ΌΗ) were used. While the concentration of ROS scavengers used in these experiments did not show any cytotoxicity, addition of ROS scavengers did not suppress nanorod mediated virus inactivation, suggesting that ROS formation mechanism is not responsible for the virus inactivation.

[00115] It is believed that the virus inactivation is primarily caused by molecular-level perturbations of the MLV membrane, which prevents an effective binding to the host cell and subsequent fusion. This is supported by the results of the fs laser irradiation of a MLV/luc particles containing S 15-BlaM fusion protein in presence and absence of resonant nanorod. The experiments show that only laser irradiation in the presence of resonant nanorods achieves a measurable decrease (~2.5-fold) in virus fusion. Laser irradiation without nanorods had no measurable effect on fusion. Plasmonically enhanced photonic inactivation was found to inactivate viral functions responsible for early stages of the infection, including host cell binding and fusion.

[00116] The enhancement of fs-pulsed NIR radiation induced photonic virus inactivation through plasmonic nanoparticles was demonstrated. Irradiation in the presence of resonant nanorods significantly reduced viral fusion with the host cell, suggesting that the plasmonic enhancement acts on viral surface functionalities responsible for early stages of the viral infection rather than on the viral genome or enzymes. LRVs of > 3.7 were achieved with MLV in as little as 10 s of irradiation. The inactivation was highly specific to the virus while co-incubated IgG antibodies did not show any loss in functionality. Example 5 - Bipyramidal gold nanoparticles (BPs)

[00117] Preparation

[00118] Typically, Initial gold seeds were prepared by fast reduction of HAuCl 4 (10 mL, 0.25 mM) in an aqueous CTAC solution (50 mM) with freshly prepared NaBH 4 (0.25 mL, 25 mM) in the presence of citric acid (5 mM) under vigorous stirring at room temperature. After 2 minutes, the seed solution was heated in oil bath at 80 °C for 90 minutes under gentle stirring, which leads to a gradual color change from brown to red. The thermally treated seed solution was removed from the bath and stored at room temperature. The gold BPs were grown by adding gold seeds under vigorous stirring to an aqueous growth solution containing CTAB (100 mL, 100 mM), HAuCl 4 (5 mL, 10 mM), AgN0 3 (1 mL, 10 mM), HC1 (2 mL, 1M) and AA (0.8 mL, 100 mM). The mixture was kept at 30 °C overnight. Size of BPs based on SEM: L=80nm, W=25.7 nm, Aspect ratio -3.1.

Optical Properties

[00119] FIG. 10 provides the UV-Vis spectra of the bipyramidal nanoparticles, prepared as described above, recorded after 10s exposure with 35 fs laser pulses at 805 nm, with different average powers.

[00120] FIG. 11 provides a graph showing peak wavelengths and associated spectral widths of the longitudinal bipyramidal nanoparticle plasmon modes as function of the average power of the laser irradiation.

[00121] Changing the morphology of nanoparticles, for example, from nanorods to bipyramidal nanoparticles, is an example of geometric tuning, which corresponds to engineering of the quantum states of the plasmonic material. As can be seen by comparing the optical properties of above described gold nanorods (see FIGS. 7 and 8) with above described bipyramidal gold nanoparticles (see FIGS. 11 and 10), geometric tuning allows engineering the optical properties of the nanoparticles.

[00122] Desirable properties of gold nanorods include their optical high extinction cross sections and local electric field enhancements at their tips. Such optical properties can be enhanced with sharp nanostructures such as bipyramidal gold nanoparticles (e.g., comparing FIG. 8 with FIG. 10). The overall size and the sharpness of the tips of the bipyramidal gold nanoparticles affect the wavelength of the LSPR peak as well as its height.

[00123] Generally, geometric tuning together with excitation pulse paramater selection allows increasing pathogen inactivation in the methods described herein. [00124] Stability of the Bipyramidal Gold Nanoparticles

[00125] Upon 10 second exposure to 35 fs laser pulses at 805 nm (and at different average powers ranging from control (i.e., no irradiation) to 3W), the bipyramidal gold nanoparticles did not form a second band in a 2% w/v agarose gel (see FIG. 12). This is in contrast to gold nanorods, prepared using the methods described above, which showed a clear second band, particularly at higher average powers (see FIG. 13). It is believed the second band in the nanorods is due to agglomeration and that these results indicate that the bipyramidal gold nanoparticles have improved stability relative to gold nanorods. The gel electrophoresis of the nanoparticles was done as follows. The nanoparticles samples were run in 2% w/v agarose made from 0.5x TBE. The samples were loaded with ficol and 0.5x TBE was used as running buffer. The samples were run at 140v, 200 mA for 30 min.

[00126] For the following examples, the gold nanorods were prepared as described in Example 4 and the bipyramidal gold nanoparticles were prepared as described in Example 5.

Example 6 - Flow Through Mode Plasmonic Enhancement of Photonic Phi-X174 Inactivation with Nanorods and Bipyramidal Nanoparticles

[00127] Three different samples were investigated: (1) Phi-X174 with initial titer of 10 9 pfu/ml and a sample volume of 1.5 ml (see "V" in FIG. 14); (2) Phi-X174 with initial titer of 10 9 pfu/ml combined with 100-fold excess gold nanorods to form a sample volume of 1.5 ml (see "V+NR" in FIG. 14); and (3) Phi-X174 with initial titer of 10 9 pfu/ml combined with 100-fold excess gold bipyramidal nanoparticles to form a sample volume of 1.5 ml (see "V+BP" in FIG. 14). The results were obtained for flowthrough mode using programmable syringe pump with infusion rate 2ml/hr rate and withdrawal rate 100 ml/hrs . To ensure complete exposure of sample with laser light, three withdrawal and Infusion pumping cycle was used.

[00128] As shown in FIG. 14, both nanorods and bipyramids resulted in greater than million-fold reduction in the pathogen concentration.

Example 7 - Plasmonic Enhancement of Selective Photonic Mycoplasma Inactivation

[00129] Acholeplasma laidlawii (A. laidlawii) was cultured in GUB. Two flasks of 25mL volume each were incubated for 20-24 hours. After 20-24 hours the flasks were combined. [00130] A. laidlawii cultured in GHB with 2.2e9 cfu/ml concentration and sample volume of 1.5 ml was exposed with 800 nm wavelength (Red) 35 fs laser with 1kHz repetition rate, 3 W power, 30 min exposure time, 400 nm laser (Blue) with 2.5 w power and 800 nm laser in presence of PEGylated NR with OD~ 1.5. FIG. 15 shows the Log Reduction Value (LRV) results the A. laidlawii (cultured in GHB) photonic inactivation, including those in which the photonic inactivation was plasmonically enhanced with gold nanorods ("Red+NR").

[00131] A. laidlawii in buffer with 1.6e9 cfu/ml concentration was exposed under similar laser conditions as described in the preceding paragraph but in presence of ConcanavalinA functionalized NR with OD-1.25. FIG. 16 shows the Log Reduction Value (LRV) results for A. laidlawii (in buffer) photonic inactivation, including those in which the photonic inactivation was plasmonically enhanced with gold nanorods functionalized with

Concanavalin A ("Red+Conc A NR").

[00132] Before the experiments, A. laidlawii bacteria were incubated in a refrigerator for ~5 hours.

Example 8 - Plasmonic Enhancement of Selective E. coli Inactivation

[00133] E. coli sample preparation: Bacterial cells were cultured on a nutrient agar plate at 37°C overnight. Cells from well-grown colonies were then transferred to a 8-ml of tryptic soy broth with shaking (125 rpm) at 37°C for 2 h. Sample preparation: 500μΕ of purified bipyramids was added to 500 μΕ of E. coli with 3e7 CFU/ml concentration. E. coli cell counts: The cell counts were measured with the standard plate count agar method. Namely, the sample suspension was serially diluted with tryptic soy broth and 100 μΐ of the diluted samples were incubated with the standard plate count agar at 37°C overnight.

[00134] A 250 μΕ sample, prepared as described in the preceding paragraph, was irradiated with 800 nm excitation wavelength laser (Red) in presence of bipyramidal nanoparticles with OD~l .1 for 30 min. The results are provided in FIG. 17 which shows the Log Reduction Value (LRV) for E. coli photonic inactivation with plasmonic enhancement using gold bipyramidal nanoparticles.

Example 9 - Plasmonic Enhancement of Selective Photonic Bacteriophage PhiX-174 Inactivation with Polymer Matrix Embedded Plasmonic Material

[00135] COOH-PEG functionalized gold nanorods, prepared as described above, were incubated with excessive amount of neutravidin (n:n = 1 : 1000) in PBS buffer on a rocker shaker for 2 hours. The samples were washed twice by centrifugation and subsequent resuspension in PBS buffer. The gold NRs- neutravidin were then incubated with biotin functionalized agarose bead (n:n = 100: 1) on a rocker shaker for 2 hrs. The nanorods were cleaned again through repeated centrifugation and resuspension at low speed in PBS. This resulted in agarose beads with embedded gold nanorods. Two samples were prepared: (1) PhiX-174 at an initial titer of le9 pfu/ml with a sample volume of 250 μΐ, (see "V" in FIG.18), and (2) 125 μL· PhiX-174 at an initial titer of le9 pfu/ml combined with 125 μL· NR-functionalized agarose bead to form a sample volume of 250 f L.

[00136] The samples were exposed to 30 minutes of 35 fs laser pulses centered at 805 nm with an average power of 3.1 W. FIG. 18 shows the corresponding Log Reduction Value (LRV) results for PhiX-174 photonic inactivation with plasm onic enhancement using agarose bead embedded gold nanorods.

[00137] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[00138] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.