Enzymatic Biosensing Systems

• US 20140235501A1
• Filed: 07/31/2012
• Published: 08/21/2014
• Est. Priority Date: 06/01/2001
• Status: Active Grant

• Current Assignee: Colorado State University Research Foundation
• Original Assignee: Colorado State University Research Foundation
• Inventors: Reardon, Kenneth F.
• Granted Patent: US 9,493,806 B2
• Time in Patent Office: Days
• US Class Current: 506/11
• 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 having a first side and a second side, and

a matrix layer having a first side and a second side, and

a biocomponent disposed 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 is 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 quasi-elastic light-scattering spectroscopy, photon correlation spectroscopy and laser Doppler velocimetry.

3. The biosensor of claim 1 wherein said matrix layer is a polymer.

4. The biosensor of claim 1 wherein said matrix layer is selected from the group consisting of a hydrogel or other polymer, and

wherein said hydrogel is selected from the group consisting of algal polysaccharides, agarose, alginate, gelatin, collagen, pectin, poly(carbamoyl) sulfonate, locust bean gum, and gellan, and

wherein said other polymer is selected from the group consisting of polyacrylamide, polystyrene, polymethacrylate, polyvinylalcohol and polyurethane, and

wherein said biocomponent is adsorbed within said matrix layer by physisorption or chemisorption.

5. The biosensor of claim 1 wherein said biocomponent is stabilized against chemical degradation selected from the group of methods consisting of

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 selected from the group consisting 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 a layer of molecules selected from the group consisting 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 bound to said matrix layer through adding crosslinking agents to said biocomponent disposed within said matrix layer, and

wherein said crosslinking agents are selected from the group consisting of glutaraldehyde, hexamethylene diisocyanate and 1,5-dinitro-2,4-difluorobenzene.

8. The biosensor of claim 1 wherein said transducer layer is covalently bound to a layer of molecules bound to the first end of said fiber optical cable, said layer of molecules is selected from the group consisting of cellulose, cellulose derivatives, silica, glass, dextran, starch, agarose, porous silica, chitin and chitosan.

9. The biosensor of claim 1 wherein said biocomponent is immobilized within said matrix layer by crosslinking said biocomponent to said matrix layer with crosslinking agents selected from the group consisting of glutaraldehyde, polyethyleneimine, hexamethylenediamine 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 12 wherein the nucleotide coding sequence of said enzymes are part of a plasmid within a whole cell or are part of the chromosome of a whole cell and wherein said whole cell is a 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 wherein said transducer layer is an optical transducer that interacts with oxygen, protons or halide ions.

17. 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.

18. The biosensor of claim 17 wherein each biocomponent has a different substrate specificity for each member of a group of compounds, and

wherein said difference in substrate specificity of each of said biocomponents in said array is used to determine individual concentrations of each member of said group of compounds.

19. The biosensor of claim 17 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.

20. The biosensor of claim 17 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.

21. A method of measuring the concentration of a molecule in a solution comprising

communicating the interaction of a biocomponent with said molecule to a display and/or data storage device by communication means, said communication means comprising said biocomponent, said molecule, oxygen, a transducer layer, a fiber optical cable, a photon-detection device, a signal processor and said display and/or data storage device,

wherein said biocomponent interacts with said molecule and either uses or generates oxygen in said solution during said interaction, and

wherein said biocomponent is in contact with said transducer layer, and

wherein said transducer layer luminesces and wherein said luminescence is partially quenched by said oxygen, and

wherein said luminescence is communicated to said photon-detection device through said fiber optical cable having a first end and a second end, said first end of said fiber optical cable is in contact and communicates with said transducer layer and said second end of said fiber optical cable is in contact and communicates with said signal processor, and

wherein said signal processor processes said communication from said luminescence of said transducer layer into a communication comprising said concentration of said molecule in said solution, and

wherein said signal processor communicates said concentration of said molecule in said solution to said display and/or data storage device.

22. A method of measuring the concentration of a molecule in a solution comprising

communicating the interaction of a biocomponent with said molecule to a display and/or data storage device by communication means, said communication means comprising said biocomponent, said molecule, hydrogen ions, a transducer layer, a fiber optical cable, a photon-detection device, a signal processor and said display and/or data storage device,

wherein said biocomponent interacts with said molecule and either uses or generates hydrogen ions in said solution during said interaction, and

wherein said biocomponent is in contact with said transducer layer, and

wherein said transducer layer luminesces and wherein said luminescence is partially quenched or enhanced by said hydrogen ions, and

wherein said luminescence is communicated to said photon-detection device through said fiber optical cable having a first end and a second end, said first end of said fiber optical cable is in contact and communicates with said transducer layer and said second end of said fiber optical cable is in contact and communicates with said signal processor, and

wherein said signal processor processes said communication from said luminescence of said transducer layer into a communication comprising said concentration of said molecule in said solution, and

wherein said signal processor communicates said concentration of said molecule in said solution to said display and/or data storage device.