CA2511549A1 - Hydrophilic cross-linking agents for use in enzymatic sensors - Google Patents
Hydrophilic cross-linking agents for use in enzymatic sensors Download PDFInfo
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- 239000003431 cross linking reagent Substances 0.000 title claims abstract description 39
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 19
- 102000004190 Enzymes Human genes 0.000 claims abstract description 38
- 108090000790 Enzymes Proteins 0.000 claims abstract description 38
- 239000012528 membrane Substances 0.000 claims abstract description 33
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 31
- 239000008103 glucose Substances 0.000 claims abstract description 31
- 239000012491 analyte Substances 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 229940088598 enzyme Drugs 0.000 claims description 35
- 108010015776 Glucose oxidase Proteins 0.000 claims description 16
- 239000004366 Glucose oxidase Substances 0.000 claims description 15
- 229940116332 glucose oxidase Drugs 0.000 claims description 15
- 235000019420 glucose oxidase Nutrition 0.000 claims description 15
- 229920001223 polyethylene glycol Polymers 0.000 claims description 15
- XLJMAIOERFSOGZ-UHFFFAOYSA-M cyanate Chemical compound [O-]C#N XLJMAIOERFSOGZ-UHFFFAOYSA-M 0.000 claims description 8
- 239000012948 isocyanate Substances 0.000 claims description 8
- 150000002513 isocyanates Chemical class 0.000 claims description 8
- 125000005442 diisocyanate group Chemical group 0.000 claims description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims description 6
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 6
- 229920001451 polypropylene glycol Polymers 0.000 claims description 6
- 101710088194 Dehydrogenase Proteins 0.000 claims description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 4
- 229930091371 Fructose Natural products 0.000 claims description 4
- 239000005715 Fructose Substances 0.000 claims description 4
- 108010073450 Lactate 2-monooxygenase Proteins 0.000 claims description 4
- 108030001003 Long-chain-alcohol oxidases Proteins 0.000 claims description 4
- 108090000854 Oxidoreductases Proteins 0.000 claims description 4
- 102000004316 Oxidoreductases Human genes 0.000 claims description 4
- 108010060059 Sarcosine Oxidase Proteins 0.000 claims description 4
- 102000008118 Sarcosine oxidase Human genes 0.000 claims description 4
- 108090000531 Amidohydrolases Proteins 0.000 claims description 3
- 102000004092 Amidohydrolases Human genes 0.000 claims description 3
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims description 3
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims description 3
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims description 3
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims description 3
- 102000013009 Pyruvate Kinase Human genes 0.000 claims description 3
- 108020005115 Pyruvate Kinase Proteins 0.000 claims description 3
- 229920005862 polyol Polymers 0.000 claims description 3
- 150000003077 polyols Chemical class 0.000 claims description 3
- 229920001577 copolymer Polymers 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 4
- 108010046334 Urease Proteins 0.000 claims 2
- 238000009792 diffusion process Methods 0.000 abstract description 5
- 230000036571 hydration Effects 0.000 abstract description 4
- 238000006703 hydration reaction Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000010348 incorporation Methods 0.000 abstract description 3
- 230000037361 pathway Effects 0.000 abstract description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 10
- 239000001301 oxygen Substances 0.000 description 10
- 229910052760 oxygen Inorganic materials 0.000 description 10
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 4
- 150000001299 aldehydes Chemical class 0.000 description 4
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960003624 creatine Drugs 0.000 description 2
- 239000006046 creatine Substances 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 235000012209 glucono delta-lactone Nutrition 0.000 description 2
- 229960003681 gluconolactone Drugs 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 108010077078 Creatinase Proteins 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 241001633942 Dais Species 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 206010041235 Snoring Diseases 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 150000001913 cyanates Chemical class 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- -1 diimm_ides Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- RGXCTRIQQODGIZ-UHFFFAOYSA-O isodesmosine Chemical compound OC(=O)C(N)CCCC[N+]1=CC(CCC(N)C(O)=O)=CC(CCC(N)C(O)=O)=C1CCCC(N)C(O)=O RGXCTRIQQODGIZ-UHFFFAOYSA-O 0.000 description 1
- 150000002596 lactones Chemical group 0.000 description 1
- 239000012035 limiting reagent Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 229920005606 polypropylene copolymer Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
- C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Emergency Medicine (AREA)
- Biomedical Technology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
An enzymatic sensor comprising an enzyme that generates a signal upon contact with an analyte; a substrate; and a cross-linking agent that comprises one or more hydrophilic moieties. The enzyme is immobilized in the substrate via the hydrophilic cross-linking agent. A biocompatible membrane, comprising the enzyme, substrate and the hydrophilic cross-linking agent, is applied to a sensor to produce an enzymatic sensor. The enzymatic sensor provides enhanced sensing capabilities by improving the diffusion of reactive species and thereby increasing the overall sensitivity and lifetime of the sensor. The incorporation of hydrophilic moieties into the cross-linking agent optimizes hydration of the sensor and creates hydrophilic channels or pathways for reactive species, such as H202 and the analyte of interest. The membrane and enzymatic sensor can be used in a method for detecting an analyte, such as glucose.
Description
HYDROPHILIC CROSS-LINKING AGENTS FOR USE IN ENZYMATIC
SENSORS
TECHNICAL FIELD OF THE INVENTION
~0001~ This invention relates generally to cross-linking agents for use with enzymatic sensors and to methods of making and using such materials. The cross-linking agents are hydrophilic and suitable for use with enzymatic sensors for detecting analytes, such as glucose.
1o BACKGROUND OF THE INVENTION
~0002~ Biosensors are small devices that use biological recognition properties for selective detection of various analytes or biomolecules. Typically, the sensor will produce a signal that is quantitatively related to the concentration of the analyte. To achieve a quantitative signal, a recognition molecule or combination of molecules is often immobilized at a suitable transducer, which converts the biological recognition event into a quantttative response.
~0003~ The need for the continuous monitoring of biological markers (analytes) in medicine has sparked a tremendous interest in the study of biosensors in recent years.
Without question, the greatest interest has been geared toward the development of sensors to detect glucose. In particular, enzymatic (amperometric) glucose electrodes have been studied in more detail than any other biosensors. Electroenzymatic biosensors use enzymes to convert a concentration of analyte to an electrical signal. For a review of some of the operating principles of biosensors, see Bergveld, et al., Advances in Biosensors, Supplement 1, p. 31-91, Tuxner ed., and Collison, et al., Anal.
Chem.
62:425-437 (1990). Typically, the enzyme is immobilized onto tze sensor via use of a cross-linking agent, such as glutaraldehyde.
~0004~ An additional commercial application of this technology focuses on sensors that can be used to monitor fermentation reactions in the biotechnology industry. From a scientific and commercial standpoint, interest has grown beyond glucose to other analytes for the diagnosis of numerous medical conditions other than diabetes. One example of another analyte detectable via enzymatic sensors is lactate.
~0005~ A typical glucose sensor works by a reaction in which glucose reacts with oxygen in the presence of glucose oxidase (GOd) to form gluconolactone and hydrogen peroxide. The gluconolactone further reacts with water to hydrolyze the lactone ring and produce gluconic acid. The H2Oz formed is electrochemically oxidized at an electrode as shown below (Equation 1):
HZO2 --~ Oz +2e- +2H+ (I) The current measured by the sensor/potentiostat (+0.5 to +0.7 v oxidation at Pt black electrode) is the result of the two electrons generated by the oxidation of the HZOz. Alternatively, one can measure the decrease in the oxygen by amperometric measurement (-0.5 to -1 V reduction at a Pt black electrode).
~0007~ The stoichiometzy of the GOd reaction points to a challenge of developing a reliable glucose sensor. If oxygen and glucose are present in equimolax concentrations, then the HzOz is stoichiometzically related to the amount of glucose that reacts at the enzyme. In dais case, the ultimate current is also proportional to the amount of glucose that reacts with the enzyme. If there is insufficient oxygen for all of the glucose to react with the enzyme, then the current will be proportional to the oxygen concentration, not the glucose concentration. For the sensor to be a true glucose sensor, glucose must be the limiting reagent, i.e. the Oz concentration must be in excess for all potential glucose concentrations.
~0008~ For example, the glucose concentration in the body of a diabetic patient can vary from 2 to 30 mM (millimoles per liter or 36 to 540 mg/dl), whereas the typical oxygen concentration in the tissue is 0.02 to 0.~ inM (see, Fisher, et al., Biomed.
SENSORS
TECHNICAL FIELD OF THE INVENTION
~0001~ This invention relates generally to cross-linking agents for use with enzymatic sensors and to methods of making and using such materials. The cross-linking agents are hydrophilic and suitable for use with enzymatic sensors for detecting analytes, such as glucose.
1o BACKGROUND OF THE INVENTION
~0002~ Biosensors are small devices that use biological recognition properties for selective detection of various analytes or biomolecules. Typically, the sensor will produce a signal that is quantitatively related to the concentration of the analyte. To achieve a quantitative signal, a recognition molecule or combination of molecules is often immobilized at a suitable transducer, which converts the biological recognition event into a quantttative response.
~0003~ The need for the continuous monitoring of biological markers (analytes) in medicine has sparked a tremendous interest in the study of biosensors in recent years.
Without question, the greatest interest has been geared toward the development of sensors to detect glucose. In particular, enzymatic (amperometric) glucose electrodes have been studied in more detail than any other biosensors. Electroenzymatic biosensors use enzymes to convert a concentration of analyte to an electrical signal. For a review of some of the operating principles of biosensors, see Bergveld, et al., Advances in Biosensors, Supplement 1, p. 31-91, Tuxner ed., and Collison, et al., Anal.
Chem.
62:425-437 (1990). Typically, the enzyme is immobilized onto tze sensor via use of a cross-linking agent, such as glutaraldehyde.
~0004~ An additional commercial application of this technology focuses on sensors that can be used to monitor fermentation reactions in the biotechnology industry. From a scientific and commercial standpoint, interest has grown beyond glucose to other analytes for the diagnosis of numerous medical conditions other than diabetes. One example of another analyte detectable via enzymatic sensors is lactate.
~0005~ A typical glucose sensor works by a reaction in which glucose reacts with oxygen in the presence of glucose oxidase (GOd) to form gluconolactone and hydrogen peroxide. The gluconolactone further reacts with water to hydrolyze the lactone ring and produce gluconic acid. The H2Oz formed is electrochemically oxidized at an electrode as shown below (Equation 1):
HZO2 --~ Oz +2e- +2H+ (I) The current measured by the sensor/potentiostat (+0.5 to +0.7 v oxidation at Pt black electrode) is the result of the two electrons generated by the oxidation of the HZOz. Alternatively, one can measure the decrease in the oxygen by amperometric measurement (-0.5 to -1 V reduction at a Pt black electrode).
~0007~ The stoichiometzy of the GOd reaction points to a challenge of developing a reliable glucose sensor. If oxygen and glucose are present in equimolax concentrations, then the HzOz is stoichiometzically related to the amount of glucose that reacts at the enzyme. In dais case, the ultimate current is also proportional to the amount of glucose that reacts with the enzyme. If there is insufficient oxygen for all of the glucose to react with the enzyme, then the current will be proportional to the oxygen concentration, not the glucose concentration. For the sensor to be a true glucose sensor, glucose must be the limiting reagent, i.e. the Oz concentration must be in excess for all potential glucose concentrations.
~0008~ For example, the glucose concentration in the body of a diabetic patient can vary from 2 to 30 mM (millimoles per liter or 36 to 540 mg/dl), whereas the typical oxygen concentration in the tissue is 0.02 to 0.~ inM (see, Fisher, et al., Biomed.
Biochem. Acta. 48:965-971 (1989). This ratio in the body means that the sensor would be running in the Michaelis Menten limited regime and would be very insensitive to small changes in the glucose concentration. This problem has been called the "oxygen deficit problem". Accordingly, a method or system must be devised to either increase the OZ in the GOd enzyme layer, decrease the glucose concentration, or devise a sensor that does not use Oz.
There is a need for an enzymatic sensor with enhanced sensing capabilities and that overcomes difficulties associated with conventional cross-linking agents such as glutaraldehyde. The present invention fulfills these needs and provides other related advantages.
SUMMARY OF THE INVENTION
~0010~ The invention provides a biocompatible membrane comprising an enzyme and a hydrophilic cross-linking agent, which membrane can be applied to a sensor. Also provided is an enzymatic sensor comprising an enzyme that generates a signal upon contact with an analyte; a substrate; and a cross-linking agent that comprises one or snore hydrophilic moieties. The enzyme is immobilized onto the substrate via the hydrophilic cross-linking agent. The enzymatic sensor provides enhanced sensing capabilities by improving the diffusion of reactive species and thereby increasing the overall sensitivity and lifetime of the sensor. The incorporation of hydrophilic moieties into the cross-linking agent optimizes hydration of the sensor and creates hydrophilic channels or pathways for reactive species, such as H2O2 and the analyte of interest.
~0011~ In one embodiment, the one or more hydrophilic moieties comprise a polyol selected from the group consisting of polyethylene glycol, polypropylene glycol and a copolymer of polypropylene glycol and polyethylene glycol, and the cross-linking agent comprises an aldehyde, diimm_ide, cyanate, isocyanate, or diisocyanate.
A typical cross-linking agent of the invention comprises:
There is a need for an enzymatic sensor with enhanced sensing capabilities and that overcomes difficulties associated with conventional cross-linking agents such as glutaraldehyde. The present invention fulfills these needs and provides other related advantages.
SUMMARY OF THE INVENTION
~0010~ The invention provides a biocompatible membrane comprising an enzyme and a hydrophilic cross-linking agent, which membrane can be applied to a sensor. Also provided is an enzymatic sensor comprising an enzyme that generates a signal upon contact with an analyte; a substrate; and a cross-linking agent that comprises one or snore hydrophilic moieties. The enzyme is immobilized onto the substrate via the hydrophilic cross-linking agent. The enzymatic sensor provides enhanced sensing capabilities by improving the diffusion of reactive species and thereby increasing the overall sensitivity and lifetime of the sensor. The incorporation of hydrophilic moieties into the cross-linking agent optimizes hydration of the sensor and creates hydrophilic channels or pathways for reactive species, such as H2O2 and the analyte of interest.
~0011~ In one embodiment, the one or more hydrophilic moieties comprise a polyol selected from the group consisting of polyethylene glycol, polypropylene glycol and a copolymer of polypropylene glycol and polyethylene glycol, and the cross-linking agent comprises an aldehyde, diimm_ide, cyanate, isocyanate, or diisocyanate.
A typical cross-linking agent of the invention comprises:
PEG
R-(C-C-C-C)N-R;
PEG; or PEG
R-(C=C-C-C)N-R;
PEG; or PEG R
PEG
R
wherein R is an aldehyde, diimtnide, cyanate, isocyanate, oY diisocyanate, and N is an integer from 1 to about 10. In some embodiments, N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
(0012 Examples of enzymes for use with the invention include, but are not litni.ted to, glucose oxidase, a-hydYOxy oxidase, lactate oxidase, uYease, creatine ainidohydYOlase, creative amidinohydrolase, sarcosine oxidase, glutamate dehydYOgenase, pyYUVate liinase, long chain alcohol oxidase, lactate dehydYOgenase, and fructose dehydrogenase.
(0013 The invention additionally provides a method of measuring an analyte in a tissue of a subject. The method compYises introducing an enzymatic sensoY of the invention into the tissue of the subject, and detecting the signal geneYated by the enzyme.
The amount of signal corYesponds to the amount of analyte. PYefeYably, the analyte is glucose and the enzyme is glucose oxidase.
R-(C-C-C-C)N-R;
PEG; or PEG
R-(C=C-C-C)N-R;
PEG; or PEG R
PEG
R
wherein R is an aldehyde, diimtnide, cyanate, isocyanate, oY diisocyanate, and N is an integer from 1 to about 10. In some embodiments, N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
(0012 Examples of enzymes for use with the invention include, but are not litni.ted to, glucose oxidase, a-hydYOxy oxidase, lactate oxidase, uYease, creatine ainidohydYOlase, creative amidinohydrolase, sarcosine oxidase, glutamate dehydYOgenase, pyYUVate liinase, long chain alcohol oxidase, lactate dehydYOgenase, and fructose dehydrogenase.
(0013 The invention additionally provides a method of measuring an analyte in a tissue of a subject. The method compYises introducing an enzymatic sensoY of the invention into the tissue of the subject, and detecting the signal geneYated by the enzyme.
The amount of signal corYesponds to the amount of analyte. PYefeYably, the analyte is glucose and the enzyme is glucose oxidase.
DETAILED DESCRIPTION
~0014~ All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.
~0015~ As used herein, "adhered to" or "adhered thereto" means stuck to or fused with such that a substance adhered to a surface remains substantially attached to or closely associated with the surface.
[001G] As used herein, "a" or "an" means at least one, and unless clearly indicated otherwise, includes a plurality.
Overview ~0017~ The invention is based on the discovery that hydrophilic cross-linking agents can be used in enzymatic sensors, resulting in sensors with improved hydration and enhanced sensitivity. The invention provides a biocompatible membrane comprising an enzyme that generates a signal upon contact with an analyte, a substrate, and a hydrophilic cross-linking agent. The enzyme is immobilized in the substrate via the hydrophilic cross-linking agent. The cross-linking agent comprises one or more hydrophilic moieties. The membrane can be applied to a sensor to produce an enzymatic sensor.
~0018~ The enzymatic sensors of the invention provide enhanced sensing capabilities by improving the diffusion of reactive species and thereby increasing the overall sensitivity and lifetime of the sensors. The incorporation of hydrophilic moieties into the cross-linking agent optimizes hydration of the sensor and creates hydrophilic channels or pathways for reactive species, such as HzOz and the analyte of interest. The increased hycliation of the sensor environment may also reduce the formation of a skin over the sensor membrane, as occurs with use of the conventional cross-linking agent, glutaraldehyde.
~0014~ All scientific and technical terms used in this application have meanings commonly used in the art unless otherwise specified. As used in this application, the following words or phrases have the meanings specified.
~0015~ As used herein, "adhered to" or "adhered thereto" means stuck to or fused with such that a substance adhered to a surface remains substantially attached to or closely associated with the surface.
[001G] As used herein, "a" or "an" means at least one, and unless clearly indicated otherwise, includes a plurality.
Overview ~0017~ The invention is based on the discovery that hydrophilic cross-linking agents can be used in enzymatic sensors, resulting in sensors with improved hydration and enhanced sensitivity. The invention provides a biocompatible membrane comprising an enzyme that generates a signal upon contact with an analyte, a substrate, and a hydrophilic cross-linking agent. The enzyme is immobilized in the substrate via the hydrophilic cross-linking agent. The cross-linking agent comprises one or more hydrophilic moieties. The membrane can be applied to a sensor to produce an enzymatic sensor.
~0018~ The enzymatic sensors of the invention provide enhanced sensing capabilities by improving the diffusion of reactive species and thereby increasing the overall sensitivity and lifetime of the sensors. The incorporation of hydrophilic moieties into the cross-linking agent optimizes hydration of the sensor and creates hydrophilic channels or pathways for reactive species, such as HzOz and the analyte of interest. The increased hycliation of the sensor environment may also reduce the formation of a skin over the sensor membrane, as occurs with use of the conventional cross-linking agent, glutaraldehyde.
(0019 In addition, the cross-linking agents of the invention avoid or dhrunish negative consequences of e-beam sterilization of sensors that occurs with conventional cross-linking agents. For example, glutaraldehyde and other conventional cross-linking agents can leave by-products that damage the sensor during e-beam sterilization.
Biocompatible Membranes ~0020~ A glucose sensor intended for iTa vivo use requires that the supply of oxygen in the vicinity of the sensing element not be depleted. Additionally, the glucose should diffuse to the sensor at a controlled rate. This diffusion of the analyte to the sensor occurs through a membrane adhered to the surface of the sensor.
~verall, the membrane should control the relative rates of diffusion of oxygen and glucose to the sensor so that the local concentration of oxygen is not depleted.
Additionally, glucose sensors intended for in vivo use must also be biocompatible with the body.
Thus, the enzymes) used in such sensors must be protected from degradation or denaturation, while the elements of such sensors must be protected from molecules that would foul the sensors or their accuracy will decrease over tithe.
~0021~ In one aspect, the present invention provides a biocompatible membrane comprising an enzyme immobilized in a substrate by a hydrophilic cross-linking agent. The substrate is typically a polymer matrix. Suitable polymeric compositions are known in the art (see, e.g., U.S. Patent Nos. 5,777,060 and 5,786,439, both of which are incorporated herein by reference). The hydrophilic cross-linking agent comprises a cross-linking agent that includes one or more hycliophilic moieties.
Examples of cross-linking agents having hydroplvlic moieties are described below.
~0022~ In another aspect of the invention, the homogeneity of the membrane can be further enhanced by introducing hydrophilic moieties into other proteins present in the membrane. For example, pegylation of albumin, glucose oxidase, or other enzyme present in the membrane would create an even more homogeneous structure.
Hydrophilic Cross-linking Agents (0023 The biocompatible membrane of the invention comprises a polymeric composition that contains an enzyme that generates a signal upon contact with an analyte of interest. Typically, die enzyme is immobilized in the composition or membrane that covers the sensor via a cross-linking agent. Examples of cross-linking agents suitable for use with the invention include, but are not limited to, molecules that comprise aldehydes, diimm_ides, cyanates, isocyanates, and diisocyanates, into which hydrophilic moieties are incorporated. Examples of hydrophilic moieties include, but are not limited to: polyols, such as polyethylene glycol, polypropylene glycol and copolymers of polypropylene glycol and polyethylene glycol; and other non-ionic surfactants, including Tweens 20-80.
(0024 Representative embodiments of hydrophilic cross-linking agents include:
PEG
R-(C-C-C-C)N-R;
PEG;
PEG
PEG R
~\\~ PEG
R
R-(C=C-C-C)N-R;
PEG; and wherein R is an aldehyde, diin~nide, cyanate, isocyanate, or diis~cyanate, and N is an integer from 1 to about 10. In some embodiments, N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
Biosensor ~0025~ Biosensors typically include a transducer, or enzyme, that generates a signal upon contact with an analyte of interest, and is adhered to a detector, such as an electrode. For example, glucose sensors suitable for iyt vivo use can be prepared by depositing a membrane comprising a glucose sensitive enzyme, such as glucose oxidase, onto an electrode via an electromotive plating process. The membrane can be applied by immersion of the sensor in a bath comprising glucose oxidase, a stabilizing protein, a surfactant and a buffer for conductivity and stability of the protein solution, and the enzyme is then deposited onto the electrode potentiometzically. Alternatively, the membrane can be applied using a microelectrogravimetric plating method, such as is described in U.S. Patent No. 6,340,021, issued January 22, 2002.
~002G~ The invention provides a sensor for measuring an analyte of interest in biological tissue, the sensor having a coating comprising a biocompatible membrane of the invention that includes an enzyme serving as a transducer that generates a signal upon contact with the analyte. The sensor can be used in vitro, or is suitable for use as an implantable biosensor or other in vivo applications. In a preferred embodiment, the analyte is glucose and the transducer is glucose oxidase. Other enzymes can serve as transducers as appropriate for the analyte of interest and examples of such enzymes include, but are not limited to, a-hydroxy oxidase, lactate oxidase, unease, creatine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, glutamate dehydrogenase, pyruvate kinase, long chain alcohol oxidase, lactate dehydrogenase, and fructose dehydrogenase.
Methods ~0027~ The invention additionally provides a method of measuring an analyte in a tissue of a subject. The method comprises introducing an enzymatic sensor of the invention into the tissue of the subject, and detecting the signal generated by the enzyme.
The amount of signal generated corresponds to the amount of analyte.
Preferably, the analyte is glucose and the enzyme is glucose oxidase. As described herein, other analytes and other corresponding enzymes can be used. The sensor is typically introduced into the tissue iya vivo, via subcutaneous implantation, although those skilled in the art will appreciate other means for introducing the sensor into the tissue.
~0028~ The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. Merely by way of example a variety of solvents, membrane formation methods, and other materials may be used without departing from the scope of the invention. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.
~0029~ All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
Biocompatible Membranes ~0020~ A glucose sensor intended for iTa vivo use requires that the supply of oxygen in the vicinity of the sensing element not be depleted. Additionally, the glucose should diffuse to the sensor at a controlled rate. This diffusion of the analyte to the sensor occurs through a membrane adhered to the surface of the sensor.
~verall, the membrane should control the relative rates of diffusion of oxygen and glucose to the sensor so that the local concentration of oxygen is not depleted.
Additionally, glucose sensors intended for in vivo use must also be biocompatible with the body.
Thus, the enzymes) used in such sensors must be protected from degradation or denaturation, while the elements of such sensors must be protected from molecules that would foul the sensors or their accuracy will decrease over tithe.
~0021~ In one aspect, the present invention provides a biocompatible membrane comprising an enzyme immobilized in a substrate by a hydrophilic cross-linking agent. The substrate is typically a polymer matrix. Suitable polymeric compositions are known in the art (see, e.g., U.S. Patent Nos. 5,777,060 and 5,786,439, both of which are incorporated herein by reference). The hydrophilic cross-linking agent comprises a cross-linking agent that includes one or more hycliophilic moieties.
Examples of cross-linking agents having hydroplvlic moieties are described below.
~0022~ In another aspect of the invention, the homogeneity of the membrane can be further enhanced by introducing hydrophilic moieties into other proteins present in the membrane. For example, pegylation of albumin, glucose oxidase, or other enzyme present in the membrane would create an even more homogeneous structure.
Hydrophilic Cross-linking Agents (0023 The biocompatible membrane of the invention comprises a polymeric composition that contains an enzyme that generates a signal upon contact with an analyte of interest. Typically, die enzyme is immobilized in the composition or membrane that covers the sensor via a cross-linking agent. Examples of cross-linking agents suitable for use with the invention include, but are not limited to, molecules that comprise aldehydes, diimm_ides, cyanates, isocyanates, and diisocyanates, into which hydrophilic moieties are incorporated. Examples of hydrophilic moieties include, but are not limited to: polyols, such as polyethylene glycol, polypropylene glycol and copolymers of polypropylene glycol and polyethylene glycol; and other non-ionic surfactants, including Tweens 20-80.
(0024 Representative embodiments of hydrophilic cross-linking agents include:
PEG
R-(C-C-C-C)N-R;
PEG;
PEG
PEG R
~\\~ PEG
R
R-(C=C-C-C)N-R;
PEG; and wherein R is an aldehyde, diin~nide, cyanate, isocyanate, or diis~cyanate, and N is an integer from 1 to about 10. In some embodiments, N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
Biosensor ~0025~ Biosensors typically include a transducer, or enzyme, that generates a signal upon contact with an analyte of interest, and is adhered to a detector, such as an electrode. For example, glucose sensors suitable for iyt vivo use can be prepared by depositing a membrane comprising a glucose sensitive enzyme, such as glucose oxidase, onto an electrode via an electromotive plating process. The membrane can be applied by immersion of the sensor in a bath comprising glucose oxidase, a stabilizing protein, a surfactant and a buffer for conductivity and stability of the protein solution, and the enzyme is then deposited onto the electrode potentiometzically. Alternatively, the membrane can be applied using a microelectrogravimetric plating method, such as is described in U.S. Patent No. 6,340,021, issued January 22, 2002.
~002G~ The invention provides a sensor for measuring an analyte of interest in biological tissue, the sensor having a coating comprising a biocompatible membrane of the invention that includes an enzyme serving as a transducer that generates a signal upon contact with the analyte. The sensor can be used in vitro, or is suitable for use as an implantable biosensor or other in vivo applications. In a preferred embodiment, the analyte is glucose and the transducer is glucose oxidase. Other enzymes can serve as transducers as appropriate for the analyte of interest and examples of such enzymes include, but are not limited to, a-hydroxy oxidase, lactate oxidase, unease, creatine amidohydrolase, creatine amidinohydrolase, sarcosine oxidase, glutamate dehydrogenase, pyruvate kinase, long chain alcohol oxidase, lactate dehydrogenase, and fructose dehydrogenase.
Methods ~0027~ The invention additionally provides a method of measuring an analyte in a tissue of a subject. The method comprises introducing an enzymatic sensor of the invention into the tissue of the subject, and detecting the signal generated by the enzyme.
The amount of signal generated corresponds to the amount of analyte.
Preferably, the analyte is glucose and the enzyme is glucose oxidase. As described herein, other analytes and other corresponding enzymes can be used. The sensor is typically introduced into the tissue iya vivo, via subcutaneous implantation, although those skilled in the art will appreciate other means for introducing the sensor into the tissue.
~0028~ The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. Merely by way of example a variety of solvents, membrane formation methods, and other materials may be used without departing from the scope of the invention. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.
~0029~ All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.
Claims (16)
1. A biocompatible membrane comprising:
(a) an enzyme that generates a signal upon contact with an analyte;
(b) a substrate; and (c) a cross-linking agent that comprises one or more hydrophilic moieties, wherein the enzyme is immobilized in the substrate via the hydrophilic cross-linking agent.
(a) an enzyme that generates a signal upon contact with an analyte;
(b) a substrate; and (c) a cross-linking agent that comprises one or more hydrophilic moieties, wherein the enzyme is immobilized in the substrate via the hydrophilic cross-linking agent.
2. The membrane of claim 1, wherein the one or more hydrophilic moieties comprise a polyol selected from the group consisting of polyethylene glycol, polypropylene glycol and a copolymer of polypropylene glycol and polyethylene glycol.
3. The membrane of claim 1, wherein the cross-linking agent comprises an aldehyde, diimmide, cyanate, isocyanate, or diisocyanate.
4. The membrane of claim 1, wherein the cross-linking agent comprises wherein R is an aldehyde, diimmide, cyanate, isocyanate, or diisocyanate, and N is an integer from 1 to about 10.
5. The membrane of claim 4, wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
6. The membrane of claim 1, wherein the cross-linking agent comprises wherein R is an aldehyde, diimmide, cyanate, isocyanate, or diisocyanate, N is an integer from 1 to about 10.
7. The membrane of claim 6, wherein N is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
8. The membrane of claim 1, wherein the cross-linking agent comprises wherein R is an aldehyde, diimmide, cyanate, isocyanate, or diisocyanate.
9. The membrane of claim 1, wherein the enzyme is glucose oxidase, .alpha.-hydroxy oxidase, lactate oxidase, urease, creative amidohydrolase, creative amidinohydrolase, sarcosine oxidase, glutamate dehydrogenase, pyruvate kinase, long chain alcohol oxidase, lactate dehydrogenase, or fructose dehydrogenase.
10. The membrane of claim 1, wherein the analyte is glucose and the enzyme is glucose oxidase.
11. The membrane of claim 1, wherein the substrate comprises a polymer matrix.
12. An enzymatic sensor comprising an electrode and the membrane of claim 1 adhered to the electrode.
13. The sensor of claim 12, wherein the analyte is glucose and the enzyme is glucose oxidase.
14. A method of measuring an analyte in a tissue of a subject, the method comprising introducing a sensor of claim 12 into the tissue of the subject and detecting the signal generated by the enzyme, wherein the amount of signal corresponds to the amount of analyte.
15. The method of claim 14, wherein the enzyme is glucose oxidase, .alpha.-hydroxy oxidase, lactate oxidase, urease, creative amidohydrolase, creative amidinohydrolase, sarcosine oxidase, glutamate dehydrogenase, pyruvate kinase, long chain alcohol oxidase, lactate dehydrogenase, or fructose dehydrogenase.
16. The method of claim 14, wherein the analyte is glucose and the enzyme is glucose oxidase.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US33550602A | 2002-12-31 | 2002-12-31 | |
| US10/335,506 | 2002-12-31 | ||
| PCT/US2003/041060 WO2004060297A2 (en) | 2002-12-31 | 2003-12-19 | Hydrophilic cross-linking agents for use in enzymatic sensors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2511549A1 true CA2511549A1 (en) | 2004-07-22 |
Family
ID=32710917
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002511549A Abandoned CA2511549A1 (en) | 2002-12-31 | 2003-12-19 | Hydrophilic cross-linking agents for use in enzymatic sensors |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP1587546A4 (en) |
| AU (1) | AU2003297503A1 (en) |
| CA (1) | CA2511549A1 (en) |
| WO (1) | WO2004060297A2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9493806B2 (en) | 2001-06-01 | 2016-11-15 | Colorado State University Research Foundation | Enzymatic biosensing systems |
| US9493805B2 (en) | 2001-06-01 | 2016-11-15 | Colorado State University Research Foundation | Enzymatic biosensors with enhanced activity retention for detection of organic compounds |
| US9796998B2 (en) | 2007-04-09 | 2017-10-24 | Colorado State University Research Foundation | Oxygenase-based biosensing systems for measurement of halogenated alkene concentrations |
| US20110082356A1 (en) * | 2009-10-01 | 2011-04-07 | Medtronic Minimed, Inc. | Analyte sensor apparatuses having interference rejection membranes and methods for making and using them |
| WO2012071471A2 (en) | 2010-11-22 | 2012-05-31 | Colorado State University Research Foundation | Biosensing systems for measurement of lactose |
| WO2013019982A2 (en) | 2011-08-02 | 2013-02-07 | Colorado State University Research Foundation | Biosensing system with extended lifetime via cofactor recycling |
| US10669153B2 (en) | 2017-05-23 | 2020-06-02 | International Business Machines Corporation | Neuro-chemical sensor with selectively permeable membrane on nano-electrode |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3106456A1 (en) * | 1981-02-21 | 1982-10-07 | Röhm GmbH, 6100 Darmstadt | METHOD FOR THE PRODUCTION OF PEARL-SHAPED, HYDROPHILIC, POLYMER-BASED POLYMERS TO PROTEINS |
| US5786439A (en) * | 1996-10-24 | 1998-07-28 | Minimed Inc. | Hydrophilic, swellable coatings for biosensors |
| US5874165A (en) * | 1996-06-03 | 1999-02-23 | Gore Enterprise Holdings, Inc. | Materials and method for the immobilization of bioactive species onto polymeric subtrates |
| US6485703B1 (en) * | 1998-07-31 | 2002-11-26 | The Texas A&M University System | Compositions and methods for analyte detection |
| WO2001022820A1 (en) * | 1999-09-30 | 2001-04-05 | The General Hospital Corporation | Use of pramipexole as a treatment for cocaine craving |
| CA2395254C (en) * | 1999-12-24 | 2010-05-11 | Kyowa Hakko Kogyo Co., Ltd. | Branched polyalkylene glycols |
-
2003
- 2003-12-19 AU AU2003297503A patent/AU2003297503A1/en not_active Abandoned
- 2003-12-19 WO PCT/US2003/041060 patent/WO2004060297A2/en not_active Application Discontinuation
- 2003-12-19 EP EP03814924A patent/EP1587546A4/en not_active Withdrawn
- 2003-12-19 CA CA002511549A patent/CA2511549A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| AU2003297503A1 (en) | 2004-07-29 |
| WO2004060297A3 (en) | 2005-05-06 |
| AU2003297503A8 (en) | 2004-07-29 |
| WO2004060297A2 (en) | 2004-07-22 |
| EP1587546A4 (en) | 2007-02-14 |
| EP1587546A2 (en) | 2005-10-26 |
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