Enzymatic biosensing systems

• US 9,493,806 B2
• Filed: 07/31/2012
• Issued: 11/15/2016
• Est. Priority Date: 06/01/2001
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation (Colorado State University System)
• Original Assignee: Colorado State University Research Foundation (Colorado State University System)
• Inventors: Reardon, Kenneth F.
• Primary Examiner(s): Conley, Sean E
• Assistant Examiner(s): Brazin, Jaqueline
• Application Number:
  US13/562,592
• Publication Number:
  US 20140235501A1
• Time in Patent Office: 1,568 Days
• Field of Search: 422/72
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/002 Electrode membranes
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: G01N 2333/90241 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: G01N 2333/90245 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: G01N 2333/914 Hydrolases (3)
• CPC Class Codes: G01N 2333/988 Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase

Abstract

Biosensors and methods of producing biosensors for use in detecting one or more analytes in a solution are disclosed herein.

Claims

1. A biosensor for measuring the concentration of a molecule in a solution comprising:

a fiber optical cable having a first end and a second end, and

a transducer layer comprising an optical transducer capable of interacting with oxygen, protons, or halide ions, the transducer layer having a first side and a second side, and

a matrix layer comprising a hydrogel or polymer matrix having a first side and a second side, and

a biocomponent immobilized within said matrix layer,

wherein said first end of said fiber optical cable is bound to said second side of said transducer layer, and

wherein said first side of said transducer layer is bound to said second side of said matrix layer, and

wherein said first side of said matrix layer is in contact with said solution, and

wherein said second end of said fiber optical cable is coupled to an optical excitation source, a photon detection device, an electronic amplification device, signal processing circuitry and a signal output device.

2. The biosensor of claim 1 wherein said transducer layer comprises an optical transducer selected from the group consisting of:

an ultraviolet-visible absorption transducer,

a luminescence transducer selected from the group consisting of trisodium 8-hydroxy-1,3,6-trisulphonate, fluoro (8-anilino-1-naphthalene sulphonate), tris(bipyridine)ruthenium(II) complex, ruthenium complexes, and acridinium- and quinidinium-based reagents,

a fluorescence transducer,

a phosphorescence transducer,

an emission transducer,

a bioluminescence transducer,

a chemiluminescence transducer, an internal reflection spectroscopy transducer, and

a laser light scattering methods transducer selected from the group consisting of quasielastic light-scattering spectroscopy, photon correlation spectroscopy and laser Doppler velocimetry.

3. The biosensor of claim 1 wherein said polymer matrix in said matrix layer comprises one or more of polyacrylamide, polystyrene, polymethacrylate, polyvinylalcohol and polyurethane.

4. The biosensor of claim 1 wherein said hydrogel in said matrix layer comprises one or more of algal polysaccharides, agarose, alginate, gelatin, collagen, pectin, poly(carbamoyl) sulfonate, locust bean gum, and gellan.

5. The biosensor of claim 1 wherein said biocomponent is treated to stabilize said biocomponent against degradation using one or more methods comprising:

adding inhibitors of protein synthesis to said biocomponent,

adding protease inhibitors to said biocomponent,

freeze drying said biocomponent,

dry heating said biocomponent, and

grafting a molecule onto said biocomponent, said molecule one or more of polysaccharides, polyalcohols, trehalose, maltose, lactose, sucrose, glucose and galactose.

6. The biosensor of claim 1 wherein said first end of said fiber optical cable is coated with one or more agents comprising one or more of cellulose acetate, polycarbonate, collagen, acrylate copolymers, poly(ethylene glycol), polytetrafluoroethylene, and alginate-polylysine-alginate microcapsules.

7. The biosensor of claim 1 wherein said biocomponent is treated through adding one or more gel-hardening agents to said biocomponent immobilized within said matrix layer.

8. The biosensor of claim 1 wherein said transducer layer is covalently bound to one or more agents bound to the first end of said fiber optical cable, said one or more agents comprising one or more of cellulose, cellulose derivatives, silica, glass, dextran, starch, agarose, porous silica, chitin and chitosan.

9. The biosensor of claim 7 wherein said one or more gel-hardening agents comprise one or more of glutaraldehyde, polyethyleneimine, hexamethylenediamine, hexamethylene diisocyanate, 1,5-dinitro-2,4-difluorobenzene and formaldehyde.

10. The biosensor of claim 1 comprising a capillary tube having a first end and second end, wherein said second end of said capillary tube is coupled to a source of a molecule, said molecule is selected from the group consisting of oxygen, hydrogen peroxide, NADH, NADPH, a cofactor, or a substrate of said biocomponent, and

wherein said first end of said capillary tube is coupled to said matrix layer, and wherein said molecule is delivered into said matrix layer through said capillary tube.

11. The biosensor of claim 1 wherein said optical excitation source is selected from the group consisting of a halogen lamp, laser, and light emitting diode.

12. The biosensor of claim 1 wherein said electronic amplification device comprises a photomultiplier tube, an avalanche photodiode, and a p-i-n photodiode having an integrated amplifier.

13. The biosensor of claim 1 wherein said biocomponent is an enzyme.

14. The biosensor of claim 13 wherein a nucleotide coding sequence of said enzyme is part of a plasmid within a whole cell or is part of a chromosome of a whole cell and wherein said whole cell is within said matrix layer.

15. The biosensor of claim 1 wherein said biocomponent is selected from the group consisting of purified enzymes, live whole cells, and dead whole cells.

16. The biosensor of claim 1 arranged as an array of biosensors wherein each biosensor comprises a different biocomponent or mixture of biocomponents,

wherein each of said biocomponent interacts with one type of transducer.

17. The biosensor of claim 1 arranged as an array of biosensors wherein each biosensor comprises a different biocomponent or mixture of biocomponents, wherein said molecule is an analyte and said solution comprises a group of analytes, and wherein each biocomponent has a different substrate specificity for each analyte among said group of analytes, and wherein said difference in substrate specificity of each of said biocomponents in said array is used to determine individual concentrations of each analyte among said group of analytes.

18. The biosensor of claim 16 wherein each of said biosensors in said array are individually coupled to an optical excitation source, a photon detection device, an electronic amplification device, and signal processing circuitry through an optical fiber and wherein said signal processing circuitry detects the signal from each of said biosensors in said array.

19. The biosensor of claim 16 wherein each of said biosensors in said array are coupled to an optical excitation source, a photon detection device, an electronic amplification device, and signal processing circuitry through an optical fiber and wherein said signal processing circuitry detects the signal from each of said biosensors in said array.