EP2780709A2 - Pathogen detection using metal enhanced fluorescence - Google Patents

Pathogen detection using metal enhanced fluorescence

Info

Publication number
EP2780709A2
EP2780709A2 EP12852456.8A EP12852456A EP2780709A2 EP 2780709 A2 EP2780709 A2 EP 2780709A2 EP 12852456 A EP12852456 A EP 12852456A EP 2780709 A2 EP2780709 A2 EP 2780709A2
Authority
EP
European Patent Office
Prior art keywords
pathogen
seq
peptide ligand
labeling
sensor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12852456.8A
Other languages
German (de)
French (fr)
Inventor
Melanie TOMCZAK
David C. LIPTAK
Melinda A. OSTENDORF
Clint B. SMITH
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
UES Inc
Original Assignee
UES Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by UES Inc filed Critical UES Inc
Publication of EP2780709A2 publication Critical patent/EP2780709A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553Metal or metal coated
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Inorganic Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

Pathogen sensors, systems for pathogen detection, peptide ligands, and methods for detecting pathogens are disclosed. The pathogen sensors include silver particles attached to optically clear substrates, immobilized capture peptide or glycan ligands attached to the silver particles, and free labeling peptide or glycan ligands having fluorophores. Binding of the immobilized capture ligands and the free labeling ligands to the pathogen positions the fluorophore at a distance from the silver particles sufficient to initiate metal enhanced fluorescence.

Description

PATHOGEN DETECTION USING METAL ENHANCED FLUORESCENCE
[0001] This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application Serial No. 61/560,501, filed November 16, 2011, the contents of which are hereby incorporated by reference in their entirety.
Statement of Government Interests
[0002] The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Contract Nos. W912HZ-09-C-0046 and W9132V-10-C-0001 awarded by the United States Army.
Technical Field
[0003] 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.
Background
[0004] The need for rapid, selective, and highly sensitive biological sensors to detect biological warfare agents (e.g., pathogens) in complex samples is ever-present in both the military and private industries. Current sensors for such agents are complex, requiring extensive blocking and/or washing of samples in order to positively identify target pathogens. More particularly, current sensors may involve the use of capture ligands, (e.g., nucleotide probes and/or antibodies), which bind to a target pathogen in a sample upon contact. However, the practical application of such capture ligands in the context of battlefield and/or real-world conditions is limited. For example, with regard to nucleotide probes, personnel must pre-treat samples under lysis conditions in order to provide access to complementary nucleotides within the target pathogen. Additionally, with regard to antibodies, such proteins are not inherently stable in battlefield and/or real- world conditions. Accordingly, additional embodiments for pathogen sensors are desired. Summary
[0005] In one embodiment, a pathogen sensor for detecting the presence of a target pathogen in a sample is disclosed. 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.
[0006] In another embodiment, a pathogen sensor for detecting the presence of pathogenic Escherichia coli in a sample is disclosed. 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:
or.
. When the at least one first capture glycan ligand binds specifically to a first region of the pathogenic Escherichia coli, and the at least one first labeling glycan ligand binds specifically to a second region of the same pathogenic Escherichia coli: 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. [0007] Other embodiments are directed to systems incorporating the pathogen sensors.
[0008] In yet another embodiment, 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.
[0009] Additional features and advantages of the embodiments described herein will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments described herein, including the detailed description which follows, the claims, as well as the appended drawings.
[0010] It is to be understood that both the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated into and constitute a part of this specification. The drawings illustrate the various embodiments described herein, and together with the description serve to explain the principles and operations of the claimed subject matter.
Brief Description of the Drawings
[0011] FIG. 1 is a schematic view of a pathogen sensor according to one or more embodiments of the present invention;
[0012] FIG. 2 is a graph of known quantum yields (Qo) of Rhodamine B, ErB, [Ru(bpy)3)]2+, basic fucsin, and [Ru(phen)2dppz]2+ in solution with respect to intensity ratio
[0013] FIG. 3 is a schematic view of a sensor board according to one or more embodiments of the present invention;
[0014] 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; [0015] 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;
[0016] 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;
[0017] 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;
[0018] FIG. 8 is a bar graph of TAMRA-labeled peptide ^g/mL) immobilized on silver nanoparticles with respect to fluorescence enhancement (% Enhancement) wherein n=3;
[0019] 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;
[0020] 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;
[0021] 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);
[0022] 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;
[0023] FIG. 13 is a bar graph illustrating the target capture density (μιη 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; [0024] 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;
[0025] 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;
[0026] 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;
[0027] FIG. 17A-17D are micrographs (with and without fluorescence) which
comparatively depicts the binding of the peptide ligand to B. subtilis spores (FIGS. 17A-17B) versus binding with E.coli (FIGS. 17C-17D);
[0028] 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);
[0029] 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); and
[0030] 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).
[0031] Skilled artisans appreciate that elements in the figures are illustrated for simplicity and clarity and are not necessarily drawn to scale. For example, the dimensions of some of the elements in the figures may be exaggerated relative to other elements, as well as conventional parts removed, to help to improve understanding of the various embodiments of the present invention. Detailed Description
[0032] The following terms are used in the present application:
[0033] As used herein, the term "pathogen" refers to a microorganism which can cause disease in its host. Examples of pathogens suitable for detection in accordance with the present disclosure include bacteria, viruses, fungi, prions, and combinations thereof.
[0034] As used herein, the terms "peptide" and "peptides" refer to short polymer chains having equal to or less than about 50 amino acids.
[0035] As used herein, the terms "glycan" or "glycans" refer to polysaccharides and/or oligosaccharides.
[0036] 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.
Pathogen Sensors
[0037] In one embodiment, a pathogen sensor for detecting the presence of a target pathogen in a sample is disclosed. Referencing FIG. 1, 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. In one embodiment, the target pathogen 62, 64 includes bacteria, viruses, fungus, and combinations thereof. In one particular embodiment, the size of the target pathogen 62, 64 is from about 10s nm to about 100 μιη. Examples of suitable 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.
[0038] In one particular embodiment, the target pathogen 62, 64 is bacteria. Examples of 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.
Table I
1 I'allio.ncn Sl raiiK M
Bacillus subtilis (vegetative) 168, 6051
Spores of Bacillus subtilis 168, 6051
Bacillus cereus 569
Bacillus thuringiensis CGB 14-4
Francisella philomiragia ATCC Cat. No. 25015
Vibrio cholerae M045
Bacillus coagulans Hammer
Bacillus anthracis Sterne ApagA
Escherichia coli ORN 178, ORN 208, MDL 3562
(0157:H7), 15597, ATCC Cat No.
25922
[0039] In another particular embodiment, the target pathogen 62, 64 is a virus. Examples of 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
[0040] In yet another particular embodiment, the target pathogen 62, 64 is a fungus. Examples of 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.
Table III
[0041] With regard to the sample, 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. In one particular embodiment, the sample is provided in liquid form. In another particular embodiment, 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. Substrate
[0042] In one embodiment, 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. In one particular embodiment, the optically clear substrate 10 is glass. In another particular embodiment, the optically clear substrate 10 is a plastic. With specific regard to plastics, examples of suitable plastics include but should not be limited to, poly(methyl methacrylate), polycarbonate, polystyrene, and combinations thereof.
[0043] In one embodiment, 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. In one embodiment, the silver particles 14 are deposited on the surface 12 of the optically clear substrate 10 such that they are immobilized thereon. In one particular embodiment, deposition of the silver particles 14 on the surface 12 of the substrate 10 forms a silver island film 20. Alternatively, in another particular embodiment, 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. 15(5): 643-54 (2005), the contents of which are incorporated by reference herein. Alternatively, in still another particular embodiment, deposition of the silver particles 14 on the surface 12 of the substrate 10 forms a silver nanostructured surface. While silver particles 12 are utilized in the present embodiment, it is contemplated that other metals suitable for metal enhanced fluorescence may also be utilized. For example, in one particular embodiment, 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.
[0044] With regard to the surface 12 of the substrate 10, the surface 12 may be modified with silanes. For example, 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. In one particular embodiment, the substrate 10 is glass modified with aminopropylsilane. In another particular embodiment, the substrate 10 is a plastic modified with a silane coating. Without being bound by the theory, it is believed that 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.
2. Immobilized Capture Ligands
[0045] In one embodiment, 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. For example, 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. Similarly, as another example, 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.
a, Immobilized Capture Peptide Ligands
[0046] In one particular embodiment, 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. Alternatively, each of the plurality of immobilized capture peptide ligands 32, 34 may have different amino acid sequences. Generally, the immobilized capture peptide ligands 32, 34 may have from about 5 to about 50 amino acids. Alternatively, in another embodiment, 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. Additionally, in another embodiment, 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. Moreover, in another embodiment, the immobilized capture peptide ligands 32, 34 include at least one aromatic amino acid (e.g., Phe, Trp, His, and Tyr). For example, in one particular embodiment, 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.
[0047] 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. Examples of suitable 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).
Table IV
SEQ ID NO: 5 HHQHLSHISRSN Bacillus cereus 5 BC-3
[0048] While the present embodiment is directed specific capture peptide ligands 32, 34 which specifically bind to Enter obacteria phage MS2, Bacillus subtilis, and Bacillus cereus, it is contemplated that additional capture peptide ligands 32, 34 may also be designed and synthesized. Generally, such 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). Specific binding of such amino acid sequences and/or structures to 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. Upon identification of potential target capture peptide ligands, 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.
[0049] With reference to the at least one first capture peptide ligand, 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).
[0050] In one particular embodiment, 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).
[0051] 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. b. Immobilized Capture Glycan Ligands
[0052] In another particular embodiment, 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. Alternatively, each of the plurality of immobilized capture glycan ligands 32, 34 may have different polysaccharides and/or oligosaccharides. In one embodiment, 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. .
[0053] In one particular embodiment, each of the plurality of immobilized capture glycan ligands 32, 34 is respectively configured to bind specifically to Escherichia coli. In this particular embodiment, suitable examples of immobilized capture glycan ligands 32, 34 include bi-antennary biotinylated glycoconjugates and tetra-antennary biotinylated glycoconjugates. Generally, such bi-antennary biotinylated glycoconjugates and tetra- antennary biotinylated glycoconjugates have the following structure:
;;.f: mas «-S-S¾fes*J*
TO
Formula (I)
[0054] As shown in Formula (I), such bi-antennary biotinylated glycoconjugates and tetra- antennary biotinylated glycoconjugates include a carbohydrate recognition element, a variable oligoethylene glycol spacer, and a biotinylated scaffold. In one embodiment, 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.
[0055] In one particular embodiment, the bi-antennary biotinylated glycoconjugates are bi- antennary biotinylated a-mannoside. In another particular embodiment, 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.
Table V ίΙΙΙΚ' (, liissiiK iMum
Bi-antennary
MD biotinylated a- mannoside
Tetra-antennary
MT biotinylated a- mannoside
[0056] The design and synthesis of Formula I and the compounds MD and MT disclosed in Table V is described in Chembiochem 9(15):2433-42 (2008), the contents of which are incorporated by reference herein.
[0057] With regard to the at least one first capture glycan ligand, the first capture glycan ligand may be MD or MT, as set forth in Table V. In one particular embodiment, 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
Table V.
[0058] Alternatively, in another embodiment, 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.
3. Free Labeling Ligands
[0059] In one embodiment, 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. For example, 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. Similarly, as another example, 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.
[0060] In one embodiment, 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. Similarly, 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. Such principle may also be extended to embodiments wherein the pathogen sensor includes a second capture ligand 32, 34 and a second labeling peptide ligand 42, 44.
a, Free Labeling Peptide Ligands
[0061] In one particular embodiment, 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. Alternatively, each of the plurality of free labeling peptide ligands 42, 44 may have different amino acid sequences. Generally, as previously discussed with regard to the immobilized capture peptide ligands 32, 34, 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. Additionally, in another embodiment, 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. Moreover, in another embodiment, the free labeling peptide ligands 32, 34 include at least one aromatic amino acid (e.g., Phe, Trp, His, and Tyr). For example, in one particular embodiment, 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.
[0062] 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. Examples of 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.
[0063] With reference to the at least one first labeling peptide ligand, 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).
[0064] In one particular embodiment, 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).
b. Free Labeling Glycan Ligands
[0065] In another particular embodiment, 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. Alternatively, 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. [0066] In one particular embodiment, each of the plurality of free labeling glycan ligands 42, 44 is respectively configured to bind specifically to Escherichia coli. In this particular embodiment, suitable examples of free labeling glycan ligands 42, 44 include bi-antennary biotinylated glycoconjugates and tetra-antennary biotinylated glycoconjugates. Generally, such 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.
[0067] In one particular embodiment, the bi-antennary biotinylated glycoconjugates are bi- antennary biotinylated a-mannosides. In another particular embodiment, the tetra-antennary biotinylated glycoconjugates are tetra-antennary biotinylated a-mannosides. Suitable examples of such bi-antennary and tetra-antennary biotinylated α-mannosides include MD and MT, as set forth in Table V.
[0068] With reference to the at least one first labeling glycan ligand, the first labeling glycan ligand may be MD or MT, as set forth in Table V. In one particular embodiment, 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.
[0069] In an alternative embodiment, 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.
c. Fluorophores
[0070] In one embodiment, each of the plurality of free labeling ligands 42, 44 includes a fluorophore 52, 54. Alternatively, in another embodiment, 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). For example, 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.
[0071] In one particular embodiment, 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)2dppz]2+, Cy3, Cy5, and combinations thereof. With regard to 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. In one particular embodiment, fluorophores 52, 54 include Rhodamine B, Ru(bipy)3, or TAMRA.
[0072] With specific regard to free labeling peptide ligands 42, 44, in one particular embodiment, the fluorophores 52, 54 are conjugated to the N-terminus or C-terminus of such free labeling peptide ligands 42, 44.
4. Metal Enhanced Fluorescence
[0073] In operation, 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. Such effect is disclosed in /. Fluoresc. 15(5): 643-54 (2005) and in /. Mater. Chem. , 16: 2846-52 (2006), the contents of which are incorporated by reference herein.
[0074] Referring again to FIG. 1, in one embodiment, the at least one first capture ligand binds 32, 34 specifically to a first region 72, 74, 76 of the target pathogen 62, 64. Similarly, the at least one first labeling ligand 42, 44 binds specifically to a second region 82, 84, 86 of the target pathogen 62, 64. Upon specific binding of the first capture ligand 32, 34 to the first region 72, 74, 76 of the target pathogen 62, 64 and the first labeling ligand 42, 44 to the 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.
[0075] Sandwiching of the target pathogen 62, 64 in between the first capture ligand 32, 34 and the first labeling ligand 42, 44, wherein the fluorophore 52, 54 of the first labeling ligand 42, 44 positioned at a metal enhancing distance D results in a stable fluorescence signal upon excitation. In one embodiment, 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.
[0076] Excitation of the fluorophore 52, 54 may be accomplished with a light source. Examples of 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. In one particular embodiment, the fluorescence emission of the fluorophore 52, 54 is enhanced relative to a control. For example, in one embodiment, the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 75%. In another embodiment, the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 100%. In still another embodiment, the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 150%.
[0077] In one particular embodiment of the pathogen sensor 1, 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%. In another particular embodiment of the pathogen sensor 1, 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), and the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 100%. In still another particular embodiment of the pathogen sensor 1, 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, and the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 75%.
[0078] In yet still another embodiment of the pathogen sensor 1, 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%. In another embodiment of the pathogen sensor 1, the first capture peptide ligand 32, 34 is Peptide Ligand No. 5 (SEQ ID NO: 5), the target pathogen is Bacillus cereus, the first labeling peptide ligand 42, 44 is Peptide Ligand No. 4 (SEQ ID NO: 4), and the fluorescence emission of the fluorophore 52, 54 is enhanced by at least about 75 %. In still another embodiment of the pathogen sensor 1, the pathogen sensor 1 has a limit of detection of less than about 10 pfu/mL.
[0079] Embodiments of the pathogen sensor 1 have been described in detail. Further embodiments directed to systems for pathogen detection will now be described.
II. System for Pathogen Detection
[0080] The pathogen sensors 1 may be incorporated into various systems or applications which require pathogen detection.
[0081] In general, 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. Referring again to FIG. 3, the sensor board 100 comprises at least one sample chamber 150 configured to house the pathogen sensors 1 described above. In one or exemplary embodiments, the sample chamber 150 may comprise a disposable sample cartridge, which is removable from the sensor board 100.
[0082] As described herein, there are additional components which can deliver the sample and/or the detection ligands (e.g., the peptides or glycans) to the sample chamber 150 during use. Prior to use, the sample chamber 150 may comprise the optically clear substrate 10, and optionally the immobilized ligands.
[0083] As shown in FIG. 3, 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.
[0084] Additionally, 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.
[0085] Further, 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. Various suitable pumps are contemplated. For example, the pump 130 may comprise a micropump, for example, a peristaltic pump. In one embodiment, 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.
Additionally, while the pump and associated flow system describes liquid samples, it is contemplated that the flow system may deliver solid samples, liquid samples, gaseous samples, or combinations thereof for detection by the pathogen sensor.
[0086] Moreover, the sensor board 100 also comprises at least one light source 170 configured to induce fluorescence inside the sample chamber 150. In one embodiment, the light source 170 may comprise at least one light emitting diode (LED) module. In a further embodiment, 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.
[0087] Referring again to FIG. 3, 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. For example, the spectrometer 140 may be a CCD spectrometer. Commercially suitable spectrometers may include the USB2000+ miniature CCD spectrometer available from Ocean Optics Inc. The detector could also be a photodiode or an avalanche phototide. [0088] Moreover, the sensor board 100 also comprises a computer unit 110, and at least one power source 120. Various power sources are considered suitable for the present system. In one embodiment, the power source is a battery. In an exemplary embodiment, 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.
[0089] The computer unit 110 may encompass various hardware and software components. For example, 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
communicate with other devices, for example, a network of sensors and/or a network controller. The computer unit may be equipped to receive commands from a network controller and also to transmit status information to that network controller. To communicate with the sensor network or a network controller, various communication protocols are considered suitable. For example, ZigBee communication protocol, e.g., ZigBee Pro
802.15.4, may be utilized. In one commercial embodiment, the computer unit 110 may comprise a ZigBee enabled Rabbit BL4S150 distributed by Digi International Inc.
[0090] In operation, 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. In one embodiment, following each acquisition of data, 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. Next, 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. In further embodiments, it is contemplated that 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. In an additional embodiment, 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.
[0091] While not depicted in FIG. 3, various additional components are contemplated for use with the sensor board 100. For example, it is contemplated that additional sensors may be included, for example, radiation sensors, temperature sensors, global positioning sensors (GPS), or combinations thereof. Generally, the present system will have the flexibility to detect pathogens while also monitoring other potential health concerns, such as radiation. With the use of GPS, the user may pinpoint the exact location of pathogens, or the exact locations of pathogens if there is a network of sensors.
[0092] Moreover, this present system is contemplated for use in various environments, i.e., land or sea. For example, the pathogen sensor may be disposed in a buoy system as shown in FIG. 4 for open water or well water monitoring. As shown, 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. While not shown, it is contemplated that 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. Additionally, as shown in FIG. 7, 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.
[0093] Additionally, the present pathogen detection system may also be utilized in the detection of airborne agents as shown in FIGS. 5 and 6. Referring to FIG. 6, the portable airborne pathogen detection system may utilize an air-to-water mixing impinger 480. In operation, 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.
[0094] Various other aerosolization and intake platforms may also be used in airborne detection. For example, 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
[0095] Embodiments of systems for pathogen detection have been described in detail. Further embodiments directed to peptide ligands will now be described.
Peptide Ligands
[0096] In one embodiment, peptide ligands are disclosed. In one embodiment, 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).
[0097] Embodiments of peptide ligands have now been described in detail. Methods for detecting pathogens will now be described in detail.
IV. Method for Detecting Pathogens
[0098] In one embodiment, a method for detecting pathogens is disclosed. In one particular embodiment, the method 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.
Examples
[0099] The following non-limiting examples illustrate the pathogen sensor, peptide ligands, and methods of the present disclosure. Example 1 : Effect of Concentration on Fluorescent Enhancement of TAMRA Labeled Peptide Ligand No. 2 (SEQ ID NO: 2) Bound to Silver Island Film
[00100] Experimental Protocol. Fluorescent enhancement of fluorescently labeled Peptide Ligand No. 2 bound to the surface of silver nanoparticles was investigated. The fluorescently labeled Peptide Ligand No. 2 included a TAMRA-label at the N-terminus and a thiol linker at the C-terminus. Unlabeled Peptide Ligand No. 2 was employed as a negative control. The unlabeled Peptide Ligand No. 2 also included a thiol linker at the C-terminus. The fluorescently labeled Peptide Ligand No. 2 and the unlabeled Peptide Ligand No. 2 were bound to surfaces of the silver nanoparticles through the thiol linkers. Fluorescence of the labeled Peptide Ligand No. 2 was monitored upon excitation and measured against the negative control.
[00101] Experimental Results. As shown in FIG. 8, optimal fluorescent enhancement via the silver nanoparticles was demonstrated at a concentration of 0.1 μΜ for Peptide Ligand No. 2. Additionally, concentrations of Peptide Ligand No. 2 below 0.01 μΜ did not exhibit enhancement over the negative control.
Example 2: Effect of Enterobacteria phage MS2 Concentration on Fluorescent
Enhancement of Ru(bipy)3 Labeled Peptide Ligand No. 2 (SEQ ID NO: 2) with Silver Nanoparticles
[00102] Experimental Protocol. The effect of Enterobacteria phage MS2 concentration on fluorescent enhancement of Ru(bipy)3 labeled free Peptide Ligand No. 2 with silver nanoparticles was investigated. Capture Peptide Ligand Nos. 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) were bound to silver nanoparticles. More particularly, Peptide Ligand Nos. 1 and 2 were bound to the silver nanoparticles via thiol linkers which were included at the C- termini thereof. Capture Peptide Ligand Nos. 1 and 2 were immobilized on the silver nanoparticles, forming a functionalized surface. Ru(bipy)3 label was added to the N-terminus of free Peptide Ligand No. 2.
[00103] To evaluate the effect of Enterobacteria phage MS2 concentration, samples containing varying concentrations (i.e., 7.40E+06, 1.50E+07, 2.20E+07, and 3.00E+07 pfu/mL) of Enterobacteria phage MS2 were applied to the functionalized surface 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 Ru(bipy)3 labeled free Peptide Ligand No. 2 was applied to the functionalized surface and allowed to incubate for from about 30 seconds to about 2 minutes. 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).
[00104] Experimental Results. As shown in FIGS. 9 and 10, fluorescence enhancement of Ru(bipy)3 labeled free Peptide Ligand No. 2 is dependent upon the concentration of Enterobacteria phage MS2. More particularly, fluorescence enhancement of Ru(bipy)3 labeled free peptide ligands increases with increasing concentration of Enterobacteria phage MS2.
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)
[00105] Experimental Protocol. Fluorescent enhancement of TAMRA labeled free Peptide Ligand No. 2 and Ru(bipy)3 labeled free Peptide Ligand No. 2 was compared. Capture Peptide Ligand No. 2 was bound to silver island film. More particularly, capture Peptide Ligand No. 2 was bound to the silver island film via thiol linkers included at the C-terminus thereof. The capture Peptide Ligand No. 2 was immobilized on the silver island film, forming a functionalized surface. TAMRA and Ru(bipy)3 labels were respectively added to the N-termini of free Peptide Ligand No. 2.
[00106] To evaluate the effect of employing TAMRA labeled free Peptide Ligand No. 2 versus Ru(bipy)3 labeled free Peptide Ligand No. 2, samples containing Enterobacteria phage MS2 were applied to the functionalized surface 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 No. 2 and Ru(bipy)3 labeled free Peptide Ligand No. 2 were respectively applied to the functionalized surface and allowed to incubate for from about 30 seconds to about 2 minutes. Unbound TAMRA labeled free Peptide Ligand No. 2 and unbound Ru(bipy)3 labeled Peptide Ligand No. 2 were removed by washing with either water or with buffer. Fluorescence of TAMRA labeled free Peptide Ligand No. 2 and Ru(bipy)3 labeled 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).
[00107] Experimental Results. As shown in FIG. 11, Ru(bipy)3 labeled free Peptide Ligand No. 2 exhibited significantly higher metal enhanced fluorescence yield compared with TAMRA labeled free Peptide Ligand No. 2. Without being bound by the theory, such data supports the theory that fluorophores having lower quantum yields will result in increased fluorescence enhancement. Further, without being bound by the theory, it is believed that such effect could increase the signal-to-noise reporting of target pathogens.
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
[00108] Experimental Protocol. The specificity of Peptide Ligand Nos. 1 and 2 for Enterobacteria phage MS2 was investigated. Capture Peptide Ligand Nos. 1 and 2 were respectively bound to silver island film. More particularly, capture Peptide Ligand Nos. 1 and 2 was bound to the silver island film via thiol linkers included at the C-terminus thereof. The capture Peptide Ligand Nos. 1 and 2 were immobilized on the silver island film, forming a functionalized surface. Ru(bipy)3 labels was respectively added to the N-termini of free Peptide Ligand Nos. 1 and 2.
[00109] 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.
[00110] Samples containing Enterobacteria phage MS2 were respectively applied to the functionalized surfaces of the silver island film and the gold particles 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 Ru(bipy)3 labeled free Peptide Ligand Nos. 1 and 2 and anti-MS2 IgG antibodies were respectively applied to the functionalized surfaces of the silver island film and the gold particles. Such compositions were allowed to incubate for from about 30 seconds to about 2 minutes. Unbound Ru(bipy)3 labeled free Peptide Ligand Nos. 1 and 2 and unbound anti-MS2 IgG antibodies were respectively removed by washing with either water or with buffer. Fluorescence of Ru(bipy)3 labeled free Peptide Ligand Nos. 1 and 2 and anti-MS2 IgG antibodies was monitored upon excitation and measured against a negative control.
[00111] Experimental Results . Referring to FIGS. 12A-C and 13, 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.
Example 5: Capture of Spores of Bacillus subtilis on Silver Island Film
[00112] Experimental Protocol. 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.
[00113] Samples containing spores of Bacillus subtilis were applied to the functionalized surface 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 Ru(bipy)3 labeled free Peptide Ligand No. 3 was applied to the functionalized surface and allowed to incubate for from about 30 seconds to about 2 minutes. Unbound Ru(bipy)3 labeled Peptide Ligand No. 3 was removed by washing with either water or with buffer. Fluorescence of Ru(bipy)3 labeled Peptide Ligand No. 3 was monitored upon excitation and measured against a negative control.
[00114] Experimental Results. Referring to FIG. 14, capture of spores of Bacillus subtilis was achieved. Example 6: Capture of Bacillus subtilis (vegetative) and Bacillus cereus (vegetative) on Silver Island Film
[00115] Experimental Protocol. 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).
[00116] 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.
[00117] Experimental Results. Referring to FIG. 15, capture of Bacillus subtilis and Bacillus cereus were respectively achieved.
Example 7: Preferential Binding to Bacillus cereus over Escherichia coli
[00118] Experimental Protocol. 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).
[00119] 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.
[00120] Experimental Results. Referring to FIG. 16, 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.
Example 8: Cross-Reactivity of Peptide Ligand No. 3 (SEQ ID NO: 3) with Escherichia coli (Vegetative Cells)
[00121] Experimental Protocol. 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).
[00122] 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.
[00123] Experimental Results. Referring to FIG. 17A-D, Peptide Ligand No. 3 did not cross-react with Escherichia coli (Vegetative).
Example 9: Detection of Escherichia coli ORN178 with Glycan MD modified with lipoic acid [00124] 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.
[00125] Samples containing varying concentrations (i.e., 105 CFU/mL, 106 CFU/mL, 107 CFU/mL, 108 CFU/mL) 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 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. Fluorescence of glycan MD modified with lipoic acid was monitored upon excitation and measured against a negative control. Negative controls included galactose ligands and a sample containing Escherichia coli ORN 208 strain.
[00126] Experimental Results. As shown in FIGS. 18 and 19, 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.
Example 10: Detection of Escherichia coli ORN178 in Complex Samples
[00127] 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.
[00128] Complex samples (e.g., apple juice, grape juice, and media) containing 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 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. Fluorescence of Ru(bipy)3 labeled glycan MD modified with lipoic acid was monitored upon excitation and measured against a negative control. Negative controls included a sample of phosphate buffered saline containing Escherichia coli ORN178.
[00129] Experimental Results.
[00130] As shown in FIG. 20, Escherichia coli ORN178 was detected in complex solutions. V. Use of Li fetime Decay Analysis
[00131] In addition to the improved specificity achieved by the metal enhanced fluorescence pathogen detection methodology described above, using lifetime decay analysis in conjunction with fluorescence emission data may be used to pinpoint specific pathogens. For example, and not by way of limitation, 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. In general, the 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. With this data alone, it often proves difficult to highlight Bacillus endospore species, especially in complex biological fluids and suspensions. However, specific species, such as B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain may be pinpointed by analyzing lifetime decay data. Using the Multi-Exponential fit method, 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. Thus, 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.
[00132] It is noted that terms like "preferably," "generally," "commonly," and "typically" are not utilized herein to limit the scope of the claims or to imply that certain features are critical, essential, or even important to the structure or function of the claims. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure.
[00133] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
[00134] For the purposes of describing and defining the present disclosure it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
[00135] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

Claims

1. A pathogen sensor for detecting the presence of a target pathogen in a sample, the pathogen sensor comprising:
an optically clear substrate comprising silver particles deposited on a surface thereof, wherein the silver particles are immobilized on the surface;
a plurality of immobilized capture peptide ligands attached to the silver particles, wherein the plurality of immobilized capture peptide ligands comprises at least one first capture peptide ligand configured to bind specifically to the target pathogen, and wherein the target pathogen is selected from the group consisting of bacteria, viruses, fungus, and combinations thereof; and
a plurality of free labeling peptide ligands comprising fluorophores, wherein the plurality of free labeling peptide ligands comprises at least one first labeling peptide ligand configured to bind specifically to the target pathogen, and wherein the at least one first labeling peptide ligand comprises a fluorophore, such that when the at least one first capture peptide ligand binds specifically to a first region of the target pathogen and the at least one first labeling peptide ligand binds specifically to a second region of the same target pathogen:
the target pathogen is sandwiched in between the at least one first capture peptide and the at least one first labeling peptide ligand bound thereto,
the fluorophore of the at least one 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 at least one first labeling peptide ligand is enhanced upon excitation, indicating the presence of the target pathogen.
2. The pathogen sensor of claim 1, wherein the sample is complex.
3. The pathogen sensor of claim 1, wherein the optically clear substrate is selected from the group consisting of glass, silica, quartz, plastics, and combinations thereof.
4. The pathogen sensor of claim 1 , wherein the silver particles immobilized on the surface of the optically clear substrate comprises silver island film.
5. The pathogen sensor of claim 1, wherein the plurality of immobilized capture peptide ligands comprises at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
6. The pathogen sensor of claim 1 , wherein the at least one first capture peptide ligand comprises SEQ ID NO: 1.
7. The pathogen sensor of claim 1, wherein the target pathogen is selected from the group consisting of Bacillus subtilis, Bacillus cereus, Bacillus thuringiensis, Francisella philomiragia, Vibrio cholerae, Enter obacteria phage MS2, and combinations thereof.
8. The pathogen sensor of claim 1, wherein the target pathogen is Enterobacteria phage MS2.
9. The pathogen sensor of claim 8, wherein the pathogen sensor comprises a limit of detection of less than about 10 pfu/mL of the target pathogen.
10. The pathogen sensor of claim 1, wherein the plurality of free labeling peptide ligands comprises at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
11. The pathogen sensor of claim 1 , wherein the at least one first labeling peptide ligand comprises SEQ ID NO: 2.
12. The pathogen sensor of claim 1, wherein the fluorophore of the at least one first labeling peptide ligand is rhodamine or tris(bipyridine)ruthenium (II) chloride.
13. The pathogen sensor of claim 1, wherein:
the fluorophore of the at least one first labeling peptide ligand is
tris(bipyridine)ruthenium (II) chloride; and
the fluorescence emission of the fluorophore is enhanced by at least about 75 %.
14. The pathogen sensor of claim 1, wherein the metal enhancing distance is less than about 10 nm.
15. The pathogen sensor of claim 1, wherein the at least one first capture peptide ligand comprises a different amino acid sequence from the at least one first labeling peptide ligand.
16. The pathogen sensor of claim 1, wherein:
the at least one first capture peptide ligand comprises SEQ ID NO: 1 and SEQ ID NO:
2;
the target pathogen is Enterobacteria phage MS2; the at least one first labeling peptide ligand comprises SEQ ID NO: 1 and SEQ ID NO: 2; and
the fluorescence emission of the fluorophore is enhanced by at least about 100 %.
17. The pathogen sensor of claim 1, wherein:
the at least one first capture peptide ligand comprises SEQ ID NO: 3;
the target pathogen is Bacillus subtilis;
the at least one first labeling peptide ligand comprises SEQ ID NO: 3;
the metal enhancing distance is less than about 50 nm; and
the fluorescence emission of the fluorophore is enhanced by at least about 75 %.
18. The pathogen sensor of claim 1, wherein:
the at least one first capture peptide ligand comprises SEQ ID NO: 4;
the target pathogen is Bacillus cereus;
the at least one first labeling peptide ligand comprises SEQ ID NO: 5;
the metal enhancing distance is less than about 50 nm; and
the fluorescence emission of the fluorophore is enhanced by at least about 75 %.
19. The pathogen sensor of claim 1, wherein:
the at least one first capture peptide ligand comprises SEQ ID NO: 5;
the target pathogen is Bacillus cereus;
the at least one first labeling peptide ligand comprises SEQ ID NO: 4; and the fluorescence emission of the fluorophore is enhanced by at least about 75 %.
20. The pathogen sensor of claim 1, wherein the pathogen sensor is operable to pinpoint specific Bacillus endospores in the sample based on fluorescence emission and lifetime decay analysis.
21. The pathogen sensor of claim 20, wherein the specific Bacillus endospores are B. subtilis, B. megaterium, B. coagulans, and B. anthracis Sterne strain.
22. A system for pathogen detection via metal enhanced fluorescence comprising at least one sensor board, the sensor board comprising at least one sample chamber which houses at least one of the pathogen sensors of claim 1.
23. A system for pathogen detection via metal enhanced fluorescence comprising at least one sensor board, the sensor board comprising:
at least one sample chamber which houses at least one of the pathogen sensors of claim 1. at least one sample reservoir; at least one detection reservoir operable to store immobilized free labeling peptide ligands, capture peptide ligands, or both; at least one pump in fluid communication with the at least one sample reservoir, wherein the pump is configured to deliver sample to the sample chamber; at least one light source configured to induce fluorescence inside the sample chamber; a spectrometer configured to detect the target pathogen within the sample based on the fluorescence emission delivered by the fluorophore; a computer unit; and at least one power source.
24. The system of claim 23 wherein the at least one pump is also in fluid communication with the detection reservoir.
25. The system of claim 23 wherein the pump is a micropump
26. The system of claim 23 wherein the pump is a peristaltic pump.
27. The system of claim 23 wherein the power source comprises a battery.
28. The system of claim 23 wherein the light source comprises at least one light emitting diode.
29. The system of claim 23 further comprising an optical probe.
30. The system of claim 23 wherein the sample chamber comprises a disposable sample cartridge.
31. The system of claim 23 wherein the computer unit comprises one or more components selected from the group consisting of a microprocessor, a display unit, wireless communication radios, circuit boards, a user interface, and combinations thereof.
32. The system of claim 23, wherein the system is configured for use in an automatic inline water monitoring system, a buoy system for open water or well water monitoring, or an airborne detection system.
33. The system of claim 23 further comprising additional sensors.
34. The system of claim 33 wherein the additional sensors comprise radiation sensors, temperature sensors, global positioning sensors, or combinations thereof.
35. A pathogen sensor for detecting the presence of pathogenic Escherichia coli in a sample, the sensor comprising: an optically clear substrate comprising silver particles deposited on a surface thereof, wherein the silver particles are immobilized on the surface;
a plurality of immobilized capture glycan ligands attached to the silver particles, wherein the plurality of immobilized capture glycan ligands comprises at least one first capture glycan ligand configured to bind specifically to the pathogenic Escherichia coli; and a plurality of free labeling glycan ligands comprising fluorophores, wherein the plurality of free labeling glycan ligands comprises at least one first labeling glycan ligand configured to bind specifically to the pathogenic Escherichia coli, wherein the at least one first labeling glycan ligand comprises a fluorophore, and wherein the at least one first capture glycan and the at least one first labeling glycan ligand individually comprises:
or
such that when the at least one first capture glycan ligand binds specifically to a first region of the pathogenic Escherichia coli, and the at least one first labeling glycan ligand binds specifically to a second region of the same pathogenic Escherichia coli:
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.
36. A system for pathogen detection via metal enhanced fluorescence comprising at least one sensor board, the sensor board comprising at least one sample chamber which houses at least one of the pathogen sensors of claim 35.
37. A system for pathogen detection via metal enhanced fluorescence comprising at least one sensor board, the sensor board comprising: at least one sample chamber which houses at least one of the pathogen sensors of claim 35; at least one sample reservoir; at least one detection reservoir operable to store free labeling glycan ligands, immobilized capture glycan ligands, or both; at least one pump in fluid communication with the at least one sample reservoir, wherein the pump is configured to deliver sample to the sample chamber; at least one light source configured to induce fluorescence inside the sample chamber; a spectrometer configured to detect the target pathogen within the sample based on the fluorescence emission delivered by the fluorophore; a computer unit; and at least one power source.
38. The system of claim 37 wherein the at least one pump is also in fluid communication with the detection reservoir.
39. The system of claim 37 wherein the pump is a micropump
40. The system of claim 37 wherein the pump is a peristaltic pump.
41. The system of claim 37 wherein the power source comprises a battery.
42. The system of claim 37 wherein the light source comprises at least one light emitting diode.
43. The system of claim 37 further comprising an optical probe.
44. The system of claim 37 wherein the sample chamber comprises a disposable sample cartridge.
45. The system of claim 37 wherein the computer unit comprises one or more components selected from the group consisting of a microprocessor, a display unit, wireless communication radios, circuit boards, a user interface, and combinations thereof.
46. The system of claim 37, wherein the system is configured for use in an automatic inline water monitoring system, a buoy system for open water or well water monitoring, or an airborne detection system.
47. The system of claim 37 further comprising additional sensors.
48. The system of claim 47 wherein the additional sensors comprise radiation sensors, temperature sensors, global positioning sensors, or combinations thereof.
49. A peptide ligand 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.
50. The peptide ligand of claim 49, wherein the peptide ligand is SEQ ID NO: 1.
51. The peptide ligand of claim 49, wherein the peptide ligand is SEQ ID NO: 2.
52. The peptide ligand of claim 49, wherein the peptide ligand is SEQ ID NO: 3.
53. The peptide ligand of claim 49, wherein the peptide ligand is SEQ ID NO: 4.
54. The peptide ligand of claim 49, wherein the peptide ligand is SEQ ID NO: 5.
EP12852456.8A 2011-11-16 2012-11-16 Pathogen detection using metal enhanced fluorescence Withdrawn EP2780709A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161560501P 2011-11-16 2011-11-16
PCT/US2012/065655 WO2013115897A2 (en) 2011-11-16 2012-11-16 Pathogen detection using metal enhanced fluorescence

Publications (1)

Publication Number Publication Date
EP2780709A2 true EP2780709A2 (en) 2014-09-24

Family

ID=48483182

Family Applications (1)

Application Number Title Priority Date Filing Date
EP12852456.8A Withdrawn EP2780709A2 (en) 2011-11-16 2012-11-16 Pathogen detection using metal enhanced fluorescence

Country Status (2)

Country Link
EP (1) EP2780709A2 (en)
WO (1) WO2013115897A2 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106442479B (en) * 2016-08-30 2019-10-22 华南师范大学 The method that paper base bipolar electrode electrochemical luminescence molecular switch system is used for rapid sensitive genetic test pathogenic bacteria
GB202019024D0 (en) * 2020-12-02 2021-01-13 Paraytec Ltd Diagnostic testing
WO2023187760A1 (en) * 2022-04-01 2023-10-05 Genevant Sciences Gmbh Mannose-targeted compositions

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MXPA01006106A (en) * 1998-12-15 2003-07-21 Exiqon As Coupling of lipopolysaccharide-derived carbohydrates onto solid surfaces.
ATE479775T1 (en) 2002-11-26 2010-09-15 Univ Maryland Biotechnology HIGHLY SENSITIVE ASSAY FOR PATHOGENE DETECTION USING METAL ENHANCED FLUORESCENCE
US7906343B2 (en) * 2005-01-24 2011-03-15 Sri International Surface-enhanced lanthanide chelates
US8735175B2 (en) * 2011-03-18 2014-05-27 Chris D. Geddes Multicolor microwave-accelerated metal-enhanced fluorescence (M-MAMEF)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2013115897A2 *

Also Published As

Publication number Publication date
WO2013115897A3 (en) 2013-11-28
WO2013115897A2 (en) 2013-08-08
WO2013115897A9 (en) 2013-09-26

Similar Documents

Publication Publication Date Title
US20200150120A1 (en) Portable electronic device, system, and method for analyte detection
Myndrul et al. Photoluminescence label-free immunosensor for the detection of Aflatoxin B1 using polyacrylonitrile/zinc oxide nanofibers
Charbonniere et al. Lanthanides to quantum dots resonance energy transfer in time-resolved fluoro-immunoassays and luminescence microscopy
Tay et al. Silica encapsulated SERS nanoprobe conjugated to the bacteriophage tailspike protein for targeted detection of Salmonella
Herland et al. Conjugated polymers as optical probes for protein interactions and protein conformations
US20110177585A1 (en) Rapid detection nanosensors for biological pathogens
US20090004670A1 (en) Methods for fabricating surface enhanced fluorescent (sef) nanoparticles and their applications in bioassays
Verma et al. Synthesis and characterization of ZnS quantum dots and application for development of arginine biosensor
CN101281137A (en) Light activating chemical luminescence luminous immune detecting method
US20160209410A1 (en) Test piece for immunochromatography, developing fluid used therefor, and immunochromatography using the same
US20090270269A1 (en) Nano-scale fluoro-biosensors exhibiting a low false alarm rate for rapid detection of biological contaminants
US20150148262A1 (en) Microparticle assembly
Zhang et al. Small-molecule ligands strongly affect the Förster resonance energy transfer between a quantum dot and a fluorescent protein
WO2015055708A1 (en) Sensitive qualitative bioassay using graphene oxide as analyte revealing agent
EP2780709A2 (en) Pathogen detection using metal enhanced fluorescence
Cheng et al. Au nanocluster-embedded chitosan nanocapsules as labels for the ultrasensitive fluorescence immunoassay of Escherichia coli O157: H7
Hering et al. Energy transfer between CdSe/ZnS core/shell quantum dots and fluorescent proteins
Yi et al. Direct immobilization of oxyamine-modified proteins from cell lysates
US7220596B2 (en) Real time detection of antigens
Wang et al. Conjugation and fluorescence quenching between bovine serum albumin and L-cysteine capped CdSe/CdS quantum dots
US9193990B2 (en) Bioluminescent metal ion assay
Kim et al. Ni–nitrilotriacetic acid-modified quantum dots as a site-specific labeling agent of histidine-tagged proteins in live cells
US20150323530A1 (en) Quantum dot-protein complexes, films, and methods of use
US11597962B2 (en) Rapid selective detection of bacteria
CA3165161A1 (en) Ultrabright fluorescent nanocomposite structures for enhanced fluorescent bioassays

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20140604

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20150429

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20150910