Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
  • G01N33/553—Metal or metal coated
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

• the present disclosure relates to pathogen detection using metal enhanced fluorescence, and more specifically, relates to pathogen sensors, systems for pathogen detection, peptide ligands, and methods for detecting pathogens using metal enhanced fluorescence.
• nucleotide probes personnel must pre-treat samples under lysis conditions in order to provide access to complementary nucleotides within the target pathogen.
• antibodies such proteins are not inherently stable in battlefield and/or real- world conditions. Accordingly, additional embodiments for pathogen sensors are desired. Summary
• a pathogen sensor for detecting the presence of a target pathogen in a sample.
• the pathogen sensor includes an optically clear substrate including silver particles deposited on a surface thereof, a plurality of immobilized capture peptide ligands attached to the silver particles, and a plurality of free labeling peptide ligands including fluorophores.
• the silver particles are immobilized on the surface.
• the immobilized capture peptide ligands include at least one first capture peptide ligand configured to bind specifically to the target pathogen.
• the target pathogen is selected from the group consisting of bacteria, viruses, fungi, and combinations thereof.
• the free labeling peptide ligands include at least one first labeling peptide ligand configured to bind specifically to the target pathogen.
• the first labeling peptide ligand includes a fluorophore, such that when the first capture peptide ligand binds specifically to a first region of the target pathogen and the first labeling peptide ligand binds specifically to a second region of the same target pathogen: the target pathogen is sandwiched in between the first capture peptide and the first labeling peptide ligand bound thereto, the fluorophore of the first labeling peptide ligand is positioned at a metal enhancing distance from the silver particles immobilized on the surface, and fluorescence emission of the fluorophore of the first labeling peptide ligand is enhanced upon excitation, indicating the presence of the target pathogen.
• a pathogen sensor for detecting the presence of pathogenic Escherichia coli in a sample.
• the pathogen sensor includes an optically clear substrate including silver particles deposited on a surface thereof, a plurality of immobilized capture glycan ligands attached to the silver particles, and a plurality of free labeling glycan ligands including fluorophores.
• the silver particles are immobilized on the surface.
• the immobilized capture glycan ligands include at least one first capture glycan ligand configured to bind specifically to the pathogenic Escherichia coli.
• the plurality of free labeling glycan ligands includes at least one first labeling glycan ligand configured to bind specifically to the pathogenic Escherichia coli.
• the at least one first labeling glycan ligand includes a fluorophore.
• the first capture glycan ligand and the first labeling glycan ligand individually include:
• the pathogenic Escherichia coli is sandwiched in between the at least one first capture glycan ligand and the at least one first labeling glycan ligand bound thereto, the fluorophore of the at least one first labeling glycan ligand is positioned at a metal enhancing distance from the silver particles immobilized on the surface, and fluorescence emission of the fluorophore of the at least one first labeling glycan ligand is enhanced upon excitation, indicating the presence of the pathogenic Escherichia coli.
• Other embodiments are directed to systems incorporating the pathogen sensors.
• a peptide ligand is disclosed.
• the peptide ligand is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4, and SEQ ID NO: 5.
• FIG. 1 is a schematic view of a pathogen sensor according to one or more embodiments of the present invention.
• FIG. 2 is a graph of known quantum yields (Qo) of Rhodamine B, ErB, [Ru(bpy) 3 )] 2+ , basic fucsin, and [Ru(phen) 2 dppz] 2+ in solution with respect to intensity ratio
• FIG. 3 is a schematic view of a sensor board according to one or more embodiments of the present invention.
• FIG. 4 depicts a buoy system used for pathogen detection in open water or well water monitoring according to one or more embodiments of the present invention
• FIG. 5 depicts a portable pathogen detection system utilized in the detection of airborne agents, wherein the portable container is in the closed position according to one or more embodiments of the present invention
• FIG. 6 also depicts a portable pathogen detection system utilized in the detection of airborne agents like FIG. 5; however, the portable container is in the closed position according to one or more embodiments of the present invention
• FIG. 7 is a portable automatic in-line water monitoring unit used for pathogen detection according to one or more embodiments of the present invention.
• FIG. 9 is a graph of wavelength (nm) with respect to Arbitrary Fluorescence Units (AFU) resulting from detection of varying concentrations (pfu/mL) of Enterobacteria phage MS2 with Ru(bipy)-labeled peptide ligands;
• FIG. 10 is a bar graph of Enterobacteria phage MS2 concentration (pfu/mL) with respect to Arbitrary Fluorescence Units (AFU) resulting from detection of Enterobacteria phage MS2 with Ru(bipy)-labeled peptide ligands;
• FIG. 11 is a bar graph of Enterobacteria phage MS2 detected with Ru(bipy) -labeled MS2-14 peptide ligands (SEQ ID NO: 2) and Enterobacteria phage MS2 detected with TAMRA-labeled peptide ligands (SEQ ID NO: 2) with respect to Arbitrary Fluorescence Units (AFU);
• FIGS. 12A-12C are schematic views and associated SEM micrographs which compare the surface capture of pathogens when using surface peptide ligands versus surface antibody ligands;
• FIG. 13 is a bar graph illustrating the target capture density ( ⁇ 2 ) of Enterobacteria phage MS2 when using surface peptide ligands, Peptide 1 (SEQ ID NO: 1), Peptide 2 (SEQ ID NO: 2) versus surface antibody ligands;
• FIG. 14 is a graph plotting the concentration of B. subtilis spores against Absolute Relative Fluorescence for the peptide ligand of SEQ ID NO. 3;
• FIG. 15 are bar graph plotting the percent enhancement for B. subtilis and B.cereus spores when binding with free peptide ligands SEQ ID NO 3 and SEQ ID NO 3, respectively;
• FIG. 16 are bar graphs depicting how peptide ligand BC-3 (SEQ ID NO. 5) binds preferentially to B. cereus over E. coli regardless of peptide ligand BC-3 concentration;
• FIG. 17A-17D are micrographs (with and without fluorescence) which
• FIGS. 17A-17B comparatively depicts the binding of the peptide ligand to B. subtilis spores (FIGS. 17A-17B) versus binding with E.coli (FIGS. 17C-17D);
• FIG. 18 is a graph of wavelength (nm) with respect to Fluorescence Intensity (AFU) resulting from detection of E.coli strain ORN 178 at different concentrations using Ru(bipy)- labeled glycan (Compound MD modified with lipoic acid);
• FIG. 19 is a graph of fluorescence intensities at different concentrations of E.coli strain ORN 178 using Ru(bipy)-labeled glycan (Compound MD modified with lipoic acid).
• FIG. 20 is a bar graph illustrating the difference of intensities when E.coli is detected in different sample media using Ru(bipy)-labeled glycan (Compound MD modified with lipoic acid).
• pathogen refers to a microorganism which can cause disease in its host.
• pathogens suitable for detection in accordance with the present disclosure include bacteria, viruses, fungi, prions, and combinations thereof.
• peptide and “peptides” refer to short polymer chains having equal to or less than about 50 amino acids.
• glycan or “glycans” refer to polysaccharides and/or oligosaccharides.
• Embodiments of the present disclosure relate to pathogen sensors, systems for pathogen detection, peptide ligands, and methods for detecting pathogens. Reference will now be made in detail to embodiments of pathogen sensors with reference to FIG. 1.
• a pathogen sensor for detecting the presence of a target pathogen in a sample.
• the pathogen sensor 1 includes a substrate 10, a plurality of immobilized capture ligands 32, 34, and a plurality of free labeling ligands 42, 44 including fluorophores 52, 54.
• the target pathogen 62, 64 includes bacteria, viruses, fungus, and combinations thereof.
• the size of the target pathogen 62, 64 is from about 10s nm to about 100 ⁇ .
• target pathogens 62, 64 for detection with the pathogen sensor 1 include Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Francisella philomiragia, Vibrio cholerae, Bacillus coagulans, Bacillus anthracis, Escherichia coli, Pseudomonas syringae, Enterobacteria phage MS2, Influenza A Virus, Influenza B Virus, Cladosporium sphaerospermum, Sacchromyces cerevisiae, and combinations thereof.
• the target pathogen 62, 64 is bacteria.
• suitable bacteria for detection with the pathogen sensor 1 include but should not be limited to Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Francisella philomiragia, Vibrio cholerae, Bacillus coagulans, Bacillus anthracis, Escherichia coli, Pseudomonas syringae, and combinations thereof. More particularly, with regard to Bacillus subtilis, Bacillus subtilis (vegetative) and spores of Bacillus subtilis represent suitable examples of target pathogens 62, 64 for detection with the pathogen sensor 1. Additionally, examples of suitable bacterial strains for detection with the pathogen sensor 1 are provided in Table I below.
• the target pathogen 62, 64 is a virus.
• suitable viruses for detection with the pathogen sensor 1 include but should not be limited to Enterobacteria phage MS2, Influenza A Virus, Influenza B Virus, and combinations thereof. Additionally, examples of suitable viral strains for detection with the pathogen sensor 1 are provided in Table II below. Table II
• the target pathogen 62, 64 is a fungus.
• suitable fungi for detection with the pathogen sensor 1 include but should not be limited to Cladosporium sphaerospermum, Sacchromyces cerevisiae, and combinations thereof. Additionally, examples of suitable fungal strains for detection with the pathogen sensor 1 are provided in Table III below.
• the sample may include a substance and/or a mixture of substances of interest to be analyzed for the presence of a target pathogen.
• the sample may be provided in liquid, solid, or gaseous form.
• the sample is provided in liquid form.
• the sample is complex. Examples of complex samples suitable for use with the pathogen sensor 1 include bulk water, air, apple juice, grape juice, media, sputum, and combinations thereof.
• the pathogen sensor 1 includes a substrate 10.
• the substrate 10 of the pathogen sensor 1 may be optically clear.
• Suitable examples of optically clear substrates 10 for use with the pathogen sensor 1 include but should not be limited to glass, silica, quartz, plastics, and combinations thereof.
• the optically clear substrate 10 is glass.
• the optically clear substrate 10 is a plastic.
• suitable plastics include but should not be limited to, poly(methyl methacrylate), polycarbonate, polystyrene, and combinations thereof.
• the optically clear substrate 10 includes silver particles deposited on a surface 12 of the substrate 10.
• the silver particles 14 may be deposited directly on the surface 12 of the substrate 10 such that the silver particles 14 directly contact at least a portion of the surface 12.
• the silver particles 14 are deposited on the surface 12 of the optically clear substrate 10 such that they are immobilized thereon.
• deposition of the silver particles 14 on the surface 12 of the substrate 10 forms a silver island film 20.
• deposition of the silver particles 14 on the surface 12 of the substrate 10 forms a silver colloid film 20. Synthesis of silver island films 20 and silver colloid films 20 is disclosed in /. Fluoresc.
• deposition of the silver particles 14 on the surface 12 of the substrate 10 forms a silver nanostructured surface.
• silver particles 12 are utilized in the present embodiment, it is contemplated that other metals suitable for metal enhanced fluorescence may also be utilized.
• gold particles may be deposited on the surface 12 of the optically clear substrate 10 in place of and/or in addition to the silver particles 14.
• the surface 12 may be modified with silanes.
• the surface 12 may be modified with a silane such as aminopropylsilane.
• the silane may take the form of a silane coating on the substrate.
• the substrate 10 is glass modified with aminopropylsilane.
• the substrate 10 is a plastic modified with a silane coating.
• modification with a silane introduces amine terminal groups to the surface 12 of the substrate 10, promoting and/or improving deposition of silver particles 14 thereon. Modification of substrates is disclosed in /. Mater. Chem. , 16: 2846-52 (2006), the contents of which are incorporated by reference herein.
• the pathogen sensor 1 includes immobilized capture ligands 32, 34 attached to the silver particles 14. Suitable examples of the immobilized capture ligands 32, 34 include peptides, glycans, antibodies, antigens, and combinations thereof. In one particular embodiment, the immobilized capture ligands 32, 34 are peptides. In another particular embodiment, the immobilized capture ligands 32, 34 are glycans. Regardless of the compositions of the immobilized capture ligands 32, 34, the pathogen sensor 1 includes at least one immobilized capture ligand 32, 34 configured to bind specifically to the target pathogen 62, 64.
• a pathogen sensor 1 including immobilized capture peptide ligands 32, 34 includes at least one first capture peptide ligand configured to bind specifically to the target pathogen 62, 64.
• a pathogen sensor 1 including immobilized capture glycan ligands 32, 34 includes at least one first capture glycan ligand configured to bind specifically to the target pathogen 62, 64.
• the pathogen sensor 1 includes a plurality of immobilized capture peptide ligands 32, 34.
• Each of the plurality of immobilized capture peptide ligands 32, 34 may have identical amino acid sequences.
• each of the plurality of immobilized capture peptide ligands 32, 34 may have different amino acid sequences.
• the immobilized capture peptide ligands 32, 34 may have from about 5 to about 50 amino acids.
• the immobilized capture peptide ligands 32, 34 have from about 40 to about 50, or from about 20 to about 40, or from about 10 to about 40, or from about 10 to about 20, or about 15 amino acids.
• each of the immobilized capture peptide ligands 32, 34 have an isoelectric point (i.e., pi) of from about 5 to about 10, or from about 6 to about 8, or about 7.
• the immobilized capture peptide ligands 32, 34 include at least one aromatic amino acid (e.g., Phe, Trp, His, and Tyr).
• the immobilized capture peptide ligands 32, 34 include from about 1 to about 5, or from about 2 to about 4, or about 3 aromatic amino acids.
• Each of the plurality of immobilized capture peptide ligands 32, 34 may be respectively configured to bind specifically to Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Francisella philomiragia, Vibrio cholerae, Bacillus coagulans, Bacillus anthracis, Escherichia coli, Pseudomonas syringae, Enterobacteria phage MS2, Influenza A Virus, Influenza B Virus, Cladosporium sphaerospermum, or Sacchromyces cerevisiae.
• capture peptide ligands 32, 34 configured to bind specifically to Enterobacteria phage MS2, Bacillus subtilis, Bacillus cereus, and/or Bacillus thuringiensis are set forth in Table IV below.
• Such capture peptide ligands 32, 34 are referred to as Peptide Ligand No. 1 (SEQ ID NO: 1), Peptide Ligand No. 2 (SEQ ID NO: 2), Peptide Ligand No. 3 (SEQ ID NO: 3), Peptide Ligand No. 4 (SEQ ID NO: 4), and Peptide Ligand No. 5 (SEQ ID NO: 5).
• capture peptide ligands 32, 34 which specifically bind to Enter obacteria phage MS2, Bacillus subtilis, and Bacillus cereus
• additional capture peptide ligands 32, 34 may also be designed and synthesized.
• capture peptide ligands 32, 34 may be selected from combinatorial libraries that contain more than a billion permutation of amino acid sequences and/or structures. Specific binding of such amino acid sequences and/or structures may be evaluated with washing steps which increase in strength (e.g., ionic strength and/or detergent concentration).
• target pathogens 62, 64 may be independently validated in assays, such as ELISA-assays, and/or with the use of a fluorescence microscope for fluorescently labeled amino acid sequences and/or structures.
• assays such as ELISA-assays
• fluorescence microscope for fluorescently labeled amino acid sequences and/or structures.
• target capture peptide ligands may be tailored to bind more specifically employing techniques including point mutations insertion, directed evolution, and/or changes to the structure.
• the first capture peptide ligand may be Peptide Ligand No. 1 (SEQ ID NO: 1), Peptide Ligand No. 2 (SEQ ID NO: 2), Peptide Ligand No. 3 (SEQ ID NO: 3), Peptide Ligand No. 4 (SEQ ID NO: 4), or Peptide Ligand No. 5 (SEQ ID NO: 5).
• the plurality of immobilized capture peptide ligands 32, 34 includes at least one second capture peptide ligand.
• the second capture peptide ligand may be Peptide Ligand No. 1 (SEQ ID NO: 1), Peptide Ligand No. 2 (SEQ ID NO: 2), Peptide Ligand No. 3 (SEQ ID NO: 3), Peptide Ligand No. 4 (SEQ ID NO: 4), or Peptide Ligand No. 5 (SEQ ID NO: 5).
• Attachment of the capture peptide ligands 32, 34 to the silver particles 14 may be accomplished by adding a cysteine amino acid residue to the C-terminus thereof. The C- terminus of each of the capture peptide ligands 32, 34 may then be conjugated to the silver particles 14 through the thiol moiety of the cysteine residue.
• the pathogen sensor 1 includes a plurality of immobilized capture glycan ligands 32, 34.
• Each of the plurality of immobilized capture glycan ligands 32, 34 may have the same polysaccharides and/or oligosaccharides.
• each of the plurality of immobilized capture glycan ligands 32, 34 may have different polysaccharides and/or oligosaccharides.
• each of the plurality of immobilized capture glycan ligands 32, 34 is respectively configured to bind specifically to Escherichia coli, Influenza A Virus, Influenza B Virus, Shiga Toxin 1, or Shiga Toxin 2. .
• each of the plurality of immobilized capture glycan ligands 32, 34 is respectively configured to bind specifically to Escherichia coli.
• suitable examples of immobilized capture glycan ligands 32, 34 include bi-antennary biotinylated glycoconjugates and tetra-antennary biotinylated glycoconjugates.
• bi-antennary biotinylated glycoconjugates and tetra- antennary biotinylated glycoconjugates have the following structure:
• such bi-antennary biotinylated glycoconjugates and tetra- antennary biotinylated glycoconjugates include a carbohydrate recognition element, a variable oligoethylene glycol spacer, and a biotinylated scaffold.
• the biotin molecule included within the biotinylated scaffold is replaced with lipoic acid. Lipoic acid provides a thiol moiety such that the immobilized capture glycan ligands 32, 34 may be attached to the silver particles 14.
• the oligoethylene glycol spacer may function to reduce nonspecific binding, to impart a degree of flexibility on the carbohydrate recognition element, and/or to connect and separate the thiol moiety within the lipoic acid of the biotinylated scaffold from the carbohydrate recognition element.
• the bi-antennary biotinylated glycoconjugates are bi- antennary biotinylated a-mannoside.
• the tetra-antennary biotinylated glycoconjugates are tetra-antennary biotinylated a-mannosides. Suitable examples of such bi-antennary and tetra-antennary biotinylated a-mannosides are respectively set forth in Table V below. Such compounds are respectively referred to as MD and MT.
• the first capture glycan ligand may be MD or MT, as set forth in Table V.
• the plurality of immobilized capture glycan ligands 32, 34 further includes at least one second capture glycan ligand.
• the second capture glycan ligand may be MD or MT, as set forth in
• each of the plurality of immobilized capture glycan ligands 32, 34 is respectively configured to bind specifically to Shiga Toxin 1 or Shiga Toxin 2 to indirectly detect the presence of Escherichia coli.
• the design and synthesis of suitable glycan ligands 32, 34 respectively configured to bind specifically to Shiga Toxin 1 or Shiga Toxin 2 is described in Angew. Chem. Int. Ed. 47: 1265-1268 (2008), the contents of which are incorporated by reference herein.
• the pathogen sensor 1 includes free labeling ligands 42, 44 including fluorophores 52, 54. Suitable examples of the free labeling ligands 42, 44 include peptides, glycans, antibodies, antigens, and combinations thereof. In one particular embodiment, the free labeling ligands 42, 44 are peptides. In another particular embodiment, the free labeling ligands 42, 44 are glycans. Regardless of the compositions of the free labeling ligands 42, 44, the pathogen sensor 1 includes at least one free labeling ligand 42, 44 configured to bind specifically to the target pathogen 62, 64.
• a pathogen sensor 1 including free labeling peptide ligands 42, 44 includes at least one first labeling peptide ligand configured to bind specifically to the target pathogen 62, 64.
• a pathogen sensor 1 including free labeling glycan ligands 42, 44 includes at least one first labeling glycan ligand configured to bind specifically to the target pathogen 62, 64.
• the at least one first capture peptide ligand 32, 34 has a different amino acid sequence from the at least one first labeling peptide ligand 42, 44.
• the at least one first capture glycan ligand 32, 34 may also have a different amino acid sequence from the at least one first labeling glycan ligand 42, 44.
• the pathogen sensor includes a second capture ligand 32, 34 and a second labeling peptide ligand 42, 44.
• the pathogen sensor 1 includes a plurality of free labeling peptide ligands 42, 44.
• Each of the plurality of free labeling peptide ligands 42, 44 may have identical amino acid sequences.
• each of the plurality of free labeling peptide ligands 42, 44 may have different amino acid sequences.
• the free labeling peptide ligands 42, 44 may have from about 5 to about 50, or from about 40 to about 50, or from about 20 to about 40, or from about 10 to about 40, or from about 10 to about 20, or about 15 amino acids.
• each of the free labeling peptide ligands 32, 34 has an isoelectric point (i.e., pi) of from about 5 to about 10, or from about 6 to about 8, or about 7.
• the free labeling peptide ligands 32, 34 include at least one aromatic amino acid (e.g., Phe, Trp, His, and Tyr).
• the free labeling peptide ligands 32, 34 include from about 1 to about 5, or from about 2 to about 4 amino acids, or about 3 aromatic amino acids.
• Each of the plurality of free labeling peptide ligands 42, 44 may be respectively configured to bind specifically to Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Francisella philomiragia, Vibrio cholerae, Bacillus coagulans, Bacillus anthracis, Escherichia coli, Pseudomonas syringae, Enterobacteria phage MS2, Influenza A Virus, Influenza B Virus, Cladosporium sphaerospermum, or Sacchromyces cerevisiae.
• Suitable capture peptide ligands 42, 44 configured to bind specifically to Enterobacteria phage MS2, Bacillus subtilis, Bacillus cereus, and/or Bacillus thuringiensis are set forth in Table IV, as previously described.
• the first labeling peptide ligand may be Peptide Ligand No. 1 (SEQ ID NO: 1), Peptide Ligand No. 2 (SEQ ID NO: 2), Peptide Ligand No. 3 (SEQ ID NO: 3), Peptide Ligand No. 4 (SEQ ID NO: 4), or Peptide Ligand No. 5 (SEQ ID NO: 5).
• the plurality of free labeling peptide ligands 42, 44 may further include at least one second labeling peptide ligand.
• the second labeling peptide ligand may be Peptide Ligand No. 1 (SEQ ID NO: 1), Peptide Ligand No. 2 (SEQ ID NO: 2), Peptide Ligand No. 3 (SEQ ID NO: 3), Peptide Ligand No. 4 (SEQ ID NO: 4), or Peptide Ligand No. 5 (SEQ ID NO: 5).
• the pathogen sensor 1 includes a plurality of free labeling glycan ligands 42, 44.
• Each of the plurality of free labeling glycan ligands 42, 44 may have the same polysaccharides and/or oligosaccharides.
• each of the plurality of free labeling glycan ligands 42, 44 may have different polysaccharides and/or oligosaccharides.
• Each of the plurality of free labeling glycan ligands 42, 44 may be respectively configured to bind specifically to Escherichia coli, Influenza A Virus, or Influenza B Virus.
• each of the plurality of free labeling glycan ligands 42, 44 is respectively configured to bind specifically to Escherichia coli.
• suitable examples of free labeling glycan ligands 42, 44 include bi-antennary biotinylated glycoconjugates and tetra-antennary biotinylated glycoconjugates.
• bi-antennary biotinylated glycoconjugates and tetra-antennary biotinylated glycoconjugates have the structure disclosed in Formula (I) as previously described.
• Such glycoconjugates may also have the thiol moiety as previously described.
• the bi-antennary biotinylated glycoconjugates are bi- antennary biotinylated a-mannosides.
• the tetra-antennary biotinylated glycoconjugates are tetra-antennary biotinylated a-mannosides.
• Suitable examples of such bi-antennary and tetra-antennary biotinylated ⁇ -mannosides include MD and MT, as set forth in Table V.
• the first labeling glycan ligand may be MD or MT, as set forth in Table V.
• the plurality of free labeling glycan ligands 42, 44 further includes at least one second labeling glycan ligand.
• the second labeling glycan ligand may be MD or MT, as set forth in Table V.
• each of the plurality of free labeling glycan ligands 42, 44 is respectively configured to bind specifically to Shiga Toxin 1 or Shiga Toxin 2 to indirectly detect the presence of Escherichia coli.
• the design and synthesis of suitable glycan ligands 32, 34 respectively configured to bind specifically to Shiga Toxin 1 or Shiga Toxin 2 is described in Angew. Chem. Int. Ed. 47: 1265-1268 (2008), the contents of which are incorporated by reference herein.
• the Shiga Toxins can be captured in the same way as the pathogens.
• the E. coli does not need to be captured first to detect Shiga toxin.
• the Shiga toxin would be free in the sample and would be detected/captured and labeled directly.
• each of the plurality of free labeling ligands 42, 44 includes a fluorophore 52, 54.
• a portion of the plurality of free labeling ligands 42, 44 includes a fluorophore 52, 54.
• the at least one first labeling ligands 42, 44 may include a fluorophore 52, 54.
• Suitable fluorophores for use with the pathogen sensor 1 are set forth in U.S. Publication No. 2006/0147927, the contents of which are incorporated by reference herein. With reference to FIG. 2, in one particular embodiment, suitable fluorophores for use with the pathogen sensor 1 are those exhibiting low quantum yield (Qo).
• suitable fluorophores may include those which exhibit a quantum yield of less than about 0.5, less than about 0.3, or less than about 0.2, or less than about 0.1. In one particular embodiment, suitable fluorophores include those which exhibit a quantum yield of less than about 0.1.
• fluorophores 52, 54 include Rhodamine B (i.e., RhB), Rose Bengal, Ru(bipy)3, 5-Carboxy-tetramethylrhodamine N-succinimidyl ester (hereinafter, "TAMRA"), Fuscin, [Ru(phen) 2 dppz] 2+ , Cy3, Cy5, and combinations thereof.
• RhB Rhodamine B
• Rose Bengal Ru(bipy)3
• Ru(bipy)3 5-Carboxy-tetramethylrhodamine N-succinimidyl ester
• Fuscin [Ru(phen) 2 dppz] 2+ , Cy3, Cy5, and combinations thereof.
• Ru(bipy)3 suitable examples include Bis(2,2'-bipyridine)-(5-isothiocyanato- phenanthroline) ruthenium bis (hexafluorophosphate), Bis(2,2'-bipyridine)-4,4'- dicarboxybipyridine-ruthenium di(N-succinimidyl ester) bis(hexafluorophosphate), Tris(bipyridine)ruthenium(II) chloride (hereinafter, "RuByp"), and combinations thereof.
• fluorophores 52, 54 include Rhodamine B, Ru(bipy)3, or TAMRA.
• the fluorophores 52, 54 are conjugated to the N-terminus or C-terminus of such free labeling peptide ligands 42, 44.
• metal enhanced fluorescence occurs when a metal, e.g. silver particles 14, is within sufficient proximity of fluorophores 52, 54 of the free labeling capture ligands resulting in a quantum effect.
• a metal e.g. silver particles 14
• fluorophores 52, 54 of the free labeling capture ligands resulting in a quantum effect.
• the at least one first capture ligand binds 32, 34 specifically to a first region 72, 74, 76 of the target pathogen 62, 64.
• the at least one first labeling ligand 42, 44 binds specifically to a second region 82, 84, 86 of the target pathogen 62, 64.
• the target pathogen 62, 64 may be sandwiched in between the first capture ligand 32, 34 and the first labeling ligand 42, 44. Additionally, such specific binding may position the fluorophore 52, 54 at a metal enhancing distance (designated by double arrow D) from the silver particles 14 immobilized on the surface 12 of the substrate 10. Such positioning of the fluorophore 52, 54 provides enhanced emission of the fluorophore 52, 54 upon excitation, which is indicative of the presence of the target pathogen 62, 64.
• the metal enhancing distance D is less than about 50 nm. Alternatively, in another embodiment, the metal enhancing distance D is less than about 10 nm. In another alternative embodiment, the metal enhancing distance D is from about 1 to about 50, or from about 5 to about 30, or from about 5 to about 10, or about 9 nm.
• Excitation of the fluorophore 52, 54 may be accomplished with a light source.
• suitable light sources include but should not be limited to light emitting diodes and lasers.
• Emission of the fluorophores 52, 54 may be determined with a spectrometer, such as described in a later section.
• the fluorescence emission of the fluorophore 52, 54 is enhanced relative to a control.
• the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 75%.
• the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 100%.
• the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 150%.
• the fluorophore 52, 54 of the first labeling peptide ligand 42, 44 is tri(bipyridine)ruthenium(II) chloride and the fluorescence emission of the fluorophore is enhanced by at least about 75%.
• the first capture peptide ligand 32, 34 is Peptide Ligand No. 1 (SEQ ID NO: 1)
• the target pathogen is Enterobacteria phage MS2
• the first labeling peptide ligand 42, 44 is Peptide Ligand No. 2 (SEQ ID NO: 2)
• the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 100%.
• the first capture peptide ligand 32, 34 is Peptide Ligand No. 3 (SEQ ID NO: 3)
• the target pathogen is Bacillus subtilis
• the first labeling peptide ligand 42, 44 is Peptide Ligand No. 3(SEQ ID NO: 3)
• the metal enhancing distance is less than about 50 nm
• the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 75%.
• the first capture peptide ligand 32, 34 is Peptide Ligand No. 4 (SEQ ID NO: 4), the target pathogen is Bacillus cereus, the first labeling peptide ligand 42, 44 is Peptide Ligand No. 5 (SEQ ID NO: 5), the metal enhancing distance is less than about 50 nm, and the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 75%.
• the first capture peptide ligand 32, 34 is Peptide Ligand No.
• the target pathogen is Bacillus cereus
• the first labeling peptide ligand 42, 44 is Peptide Ligand No. 4 (SEQ ID NO: 4)
• the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 75 %.
• the pathogen sensor 1 has a limit of detection of less than about 10 pfu/mL.
• the pathogen sensors 1 may be incorporated into various systems or applications which require pathogen detection.
• these systems for pathogen detection via metal enhanced fluorescence may comprise at least one sensor board 100 as shown in FIG. 3. While the sensor board 100 is depicted schematically in FIG. 3, FIG. 6 is also provided to illustrate the sensor board 100 incorporated into an airborne pathogen detection system. While the sensor board 100 is depicted as a platform in FIG. 3, the sensor board 100 should be construed broadly to encompass various shapes, structures, etc.
• the sensor board 100 comprises at least one sample chamber 150 configured to house the pathogen sensors 1 described above.
• the sample chamber 150 may comprise a disposable sample cartridge, which is removable from the sensor board 100.
• the sample chamber 150 may comprise the optically clear substrate 10, and optionally the immobilized ligands.
• the sensor board 100 also comprises at least one sample reservoir 160 and at least one detection reservoir 161.
• the sample reservoir 160 may store sample material which is to be detected for pathogens in the sample chamber 150. While the sample reservoir 160 is depicted as part of the sensor board 100 in FIG. 3, it is contemplated that the sample reservoir 160 is not part of the sensor board 100. For example, if the system of the present invention is disposed in a body of water, it is contemplated the body of water may essentially act as the sample reservoir which delivers a sample to the sample chamber 150.
• the detection reservoir 161 may store immobilized ligands, free ligands with fluorophore, or both. In some contemplated embodiments, it is possible that these immobilized ligands are already attached to the silver island films, so a separate reservoir is not necessary. While it is shown that the reservoirs 160 and 161 are separate from the sample chamber 150, it is contemplated that the reservoirs may be integrated inside the sample chamber 150.
• the sensor board 100 may comprise at least one pump 130 in fluid communication with the least one sample reservoir 160 and at least one detection reservoir 161, or both.
• the pump 130 may deliver sample or ligand detection agents to the sample chamber 150.
• the pump 130 may comprise a micropump, for example, a peristaltic pump.
• the peristaltic pump may include simple on/off control in order to conserve energy during non-use. Prior to each sampling event, the pump 130 may be turned on long enough to flush and fill the sample chamber 150 with a new sample. The pump 130 will be powered off at all other times.
• the flow system may deliver solid samples, liquid samples, gaseous samples, or combinations thereof for detection by the pathogen sensor.
• the sensor board 100 also comprises at least one light source 170 configured to induce fluorescence inside the sample chamber 150.
• the light source 170 may comprise at least one light emitting diode (LED) module.
• the light source may comprise two LED modules used to illuminate the sample. These two LED modules may be controlled by two separate buffered digital output lines. Consequently, it is possible that both LED modules are on simultaneously, or only one LED module will be on at a time.
• the light source 170 may comprise onboard drivers or units for supplying power to the LED modules, wherein the onboard drivers are coupled to the power source described below. Since these light sources 170 may generate a considerable amount of heat, a temperature sensor may be included to monitor the heat buildup.
• the sensor board 100 may comprise a spectrometer 140 coupled to the sample chamber 150.
• the spectrometer may detect target pathogen within the sample based on the fluorescence emission delivered by the fluorophore of the free ligand as described above.
• the spectrometer 140 works in tandem with the light source 170, the sample chamber 150, and an optical probe (not shown). There may be independent hardware components on the sensor board 100 or integrated into one discrete package.
• Various suitable spectrometers are contemplated.
• the spectrometer 140 may be a CCD spectrometer.
• Commercially suitable spectrometers may include the USB2000+ miniature CCD spectrometer available from Ocean Optics Inc.
• the sensor board 100 also comprises a computer unit 110, and at least one power source 120.
• the power source is a battery.
• the battery may comprise nickel-metal hydride (NiMH) batteries, specifically 12 V NiMH batteries, which supply electrical power to the sensor board 100 components.
• the power source 120 may deliver power to the spectrometer 140, the light source 170, the pump 130, and the computer unit 110. It is contemplated that other sensor board 100 components, such as the sample chamber 150, or additional sensor board 100 components may also derive power from the power source 120.
• the computer unit 110 may encompass various hardware and software components.
• the computer unit may comprise a microprocessor, a display unit, wireless communication radios, circuit boards, user interface, and various other hardware components.
• the computer unit 110 may be programmed to control the other components and
• the computer unit may be equipped to receive commands from a network controller and also to transmit status information to that network controller.
• various communication protocols are considered suitable. For example, ZigBee communication protocol, e.g., ZigBee Pro
• the computer unit 110 may comprise a ZigBee enabled Rabbit BL4S150 distributed by Digi International Inc.
• the computer unit 110 may perform various functions: manage electrical power from the power source 120, schedule sampling detections, energize light sources 170, retrieve data from the spectrometer 140, preprocess the data, and transmit status information.
• the computer unit may directly communicate with the spectrometer 140 through a communication interface, for example, and not by way of limitation, a RS232 interface.
• spectra will be downloaded to the computer unit 110, which will then perform basic preprocessing of the spectra such as windowing, smoothing and baseline correction.
• the computer unit 110 may perform a spectral analysis routine, which will calculate peak heights and compare them with a reference value. The result will then be transmitted to a host through the ZigBee network.
• a portable handheld device such as a cell phone or Smartphone, may communicate with the computer unit 110 to remotely control the sensor board 110 and thereby control pathogen detection.
• the computer unit 110 enables pathogen detection to be performed in real time, i.e., without any delays in analyzing the spectrometer data or delays in delivering to the user.
• sensors may be included, for example, radiation sensors, temperature sensors, global positioning sensors (GPS), or combinations thereof.
• GPS global positioning sensors
• the present system will have the flexibility to detect pathogens while also monitoring other potential health concerns, such as radiation.
• the user may pinpoint the exact location of pathogens, or the exact locations of pathogens if there is a network of sensors.
• the pathogen sensor may be disposed in a buoy system as shown in FIG. 4 for open water or well water monitoring.
• the sensor board 100 may be disposed in the upper section of the buoy, and the flotation system 300 may be disposed in a bottom section of the buoy.
• the flotation system 300 may include sampling components that draw water samples from the well or open water and deliver the water sample to the sensor board 100.
• the system may be used for automatic in-line water monitoring units 500. Specifically, these portable detection units are configured to sample water in water piping to detect for the presence of pathogens.
• the present pathogen detection system may also be utilized in the detection of airborne agents as shown in FIGS. 5 and 6.
• the portable airborne pathogen detection system may utilize an air-to-water mixing impinger 480.
• the air which is being sampled, enters via an intake of the air-to-water mixing impinger 480 and is subsequently mixed with an aqueous solution.
• the intake and exit ports for air will be arranged to create a cyclone within the mixing chamber of the impinger 480.
• the air-liquid cyclone in the funnel will facilitate the transfer of micron sized particles from the air into the liquid.
• the cyclonic force will also push the air-infused liquid to the base for sampling inside the sample chamber 150.
• Various other aerosolization and intake platforms may also be used in airborne detection.
• the dry powder aerosol generation system from Vitrocell Systems may be used to establish the concentration of the various targets required for detection from a dry sample.
• Other intake platforms which may include fans or other impinger units are contemplated herein
• peptide ligands are disclosed.
• the peptide ligand includes Peptide Ligand No. 1 (SEQ ID NO: 1), Peptide Ligand No. 2 (SEQ ID NO: 2), Peptide Ligand No. 3 (SEQ ID NO: 3), Peptide Ligand No. 4 (SEQ ID NO: 4), and Peptide Ligand No. 5 (SEQ ID NO: 5).
• a method for detecting pathogens includes providing a pathogen sensor 1 , providing a sample to the pathogen sensor 1, exciting the fluorophore 52, 54 of the free labeling ligand 42, 44, and determining fluorescence emission of the fluorophore 52, 54.
• the pathogen sensor is as described previously.
• Example 1 Effect of Concentration on Fluorescent Enhancement of TAMRA Labeled Peptide Ligand No. 2 (SEQ ID NO: 2) Bound to Silver Island Film
• Unbound Ru(bipy)3 labeled free Peptide Ligand No. 2 was removed by washing with either water or with buffer. Fluorescence of the Ru(bipy) 3 labeled free Peptide Ligand No. 2 was monitored upon excitation and measured against the negative control (i.e., Enterobacteria phage MS2 at a concentration of 0 pfu/mL).
• Example 3 Fluorescent Enhancement of TAMRA Labeled Free Peptide Ligand No. 2 (SEQ ID NO: 2) v. Ru(bipy) 3 Labeled Free Peptide Ligand No. 2 (SEQ ID NO: 2)
• Example 4 Specificity of Peptide Ligand Nos. 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) for Enterobacteria phage MS2 v. Anti-MS2 IgG Antibodies for Enterobacteria phage MS2
• Capture Peptide Ligand Nos. 1 and 2 were also bound to the gold particles, forming a functionalized surface. Additionally, anti-MS2 IgG antibodies were also bound to the gold particles, forming antibody-functionalized surfaces.
• Peptide Ligand Nos. 1 and 2 effectively capture Enterobacteria phage MS2 on the functionalized surfaces of the silver island film with a higher density than the anti-MS2 IgG antibodies. Such capture was visualized using antibody-labeled microspheres under a scanning electron microscope.
• Capture Peptide Ligand No. 3 (SEQ ID NO: 3) was bound to silver island film. More particularly, capture Peptide Ligand No. 3 was bound to the silver island film via thiol linkers which included at the C-terminus thereof. The capture Peptide Ligand No. 3 was immobilized on the silver island film, forming a functionalized surface. Ru(bipy)3 labels were added to the N-termini of free Peptide Ligand No. 3.
• Capture Peptide Ligand Nos. 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) were respectively bound to silver island films. More particularly, capture Peptide Ligand Nos. 3 and 4 were respectively bound to the silver island films via thiol linkers included at the C-terminus thereof. The capture Peptide Ligand Nos. 3 and 4 were immobilized on the silver island films, forming a functionalized surface. TAMRA labels were added to the N-termini of respective free Peptide Ligand Nos. 3 and 5 (SEQ ID NO: 5).
• Samples containing Bacillus subtilis (vegetative) and Bacillus cereus (vegetative) were respectively applied to the functionalized surfaces for from about 1 to about 5 minutes. Unbound sample was removed by washing with either water or with buffer (e.g., phosphate buffered saline at pH 7.4).
• the TAMRA labeled free Peptide Ligand Nos. 3 and 5 were applied to the functionalized surfaces and allowed to incubate for from about 30 seconds to about 2 minutes. Unbound TAMRA labeled Peptide Ligand Nos. 3 and 5 were removed by washing with either water or with buffer. Fluorescence of TAMRA labeled Peptide Ligand Nos. 3 and 5 was monitored upon excitation and measured against a negative control.
• Example 7 Preferential Binding to Bacillus cereus over Escherichia coli
• Capture Peptide Ligand No. 5 (SEQ ID NO: 5) was bound to silver island films. More particularly, capture Peptide Ligand No. 5 was bound to the silver island films via thiol linkers included at the C-terminus thereof. The capture Peptide Ligand No. 5 was immobilized on the silver island films, forming a functionalized surface. Fluorophore labels were added to the N-termini of respective free Peptide Ligand No. 5 (SEQ ID NO: 5).
• Samples containing Bacillus cereus and Escherichia coli were respectively applied to the functionalized surfaces for from about 1 to about 5 minutes. Unbound sample was removed by washing with either water or with buffer (e.g., phosphate buffered saline at pH 7.4). The labeled free Peptide Ligand No. 5 was applied to the functionalized surfaces and allowed to incubate for from about 30 seconds to about 2 minutes. Unbound labeled Peptide Ligand No. 5 was removed by washing with either water or with buffer. Fluorescence of labeled Peptide Ligand No. 5 was monitored upon excitation and measured against a negative control.
• buffer e.g., phosphate buffered saline at pH 7.4
• Peptide Ligand No. 5 preferentially captured Bacillus cereus on the functionalized surfaces of the silver island film as compared to Escherichia coli regardless of the concentration of Peptide Ligand No. 5.
• Capture Peptide Ligand No. 3 was bound to silver island films. More particularly, capture Peptide Ligand No. 3 was bound to the silver island films via thiol linkers included at the C-terminus thereof. The capture Peptide Ligand No. 3 was immobilized on the silver island films, forming a functionalized surface. Fluorophore labels were added to the N-termini of respective free Peptide Ligand No. 3 (SEQ ID NO: 3).
• Samples containing Bacillus subtilis and Escherichia coli were respectively applied to the functionalized surfaces for from about 1 to about 5 minutes. Unbound sample was removed by washing with either water or with buffer (e.g., phosphate buffered saline at pH 7.4). The labeled free Peptide Ligand No. 3 was applied to the functionalized surfaces and allowed to incubate for from about 30 seconds to about 2 minutes. Unbound labeled Peptide Ligand No. 3 was removed by washing with either water or with buffer. Fluorescence of labeled Peptide Ligand No. 3 was monitored upon excitation and measured against a negative control.
• buffer e.g., phosphate buffered saline at pH 7.4
• Example 9 Detection of Escherichia coli ORN178 with Glycan MD modified with lipoic acid
• Experimental Protocol Capture glycan MD modified with lipoic acid was bound to silver island films. More particularly, capture glycan MD modified with lipoic acid was bound to the silver island films. The capture glycan MD modified with lipoic acid was immobilized on the silver island films, forming a functionalized surface. Ru(bipy)3 labels were added to free glycan MD modified with lipoic acid.
• fluorescence enhancement of Ru(bipy)3 labeled free MD modified with lipoic acid is dependent upon the concentration of Escherichia coli. More particularly, fluorescence enhancement of labeled free glycan MD modified with lipoic acid increases with increasing concentration of Escherichia coli.
• Capture glycan MD modified with lipoic acid was bound to silver island films. More particularly, capture glycan MD modified with lipoic acid was bound to the silver island films. The capture glycan MD modified with lipoic acid was immobilized on the silver island films, forming a functionalized surface. Ru(bipy)3 labels were added to free glycan MD modified with lipoic acid.
• Complex samples e.g., apple juice, grape juice, and media
• Unbound sample was removed by washing with either water or with buffer (e.g., phosphate buffered saline at pH 7.4).
• the Ru(bipy)3 labeled free glycan MD modified with lipoic acid was applied to the functionalized surfaces and allowed to incubate for from about 30 seconds to about 2 minutes. Unbound Ru(bipy)3 labeled glycan MD modified with lipoic acid was removed by washing with either water or with buffer.
• Negative controls included a sample of phosphate buffered saline containing Escherichia coli ORN178.
• the present pathogen sensor was used to note specific Bacillus endospores in a sample based on fluorescence emission and lifetime decay analysis. Specifically, this differentiation was noted between the following Bacillus endospores, B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain.
• Bacillus endospores exhibit a dramatic blue shift of 130 nm in excitation and a smaller shift of 50 nm in emission when compared to ancillary endospore and non-endospore forming bacterial cells.
• it often proves difficult to highlight Bacillus endospore species, especially in complex biological fluids and suspensions.
• specific species such as B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain may be pinpointed by analyzing lifetime decay data.
• each of these species showed three distinct lifetimes within the following ranges, 0.2-1.3 ns; 2.5-7.0 ns; 7.5-15.0 ns, when laser induced at 307 nm.
• these four endospore species could be isolated based on lifetime decay. For more details on lifetime decay analysis, Smith et al; (2010) TCSPC Lifetime Characterization of Bacillus endospore Species. IEEE. SPIE DSS. Proc.of SPIE Vol. 7687 76870B-1 is incorporated by reference herein.

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