Colorimetric Substrates, Colorimetric Sensors, and Methods of Use

• US 20070269851A1
• Filed: 11/03/2004
• Published: 11/22/2007
• Est. Priority Date: 11/03/2003
• Status: Active Grant

• Current Assignee: Woundchek Laboratories (US), Inc.
• Original Assignee: Woundchek Laboratories (US), Inc.
• Inventors: Ellis-Busby, Diane, Havard, Jennifer, Colpas, Gerard, Sanders, Mitchell
• Granted Patent: US 8,927,204 B2
• Time in Patent Office: Days
• US Class Current: 435/34
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase

Abstract

Described herein is a substrate comprising at least one colorimetric component attached to a peptide, as well as methods for detecting a modification of said substrate. Also described are methods of detecting the presence or absence of a protein, for example, a protein produced by a microorganism, by contacting a substrate with a sample. If the sample contains the protein of interest, the substrate is modified and a visible color change results, indicating the presence or absence of the protein in the sample. The present invention also features biosensors and kits for detecting the presence or absence of microorganisms and/or proteins in a sample.

Claims

1. A method of detecting the modification of a substrate, comprising the steps of:

a) exposing an unmodified substrate to a sample under conditions that will result in a modification of the substrate, the unmodified substrate including a peptide and a first colorimetric component, the first calorimetric component coupled to the peptide; and

b) detecting a modification of the substrate or an absence of the modification of the substrate, wherein the modification comprises cleaving the first colorimetric component from the substrate and results in a visible color change.

2. A method according to claim 1, wherein the first colorimetric component is covalently bonded to the peptide.

3. A method according to claim 1, wherein the modification includes hydrolysis of a peptide bond and results in a portion of the peptide detaching from the substrate.

4. A method according to claim 1, wherein the substrate includes at least one member of the group consisting of the peptide sequence LLGDFFRKSKEKIGKEFKRIVXRIKDFLRNLVPRTES, the peptide sequence KAAHKSALKSAE, the peptide sequence KKASEAAHKSALKSAE, the peptide sequence CHHHASEAAHKSALKSAE, the peptide sequence KHLGGGALGGGAKE, the peptide sequence KHLGGGGGAKE, the peptide sequence ACCDEYLQTKE, the peptide sequence ADTVEPTGAKE, the peptide sequence KLPHKLSWSADNP, the peptide sequence PVPSTPPTPSPSTP, the peptide sequence NMLSEVERE, the peptide sequence KQNMLSEVERADTE, the peptide sequence NEAIQEDQVQYE, the peptide sequence ETKVEENEAIQK, the peptide sequence DSRPVRRRRRPRVSK, the peptide sequence KVSRRRRRGGD, the peptide sequence KKASEVSRRRRRGGK, the peptide sequence CHHHASEVSRRRRRGGK, the peptide sequence KEKIGKEFKRIVQE, the peptide sequence KVQRIKDFLRNLVE, the peptide sequence EAAGAMFLEAIPK, the peptide sequence EGAMFLEAIPMSIPK, the peptide sequence CGAMFLEAIPMSIPAAAHHHHH, the peptide sequence KARRRRRGGGAMFLEAIPMSIPCGC, the peptide sequence VSRRRRRGGDGDGC, the peptide sequence GGDGDGC, the peptide sequence VSRRRRRGGDGKGDAC, the peptide sequence NEAIQEDQVQARRAKARRAC, the peptide sequence QVQARRAKARRAC, the peptide sequence GGDGKGDAC, the peptide sequence QVQARRRAKARRRAC, the peptide sequence VSRRRRRGGKGC, the peptide sequence SVTRRRRRGGRASGGC, the peptide sequence SEAIQEDQVQYCAAAHHHHH, the peptide sequence KARRRRRGGDGDGCGC, the peptide sequence HHHHHSRRRRRGGCGC, the peptide sequence HHHHHSVQRIKDFLRNLVCGC, the peptide sequence RRRRRSVQRIKDFLRNLVCGC, the peptide sequence HHHHHAAHKSALKSACGC, the peptide sequence RRRRRAAHKSALKSACGC, the peptide sequence PGTKLYTVPW, an Alt derived peptide, a peptidoglycans, lipoteichoic acid, and a lipid vesicle.

5. A method according to claim 1, wherein the first calorimetric component is one of the members of the group consisting of a dye;

a reactive dye;

a fiber reactive dye;

a dye suitable for use in a contact lens;

a dye suitable for use in a suture;

a monohalogentriazine dye;

a dihalogentriazine dye;

a 2,4,5 trihalogenopyriminidine dye;

a 2,3 dihaloquinoxaline dye;

a N-hydroxysulfosuccinimidyl a (sulfo-NHS) ester functionalized dye;

a N-hydroxysuccinimidyl (NHS) functionalized dye;

a vinyl sulfone dye;

a sulfonyl chloride dye;

a tetrafluorophenyl ester functionalized dye;

an isothiocyanate functionalized dye; and

an iodoacetyl functionalized dyes.

6. A method according to claim 1, wherein the visible color change is a loss of color.

7. A method according to claim 1, wherein the unmodified substrate further includes a second calorimetric component that is dissimilar to the first colorimetric component.

8. A method according to claim 1, wherein the peptide is coupled to a solid support.

9. A method according to claim 8, wherein the modification of the substrate results in a hue of the solid support becoming more visible.

10. A method according to claim 8, wherein the peptide is covalently attached to the solid support.

11. A method according to claim 8, wherein the solid support is selected from the group consisting of a wound dressing, a sterilized material, an article that contains the sample, an article that collects the sample, a polymer, a membrane, a resin, glass, a sponge, a disk, a scope, a filter, a lens, a foam, a cloth, a paper, a suture, and a bag.

12. A method according to claim 1, wherein the sample is at least one of the group consisting of a wound surface on a subject, a body fluid, a piece of hair, a piece of nail, a piece of shell, a piece of scale, a piece of feather, a piece of tissue, an article implanted in the body of an animal, catheter, a urine collection bag, a blood collection bag, a plasma collection bag, a disk, a scope, a filter, a lens, foam, cloth, paper, a suture, a swab, a dipstick, a sponge, a polymeric article, an article made of a resin, a glass article, a test tube, a well of a microplate, a portion of contact lens solution, a sponge, a polymeric material, a membrane, an article made of resin, an article made of glass, and a swab.

13. A method according to claim 1, wherein modification of the substrate includes cleaving a portion of the peptide to produce a cleaved portion, the cleaved portion including the first calorimetric component, the modification resulting in the migration of the cleaved portion toward a collector, and the migration resulting in a visible color change.

14. A method according to claim 13, wherein the collector includes at least one material selected from the group consisting of a membrane, a resin, a polymer, a film, glass, or a chelating material.

15. A method according to claim 1, wherein modification of the substrate is used to indicate the presence of a bacterial enzyme selected from the group consisting of a lysin, an autolysin, a lipase, an exotoxin, a cell wall enzyme, a matrix binding enzyme, a protease, a hydrolase, a virulence factor enzyme, and a metabolic enzyme.

16. A biosensor for detecting the presence or absence of a protein, the biosensor comprising a peptide that specifically reacts with a protein and a first calorimetric component coupled to the peptide.

17. A biosensor according to claim 16, wherein the first colorimetric component is covalently bonded to the peptide.

18. A biosensor according to claim 16, wherein the substrate includes at least one member of the group consisting of the peptide sequence LLGDFFRKSKEKIGKEFKRIVXRIKDFLRNLVPRTES, the peptide sequence KAAHKSALKSAE, the peptide sequence KKASEAAHKSALKSAE, the peptide sequence CHHHASEAAHKSALKSAE, the peptide sequence KHLGGGALGGGAKE, the peptide sequence KHLGGGGGAKE, the peptide sequence ACCDEYLQTKE, the peptide sequence ADTVEPTGAKE, the peptide sequence KLPHKLSWSADNP, the peptide sequence PVPSTPPTPSPSTP, the peptide sequence NMLSEVERE, the peptide sequence KQNMLSEVERADTE, the peptide sequence NEAIQEDQVQYE, the peptide sequence ETKVEENEAIQK, the peptide sequence DSRPVRRRRRPRVSK, the peptide sequence KVSRRRRRGGD, the peptide sequence KKASEVSRRRRRGGK, the peptide sequence CHHHASEVSRRRRRGGK, the peptide sequence KEKIGKEFKRIVQE, the peptide sequence KVQRIKDFLRNLVE, the peptide sequence EAAGAMFLEAIPK, the peptide sequence EGAMFLEAIPMSIPK, the peptide sequence CGAMFLEAIPMSIPAAAHHHHH, the peptide sequence KARRRRRGGGAMFLEAIPMSIPCGC, the peptide sequence VSRRRRRGGDGDGC, the peptide sequence GGDGDGC, the peptide sequence VSRRRRRGGDGKGDAC, the peptide sequence NEAIQEDQVQARRAKARRAC, the peptide sequence QVQARRAKARRAC, the peptide sequence GGDGKGDAC, the peptide sequence QVQARRRAKARRRAC, the peptide sequence VSRRRRRGGKGC, the peptide sequence SVTRRRRRGGRASGGC, the peptide sequence SEAIQEDQVQYCAAAHHHHH, the peptide sequence KARRRRRGGDGDGCGC, the peptide sequence HHHHHSRRRRRGGCGC, the peptide sequence HHHHHSVQRIKDFLRNLVCGC, the peptide sequence RRRRRSVQRIKDFLRNLVCGC, the peptide sequence HHHHHAAHKSALKSACGC, the peptide sequence RRRRRAAHKSALKSACGC, the peptide sequence PGTKLYTVPW, an Alt derived peptide, a peptidoglycans, lipoteichoic acid, and a lipid vesicle.

19. A biosensor according to claim 16, wherein the first colorimetric component is one of the members of the group consisting of a dye;

a reactive dye;

a fiber reactive dye;

a dye suitable for use in a contact lens;

a dye suitable for use in a suture;

a monohalogentriazine dye;

a dihalogentriazine dye;

a 2,4,5 trihalogenopyriminidine dye;

a 2,3 dihaloquinoxaline dye;

a N-hydroxysulfosuccinimidyl a (sulfo-NHS) ester functionalized dye;

a N-hydroxysuccinimidyl (NHS) functionalized dye;

a vinyl sulfone dye;

a sulfonyl chloride dye;

a tetrafluorophenyl ester functionalized dye;

an isothiocyanate functionalized dye; and

an iodoacetyl functionalized dyes.

20. A biosensor according to claim 16, further including a second calorimetric component coupled to the peptide, the first calorimetric component being dissimilar to the first colorimetric component.

21. A biosensor according to claim 16, further including a solid support, the peptide coupled to the solid support.

22. A biosensor according to claim 21, wherein the peptide is covalently attached to the solid support.

23. A biosensor according to claim 21, wherein the solid support is selected from the group consisting of a wound dressing, a sterilized material, an article that contains a sample, an article that collects a sample, a polymer, a membrane, a resin, glass, a sponge, a disk, a scope, a filter, a lens, a foam, a cloth, a paper, a suture, and a bag.

24. A biosensor according to claim 16, further including a collector that includes at least one material selected from the group consisting of a membrane, a resin, a polymer, a film, glass, or a chelating material.

25. A biosensor according to claim 16, wherein the peptide includes a sequence that specifically reacts with an enzyme selected from the group consisting of a lysin, an autolysin, a lipase, an exotoxin, a cell wall enzyme, a matrix binding enzyme, a protease, a hydrolase, a virulence factor enzyme, and a metabolic enzyme.

26. A kit for detecting a protein, comprising a biosensor according to claim 16 and at least one reagent.