WO2003035891A2 - Method for formulating and immobilizing a protein matrix and a protein matrix for use in a sensor - Google Patents

Method for formulating and immobilizing a protein matrix and a protein matrix for use in a sensor Download PDF

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Publication number
WO2003035891A2
WO2003035891A2 PCT/US2002/030942 US0230942W WO03035891A2 WO 2003035891 A2 WO2003035891 A2 WO 2003035891A2 US 0230942 W US0230942 W US 0230942W WO 03035891 A2 WO03035891 A2 WO 03035891A2
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Prior art keywords
protein
formulating
mixture
protein mixture
cross
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PCT/US2002/030942
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French (fr)
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WO2003035891A3 (en
Inventor
Rajiv Shah
Bahar Reghabi
Rudolph A. Montalvo
Yanan Zhang
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Medtronic Minimed, Inc.
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Priority to AU2002327774A priority Critical patent/AU2002327774A1/en
Priority to DE60226759T priority patent/DE60226759D1/en
Priority to EP02763782A priority patent/EP1437937B1/en
Priority to CA002457545A priority patent/CA2457545A1/en
Priority to JP2003538391A priority patent/JP2005507007A/en
Publication of WO2003035891A2 publication Critical patent/WO2003035891A2/en
Publication of WO2003035891A3 publication Critical patent/WO2003035891A3/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/817Enzyme or microbe electrode

Definitions

  • Embodiments of the present invention claim priority from a U.S. Provisional Application entitled “Method for Formulating and Immobilizing a Protein Matrix and a Protein Matrix for Use in a Sensor," Serial No. 60/335,663, filed October 23, 2001, the contents of which are incorporated by reference herein.
  • the present invention relates, generally, to methods and systems for formulating and immobilizing a protein matrix and/or enzyme and, in particular embodiments, to immobilizing proteins or enzymes that are physically and chemically stable over time, for example, for use in short-term or long-term sensors and biosensors.
  • biosensors and microelectronics have resulted in the availability of portable diagnostic medical equipment and has improved the quality of life for countless people.
  • many diabetics currently utilize diagnostic medical equipment in the comfort of their own homes the vast majority of such devices still require diabetics to draw their own blood and to inject their own insulin. Drawing blood typically requires pricking a finger.
  • the sensing system illustrated in Figure 1 comprises an implantable infusion pump 12 with a catheter 20 for dispensing an infusion formulation and a lead 14 connecting the infusion pump to a sensing device 16.
  • the sensing device 16 may be inserted into a vein, an artery, or any other part of a human body where it could sense a desired parameter of the implant environment.
  • a window may be provided in the sensing device 16 to facilitate sensing.
  • An active sensing matrix 18, such as an enzyme may be placed inside of the sensing device 16.
  • the matrix 18 may be any of a variety of enzymes, proteins, or the like, that may be employed for sensing. For example, if physiological parameter sensing is desired, one or more proteins may be used as the enzyme.
  • the device is a glucose-sensing device
  • a combination of glucose oxidase (GOx) and human serum albumin (HS A) may be used concurrently in a solid matrix form to form a sensor matrix protein.
  • GOx glucose oxidase
  • HS A human serum albumin
  • the glucose oxidase reaction sequence is more thoroughly described and illustrated in Figure 5.
  • An additional problem with conventional processes for formulating protein matrices for use in sensing devices is that the sensor matrix protein may not be sufficiently stable mechanically over time.
  • the GOx In the case of glucose biosensors, for example, there has been a problem of the GOx not possessing the desired mechanical stability, i.e., that the GOx maintain its shape.
  • Enzymes produced by conventional processes can be susceptible to swelling or shrinking. For example, a conventional process of injecting a GOx solution or gel into a cavity of a sensor body and then hardening the GOx in place can result in shrinkage and disfigurement of the GOx enzyme during hardening.
  • each sensor produced according to such processes may have a different shaped GOx enzyme and, thus, may behave somewhat different than other sensors made according to the same process.
  • enzymes can be susceptible to leaching. Sensor accuracy and sensitivity can be adversely affected when the enzyme utilized in the sensor is susceptible to leaching or swelling. Indeed, swelling of the enzyme over time can cause the sensor body to deform. Deformation of the body of the sensor may alter the response or the calibration of the sensor. Moreover, a swelling or leaching of the enzyme may cause the shape of the window in the sensing device to change which also could alter the response of the sensing device. Further problems have been associated with the process of injecting an enzyme into a sensing device while the enzyme is in a gel form.
  • Embodiments of the present invention comprise a protein matrix and processes for making protein matrices having improved mechanical stability as a result of hardening and cross-linking procedures carried out before the enzyme is placed inside of a sensing device.
  • embodiments of the method could be utilized to mold proteins for uses other than sensing devices, such as, for example, the molding of biological tissue replacement, arteries, heart valves, or the like. It is a further advantage of embodiments of the present invention to provide a method for formulating and immobilizing a protein matrix to be used in an implantable sensing device that can operate in a sufficiently consistent and invariant manner, without appreciably losing its sensitivity over an extended period of time. It is a further advantage of embodiments of the present invention to provide a protein pellet that has a sufficiently high concentration of GOx. By increasing the GOx concentration, embodiments of the present invention may counteract the effects of enzyme decay and extend the life of a sensing device.
  • a protein pellet is hardened and molded into the desired shape and size before being inserted into a sensing device, for example, to ensure proper fit and to reduce the likelihood of swelling or leaching.
  • Figure 7 shows two graphs illustrating the results of a swelling analysis. The first graph shows the swelling of a glucose oxidase-human serum albumin matrix formulated with insufficient immobilization. The second graph shows the swelling properties of the matrix formulated with improved immobilization according to embodiments of the present invention.
  • a method comprises combining a protein mixture (such as, but not limited to, GOx and human serum albumin (HSA)) with a cross-linking reagent (such as, but not limited to, glutaraldehyde).
  • the mixture is injected into a mold where it is hardened.
  • the immobilized enzyme, protein, or other such matrix may be further cross-linked with glutaraldehyde or another cross-linking reagent, to give a mechanically stable protein.
  • the type of cross-linker and the extent of immobilization may vary depending on the enzyme, protein, or the like.
  • the hardened protein mixture can be cut, machined, or otherwise formed to the desired shape and size.
  • the mold is configured to shape the mixture into semi- cylindrical rope-like structures that are subsequently cut into pellets. It has been found that sensing devices utilizing a protein pellet produced by this method can exhibit improved longevity.
  • a method for formulating and immobilizing the protein matrix further comprises adding silicone particles to the protein matrix.
  • the addition of silicone particles is believed to discourage the depletion of oxygen in the sensing device. Oxygen depletion causes GOx to be in its reduced state. Reduced GOx is more vulnerable to deactivation. Accordingly, the addition of the silicone may further contribute to the chemical stability of the sensor matrix protein over time.
  • Figure la shows a fragmented cutaway view of an exemplary environment of use of an implantable sensing device (not shown to scale) in which a sensor matrix protein (not shown to scale) according to an embodiment of the present invention may be utilized.
  • Figure lb shows a two-dimensional view of an implantable infusion and sensing device.
  • Figure lc shows a cutaway expanded view of a sensor region of a sensing device.
  • Figure Id shows another fragmented cutaway view of an exemplary environment of use of an implantable sensing device.
  • Figure 2a shows flowchart diagram of a method for formulating and immobilizing a sensor matrix protein according to one embodiment of the invention.
  • Figure 2b shows a flowchart diagram of a method for formulating and immobilizing a sensor matrix protein according to one embodiment of the invention.
  • Figure 3 shows a perspective view of an example of a mold utilized in one embodiment of the invention.
  • Figures 4a through 4d show two-dimensional views of examples of molds utilized in other embodiments of the invention.
  • Figure 5 shows a flow diagram of a glucose oxidase reaction sequence.
  • Figure 6 shows a perspective view of another example of a mold that may be utilized in an embodiment of the invention.
  • Figures 7 A and 7B show two graphs depicting the results of a swelling analysis done on a glucose oxidase-human serum albumin matrix.
  • Figure 7A shows the swelling with insufficient immobilization of the matrix.
  • Figure 7B shows the swelling with the improved immobilization process according to embodiments of the invention.
  • Figure 8a shows a graph that illustrates a glucose calibration curve for a sensor without an additive.
  • Figure 8b shows a graph that illustrates a glucose calibration curve for a sensor incorporating a silicone additive according to an embodiment of the invention.
  • the present invention relates, generally, to protein matrices and/or enzymes and processes for formulating and immobilizing protein matrices and/or enzymes.
  • a protein matrix may facilitate, for example, the creation of long-lived and stable sensors.
  • the protein matrix is an immobilized glucose oxidase (GOx) and human serum albumin (HSA) matrix for use in glucose biosensors.
  • GOx immobilized glucose oxidase
  • HSA human serum albumin
  • further embodiments of the invention involve molding other proteins for a variety of purposes.
  • FIG. 1 illustrates an example of a system 10 in which a sensor matrix protein produced pursuant to an embodiment of the invention may be utilized.
  • the system 10 in Figure 1 includes an implantable infusion pump 12 which may be implanted in a patient's body, such as in a patient's abdomen.
  • the pump 12 dispenses an infusion formulation (e.g., an insulin formulation) via a catheter 20.
  • a lead 14 connects the pump 12 to a sensing device 16 for regulating the delivery of the infusion formulation dependent upon a sensed parameter of the implant environment.
  • a protein pellet 18 is employed as a protein matrix within the sensing device 16.
  • the sensing device 16 may be any suitable sensor that operates with an active protein or enzyme, including, but not limited to, the sensors described in U.S. Patent Application No. 60/318,060, which is incorporated herein by reference.
  • Figure lb shows a two-dimensional view of the system 10 that is illustrated in Figure la.
  • Figure lc shows an expanded cutaway view of the sensor region of the infusion and sensing system illustrated in Figures in la and lb.
  • Figure 2a illustrates in a flow diagram, a process according to an embodiment of the present invention for producing a protein matrix. According to the process, a protein mixture is first obtained or formulated.
  • the protein mixture may be one that is free of polymeric additives and one that is concentrated using ultrafiltration or other chromatographic techniques.
  • a protein matrix may then be formed by adding a cross-linking agent to the protein mixture and then placing the polymerizing mixture into a mold. The formed matrix is then placed in a non-liquid atmosphere containing a cross-linking agent in order to maintain compositional integrity and to achieve the desired ultimate mechanical properties. Retained activity, mechanical stability, and operational stability can be customized according to a diagnostic or continuous sensing application by changing the protein and cross-linking process parameters.
  • Figure 2b illustrates in a flow diagram, a process according to an embodiment of the present invention for producing a protein pellet 18.
  • the embodiment illustrated in Figure 2b is a process for formulating and immobilizing a sensor matrix protein.
  • the method may be employed to yield protein pellets that are mechanically and chemically stable over time and that can be inserted, in their hardened state, into sensing devices.
  • a protein mixture or suspension is obtained or formulated, as shown at 22.
  • the protein mixture that is obtained or formulated is one that is free of polymeric additives and concentrated using ultrafiltration or other chromatographic techniques.
  • the protein mixture comprises a GOx and HSA mixture. The GOx concentration may vary for different embodiments of the invention.
  • the GOx concentration may be within the range of approximately 350 mg/ml (67,000 U/ml) to approximately 700 mg/ml (150,000 U/ml).
  • the HSA concentration may vary between about 23-32.5% (w/v), depending on the GOx concentration.
  • collagen or other structural proteins could be used instead of or in addition to HSA.
  • concentrations other than those discussed herein may be utilized. For example, depending on the enzyme employed, concentrations ranging from approximately 10% weight per weight to 70% weight per weight may be suitable. The concentration may be varied not only depending on the particular enzyme being employed, but also depending on the desired properties of the resulting protein matrix.
  • a certain concentration may be utilized if the protein matrix is to be used in a diagnostic capacity while a different concentration may be utilized if certain structural properties are desired.
  • concentration utilized may be varied through experimentation to determine which concentration (and of which enzyme or protein) may yield the desired result.
  • GOx is employed in the embodiment described herein, other proteins and/or enzymes may also be used or may be used in place of GOx, including, but not limited to, hexose oxidase, lactate oxidase, and the like. Other proteins and/or enzymes may also be used, as will be evident to those skilled in the art.
  • a cross-linking reagent is added to the protein mixture as shown at 24.
  • an amine cross-linking reagent such as, but not limited to, glutaraldehyde, is added to the protein mixture.
  • the addition of a cross- linking reagent to the protein mixture creates a protein paste.
  • the concentration of the cross- linking reagent to be added may vary according to the concentration of the protein mixture.
  • the GOx concentration may be about 67,000 U/ml and the HSA concentration may be about 25% (w/v), while the final glutaraldehyde concentration in the enzyme matrix may be approximately 1.25%.
  • glutaraldehyde is utilized in the embodiment described above, other cross-linking reagents may also be used or may be used in place of glutaraldehyde, including, but not limited to, an amine reactive, homofunctional, cross- linking reagent such as Disuccinimidyl Suberate (DSS).
  • DSS Disuccinimidyl Suberate
  • EDC l-Ethyl-3 (3- Dimethylaminopropyl) Carbodumide
  • EDC forms an amide bond between carboxylic acid and amine groups.
  • Other suitable cross-linkers also may be used, as will be evident to those skilled in the art.
  • the combined mixture can be injected into or otherwise applied to a mold. This may be done, for example, by drawing the combined mixture into a syringe and injecting the mixture into a mold.
  • the combined mixture may be highly reactive, and thus, may harden quickly. Therefor, the combined mixture should be injected into, or otherwise applied to, the mold quickly before the mixture hardens.
  • the shape or form of the mold may be dependent on the desired size and shape of the resulting protein pellet.
  • FIG. 3 shows a perspective view of one example of a mold that can be utilized in accordance with an embodiment of the invention.
  • the mold may be formed to have a chemically inert surface.
  • One way to insure that the mold has a chemically inert surface is to use materials such as, but not limited to, teflon, delrin, and the like.
  • the mold in the illustrated embodiment is comprised of two opposing blocks 30 and 36. In the illustrated orientation, the bottom block 30 has intersecting channels 32 on its top surface 34.
  • the intersecting channels 32 are each formed by semicylindrical recesses in the top surface 34 of the bottom block 30.
  • a top block 36 has a flat bottom surface. In the illustrated embodiment, the top surface 34 of the bottom block 30 and the bottom surface of the top block 36 are pressed or screwed together.
  • the protein mixture may be injected into the mold through an opening or hole 38 that passes all the way through the top block 36.
  • Other embodiments may employ molds of varying configurations. Variations that may be employed in other embodiments are illustrated in Figures 4a-4d. As is illustrated in Figures 4a-4d, the recesses (or channels) in the mold may be configured differently. For example, the recesses could be parallel lines, a spiral, or multiple groups of intersecting lines.
  • the embodiment described herein employs recesses (or channels) that are semicylindrical, other suitable shapes or channel cross-section configurations could be employed.
  • the cross-section configuration may be varied according to the desired application.
  • the channel cross-sections could be rectangular, cylindrical, tubular, spherical, or any other desired or suitable configuration.
  • Other suitable application specific mold configurations will be apparent to those skilled in the art.
  • the incubation may be carried out for other suitable time periods and/or may be carried out at an elevated temperature.
  • the primary form may be imparted to the protein matrix during this initial molding operation.
  • the mold may define the ultimate shape of the protein matrix.
  • the protein matrix then may be placed in an atmosphere containing a cross-linking agent in order to maintain the compositional integrity and achieve ultimate mechanical properties.
  • the mixture is exposed to a vaporized cross-linking reagent.
  • other non-liquid-based environments may be utilized. The non-liquid cross-linking process further hardens and solidifies the mixture.
  • non-liquid cross- linking process may be done by opening the mold and exposing the mixture to a vapor phase in a reactor for a suitable time period to further harden and solidify the mixture.
  • the two blocks of the mold may be separated after which the mixture may still reside in the recesses 32 of the block 30.
  • the block 30, with the mixture still in the recesses 32, may then be placed into a chamber in a vapor phase reactor.
  • the mixture while still in the recesses 32 of the opened mold, is exposed to vapor phase for approximately 16 hours. In other embodiments, other suitable exposure time periods may be employed.
  • the vapor phase cross-linking reagent may comprise, for example, approximately 12.5% (w/w) glutaraldehyde in a water vapor. In other embodiments, other suitable vapor phase cross-linking reagents may be employed.
  • the inclusion of a water vapor in the vapor phase may be desirable in contexts in which the protein mixture should remain hydrated. For example, enzymes may cease to be active if the reactor is dry.
  • the mixture may be removed from the mold and introduced to or placed in a cross-linking solution to further cross-link the hardened mixture.
  • the mixture may, at this point in the process, resemble the configuration of the recesses in whatever type of mold was employed in the process. For example, if a mold as shown in figure 3 was employed, then at this point in the process, the hardened mixture will resemble semi- cylindrical rope-like structures.
  • the hardened ropes may be exposed to a 2.5% buffered glutaraldehyde solution.
  • the glutaraldehyde in the solution may react with remaining or residual amine groups to further cross-link the GOx-HSA matrix.
  • Other embodiments might employ glutaraldehyde solutions with suitable concentrations varying, as required, according to the proteins or enzymes utilized.
  • Further embodiments might use cross- linking solutions with cross-linking reagents other than glutaraldehyde, such as, but not limited to, EDC and DCC, and the like.
  • the hardened mixture is submerged in the cross-linking solution for approximately one hour. In other embodiments, other suitable time periods may be employed. Moreover, those skilled in the art will understand that this part of the process could be omitted in other embodiments.
  • the hardened mixture is washed.
  • the hardened mixture may be washed after the vapor phase.
  • the hardened mixture may be washed in a variety of washing solutions, including but not limited to, phosphate buffered saline (PBS), sterile de-ionized water, or any other suitable washing solution.
  • PBS phosphate buffered saline
  • the hardened mixture may be washed a single time or it may be washed more than one time.
  • the hardened mixture is washed five times with sterile deionized water, phosphate buffered saline, and or glycine buffer.
  • the purpose of washing the hardened mixture is to remove any residual glutaraldehyde.
  • methods other than washing could be employed to remove residual glutaraldehyde, or to remove residual amounts of a different cross-linking reagent that might have been employed, without deviating from the spirit or scope of the invention.
  • the washing process or the removal of residual amount of cross- linking reagents by other means could be omitted entirely.
  • the hardened mixture after the hardened mixture has been washed, it may then be cut, machined, or otherwise formed into pieces that are the desired shape and size.
  • the hardened mixture may be easily cut at this point in the process.
  • the desired form for the sensor matrix protein is a semi-cylindrical pellet structure.
  • the example mold illustrated in figure 3 yields rope-like structures that may be cut into pellet-sized pieces.
  • the primary structure or form for the protein matrix is imparted during the initial molding operation. Accordingly, the cutting or machining process could take place, for example, before the vapor phase, or at any other point in the process after the mixture has been incubated in the mold.
  • a mold may be employed which forms the mixture to the desired shape and size (i.e., a semi-cylindrical pellet structure).
  • the mixture would not need to be cut, machined, or otherwise formed as a part of the process. In that case, cutting, machining, or otherwise forming the mixture may be omitted from the process.
  • the resulting pellet is sized to be a line-to-line fit for a sensor in which it may be placed.
  • the mixture may be molded such that it is slightly oversized to insure a snug fit within a device into which it might be placed, such as, but not limited to, a sensing device. It is an advantage of an embodiment of the invention that the concentration of GOx is maximized so as to extend the life of a sensor. In other embodiments of the invention, the GOx concentration is increased to further extend the life of a sensor. For example, in another embodiment, the same procedure described above and illustrated in Figure 2 may be utilized. However, the concentrations of GOx, HSA, and glutaraldehyde may be varied. For example, an embodiment might employ a GOx concentration of approximately 100,000 U/ml with a HSA concentration of approximately 32.5% (w/v).
  • the GOx-HSA mixture may be immobilized with approximately 100 ⁇ l of a 12.5% (w/w) glutaraldehyde solution. Such an embodiment may yield a protein pellet that remains physically and chemically stable for approximately one year.
  • the same procedure described above and illustrated in Figure 2 may be utilized employing a GOx concentration of approximately 135,000 U/ml with an HSA concentration of approximately 23% (w/v).
  • the GOx-HSA mixture may be immobilized with approximately 100 ⁇ l of a 25% (w/w) glutaraldehyde solution per 1 ml GOx-HSA mixture. This embodiment may yield a protein pellet that can remain physically and chemically stable for more than one year.
  • the same procedure described above and illustrated in Figure 2 may be utilized employing a GOx concentration of approximately 150,000 U/ml with an HSA concentration of approximately 32.5% (w/v).
  • the GOx-HSA mixture may be immobilized with approximately 100 ⁇ l of a 12.5 % (w/w) glutaraldehyde solution per 1 ml GOx-HSA mixture.
  • concentrations of GOx, HSA, and glutaraldehyde incorporated in these embodiments could be varied without diverting from the scope or spirit of the invention.
  • Another embodiment of the invention involves further enhancing the operational activity of the protein matrix by utilizing additives in the protein mixture.
  • one embodiment involves adding another substance, such as a volume of silicone particles, to the protein mixture to possibly enhance operational stability.
  • the addition of the silicone particles to a protein mixture may result in the sensing device maintaining its sensitivity for a longer time. It is believed that the silicone particles prevents oxygen depletion significant enough to increase the rate of product specific enzyme deactivation.
  • the example embodiment illustrated in Figure 2 and described above remains the same in this embodiment, with the only difference being the addition of another substance, such as silicone particles, to the protein mixture.
  • the silicone particles added may be approximately 1% (w/v) to 5% (w/v) by volume so as to not change the response of a sensor in which the resultant protein pellet could ultimately be utilized.
  • the total volume of the silicone particles added is preferably less than 20% of the volume of the SMP pellet.
  • Figures 8a and 8b show glucose calibration curves for sensors employing protein matrices with no additive (8a) and with a silicone additive (8b). The tightness of the calibration bands indicates the stability of the sensor. When a sensor loses its activity, then the calibration curve may tend to drift, as illustrated in Figure 8a. The drift is not as pronounced in Figure 8b when the protein matrix includes a silicone additive.
  • the embodiments described above can be utilized to prepare any protein matrix. It has been found a protein matrix formulated by the above embodiments may be particularly well suited for use in both short term and long term biosensors.
  • the immobilization process can produce a protein matrix that is physically and chemically stable over time.
  • the final protein matrix can be formed with the advantageous qualities of being low swelling, non-leaching, and having high activity and high mechanical strength properties. These properties are essential to the creation of long lived and stable sensors.
  • the embodiments described above were described, by way of example, with reference to formulating and immobilizing glucose oxidase, other embodiments may formulate proteins and/or enzymes other than glucose oxidase.
  • proteins and/or enzymes such as hexose oxidase, lactose oxidase, and the like, could also be formulated and immobilized.
  • proteins and/or enzymes such as hexose oxidase, lactose oxidase, and the like, could also be formulated and immobilized.
  • the process is applicable to a wide variety of protein and/or enzymes.
  • embodiments described above are described, by way of example, in terms of forming or molding a protein pellet that may be used in a sensing device, those skilled in the art will appreciate and understand that embodiments of the process could be utilized to form or mold biological tissue replacements, arteries, heart valves, and the like. This may be done, for example, by immobilizing blood plasma proteins in a mold shaped as an artery, heart valve, and the like.

Abstract

A method for formulating and immobilizing a protein and a protein matrix formed by the method. The protein matrix preparation method results in a physically and chemically stable protein matrix that has low swelling, non-leaching, high activity, and high mechanical strength properties. The method includes cross-linking and hardening the protein mixture and using a mold to form a protein into a desired shape and size.

Description

METHOD FOR FORMULATING AND IMMOBILIZING A PROTEIN MATRIX AND A PROTEIN MATRIX FOR USE IN A SENSOR
CROSS-REFERENCE TO RELATED APPLICATIONS
Embodiments of the present invention claim priority from a U.S. Provisional Application entitled "Method for Formulating and Immobilizing a Protein Matrix and a Protein Matrix for Use in a Sensor," Serial No. 60/335,663, filed October 23, 2001, the contents of which are incorporated by reference herein.
BACKGROUND
1. Field of the Invention
The present invention relates, generally, to methods and systems for formulating and immobilizing a protein matrix and/or enzyme and, in particular embodiments, to immobilizing proteins or enzymes that are physically and chemically stable over time, for example, for use in short-term or long-term sensors and biosensors. 2. Description of Related Art
The combination of biosensors and microelectronics has resulted in the availability of portable diagnostic medical equipment and has improved the quality of life for countless people. Many people suffering from disease or disability who, in the past, were forced to make routine visits to a hospital or a doctor's office for diagnostic testing currently perform diagnostic testing on themselves, in the comfort of their own homes, using equipment with accuracy to rival laboratory equipment. Nonetheless, challenges in the biosensing field have remained. For example, although many diabetics currently utilize diagnostic medical equipment in the comfort of their own homes, the vast majority of such devices still require diabetics to draw their own blood and to inject their own insulin. Drawing blood typically requires pricking a finger. For someone who is diagnosed with diabetes at an early age, the number of self- induced finger-pricks and insulin injections over the course of a lifetime could reach into the tens of thousands. Drawing blood and injecting insulin thousands of times can be overtly invasive and inconvenient, as well as painful and emotionally debilitating. Diagnostic requirements of those with disease or disability may be addressed by using a sensing apparatus that may be implanted into the body and that may remain in the body for an extended period of time. For example, an implantable sensing and infusion system is disclosed in pending U.S. Patent Application No. 60/318,060, incorporated herein by reference. An example of the type of implantable sensing system described in that application is illustrated in Figure 1 herein. The sensing system illustrated in Figure 1 comprises an implantable infusion pump 12 with a catheter 20 for dispensing an infusion formulation and a lead 14 connecting the infusion pump to a sensing device 16. The sensing device 16 may be inserted into a vein, an artery, or any other part of a human body where it could sense a desired parameter of the implant environment. A window may be provided in the sensing device 16 to facilitate sensing. An active sensing matrix 18, such as an enzyme, may be placed inside of the sensing device 16. The matrix 18 may be any of a variety of enzymes, proteins, or the like, that may be employed for sensing. For example, if physiological parameter sensing is desired, one or more proteins may be used as the enzyme. More specifically, if the device is a glucose-sensing device, for example, a combination of glucose oxidase (GOx) and human serum albumin (HS A) may be used concurrently in a solid matrix form to form a sensor matrix protein. Previous processes for formulating an enzyme for use in a sensor involved placing the enzyme into a cavity within a sensing device while the enzyme was still in a liquid or gel-like form. In such processes, the gel-like enzyme would be placed into the sensing device cavity, where it would harden in place, within the cavity. A hardening or cross-linking reagent would be added to the enzyme to cause solidification of the enzyme once it was inside the sensing device. One of the difficulties associated with conventional processes is that of producing protein matrices that are sufficiently chemically stable over time. For example, in the case of glucose biosensors, it has been found that GOx undergoes oxidative inactivation by peroxide and oxygen over time. Since the lifetime of glucose sensors primarily depends on the lifetime of the GOx, the sensitivity of the sensors is lost over time as the enzyme decays. Glucose oxidase goes through a cycle of oxidation and reduction upon interaction with glucose. Glucose oxidase is most vulnerable to deactivation in its reduced state. Hydrogen peroxide, hydroperoxy radicals, and the like, can deactivate GOx, particularly in its reduced state. The glucose oxidase reaction sequence is more thoroughly described and illustrated in Figure 5. An additional problem with conventional processes for formulating protein matrices for use in sensing devices is that the sensor matrix protein may not be sufficiently stable mechanically over time. In the case of glucose biosensors, for example, there has been a problem of the GOx not possessing the desired mechanical stability, i.e., that the GOx maintain its shape. Enzymes produced by conventional processes can be susceptible to swelling or shrinking. For example, a conventional process of injecting a GOx solution or gel into a cavity of a sensor body and then hardening the GOx in place can result in shrinkage and disfigurement of the GOx enzyme during hardening. As a result, each sensor produced according to such processes may have a different shaped GOx enzyme and, thus, may behave somewhat different than other sensors made according to the same process. In addition, enzymes can be susceptible to leaching. Sensor accuracy and sensitivity can be adversely affected when the enzyme utilized in the sensor is susceptible to leaching or swelling. Indeed, swelling of the enzyme over time can cause the sensor body to deform. Deformation of the body of the sensor may alter the response or the calibration of the sensor. Moreover, a swelling or leaching of the enzyme may cause the shape of the window in the sensing device to change which also could alter the response of the sensing device. Further problems have been associated with the process of injecting an enzyme into a sensing device while the enzyme is in a gel form. When an enzyme is injected into a cavity of a sensing device, it is difficult to ensure that the enzyme has filled the volume in the sensing device completely. If there are voids left in the cavity after the enzyme has been injected, those voids can adversely affect the stability and sensitivity of the sensing device. Moreover, since the enzyme may tend to shrink as it hardens or solidifies, further voids or spaces may be left in the enzyme cavity of the sensor.
SUMMARY OF THE DISCLOSURE
Therefore, it is an advantage of embodiments of the present invention to provide a process for combining protein formulation and immobilization techniques which result in a physically and chemically stable protein matrix. It is a further advantage of the embodiments of the present invention to provide a method for formulating and immobilizing a protein matrix that is molded into a desired shape and size before it is inserted into a sensing device. Embodiments of the present invention comprise a protein matrix and processes for making protein matrices having improved mechanical stability as a result of hardening and cross-linking procedures carried out before the enzyme is placed inside of a sensing device. It is a further advantage of the embodiments of the present invention to provide a method for formulating and immobilizing a protein matrix in which retained activity, mechanical stability, and operational stability may be customized to a diagnostic or continuous sensing application by changing the enzyme or protein and by changing the cross-linking process parameters. It is a further advantage of embodiments of the present invention to provide methods for formulating and immobilizing protein matrices that have sufficiently low swelling and leaching properties as well as sufficiently high activity and mechanical strength properties. Embodiments of the formulation and immobilization methods may be applied to prepare a protein matrix for use in short-term or long-term sensors and biosensors. Moreover, embodiments of the method could be utilized to mold proteins for uses other than sensing devices, such as, for example, the molding of biological tissue replacement, arteries, heart valves, or the like. It is a further advantage of embodiments of the present invention to provide a method for formulating and immobilizing a protein matrix to be used in an implantable sensing device that can operate in a sufficiently consistent and invariant manner, without appreciably losing its sensitivity over an extended period of time. It is a further advantage of embodiments of the present invention to provide a protein pellet that has a sufficiently high concentration of GOx. By increasing the GOx concentration, embodiments of the present invention may counteract the effects of enzyme decay and extend the life of a sensing device. According to such embodiments, a protein pellet is hardened and molded into the desired shape and size before being inserted into a sensing device, for example, to ensure proper fit and to reduce the likelihood of swelling or leaching. Figure 7 shows two graphs illustrating the results of a swelling analysis. The first graph shows the swelling of a glucose oxidase-human serum albumin matrix formulated with insufficient immobilization. The second graph shows the swelling properties of the matrix formulated with improved immobilization according to embodiments of the present invention. In one embodiment of the present invention, a method comprises combining a protein mixture (such as, but not limited to, GOx and human serum albumin (HSA)) with a cross-linking reagent (such as, but not limited to, glutaraldehyde). In this embodiment, the mixture is injected into a mold where it is hardened. Once hardened, the immobilized enzyme, protein, or other such matrix, may be further cross-linked with glutaraldehyde or another cross-linking reagent, to give a mechanically stable protein. The type of cross-linker and the extent of immobilization may vary depending on the enzyme, protein, or the like. The hardened protein mixture can be cut, machined, or otherwise formed to the desired shape and size. In one embodiment, the mold is configured to shape the mixture into semi- cylindrical rope-like structures that are subsequently cut into pellets. It has been found that sensing devices utilizing a protein pellet produced by this method can exhibit improved longevity. In another embodiment of the present invention, a method for formulating and immobilizing the protein matrix further comprises adding silicone particles to the protein matrix. The addition of silicone particles is believed to discourage the depletion of oxygen in the sensing device. Oxygen depletion causes GOx to be in its reduced state. Reduced GOx is more vulnerable to deactivation. Accordingly, the addition of the silicone may further contribute to the chemical stability of the sensor matrix protein over time. These and other objects, features, and advantages of embodiments of the invention will be apparent to those skilled in the art from the following detailed description of embodiments of the invention, when read with the drawings and appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
A brief description of preferred embodiments of the invention will be made with reference to the accompanying drawings wherein: Figure la shows a fragmented cutaway view of an exemplary environment of use of an implantable sensing device (not shown to scale) in which a sensor matrix protein (not shown to scale) according to an embodiment of the present invention may be utilized. Figure lb shows a two-dimensional view of an implantable infusion and sensing device. Figure lc shows a cutaway expanded view of a sensor region of a sensing device. Figure Id shows another fragmented cutaway view of an exemplary environment of use of an implantable sensing device. Figure 2a shows flowchart diagram of a method for formulating and immobilizing a sensor matrix protein according to one embodiment of the invention. Figure 2b shows a flowchart diagram of a method for formulating and immobilizing a sensor matrix protein according to one embodiment of the invention. Figure 3 shows a perspective view of an example of a mold utilized in one embodiment of the invention. Figures 4a through 4d show two-dimensional views of examples of molds utilized in other embodiments of the invention. Figure 5 shows a flow diagram of a glucose oxidase reaction sequence. Figure 6 shows a perspective view of another example of a mold that may be utilized in an embodiment of the invention. Figures 7 A and 7B show two graphs depicting the results of a swelling analysis done on a glucose oxidase-human serum albumin matrix. Figure 7A shows the swelling with insufficient immobilization of the matrix. Figure 7B shows the swelling with the improved immobilization process according to embodiments of the invention. Figure 8a shows a graph that illustrates a glucose calibration curve for a sensor without an additive. Figure 8b shows a graph that illustrates a glucose calibration curve for a sensor incorporating a silicone additive according to an embodiment of the invention.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The following detailed description is of the best presently contemplated mode of carrying out the invention. This description is not to be taken in a limiting sense, but is made merely for the purpose of illustrating the general principles of the invention. The scope of the invention is best defined by the appended claims. The present invention relates, generally, to protein matrices and/or enzymes and processes for formulating and immobilizing protein matrices and/or enzymes. As discussed above, such a protein matrix may facilitate, for example, the creation of long-lived and stable sensors. In an example embodiment, the protein matrix is an immobilized glucose oxidase (GOx) and human serum albumin (HSA) matrix for use in glucose biosensors. However, further embodiments of the invention involve molding other proteins for a variety of purposes. Processes according to the various embodiments of the invention may be employed to produce physically and chemically stable protein matrices. Figure 1 illustrates an example of a system 10 in which a sensor matrix protein produced pursuant to an embodiment of the invention may be utilized. Other embodiments may employ other suitable system configurations. However, by way of example, the system 10 in Figure 1 includes an implantable infusion pump 12 which may be implanted in a patient's body, such as in a patient's abdomen. The pump 12 dispenses an infusion formulation (e.g., an insulin formulation) via a catheter 20. A lead 14 connects the pump 12 to a sensing device 16 for regulating the delivery of the infusion formulation dependent upon a sensed parameter of the implant environment. In the sensor system in Figure 1, a protein pellet 18 is employed as a protein matrix within the sensing device 16. The sensing device 16 may be any suitable sensor that operates with an active protein or enzyme, including, but not limited to, the sensors described in U.S. Patent Application No. 60/318,060, which is incorporated herein by reference. Figure lb shows a two-dimensional view of the system 10 that is illustrated in Figure la. Figure lc shows an expanded cutaway view of the sensor region of the infusion and sensing system illustrated in Figures in la and lb. Figure 2a illustrates in a flow diagram, a process according to an embodiment of the present invention for producing a protein matrix. According to the process, a protein mixture is first obtained or formulated. The protein mixture may be one that is free of polymeric additives and one that is concentrated using ultrafiltration or other chromatographic techniques. A protein matrix may then be formed by adding a cross-linking agent to the protein mixture and then placing the polymerizing mixture into a mold. The formed matrix is then placed in a non-liquid atmosphere containing a cross-linking agent in order to maintain compositional integrity and to achieve the desired ultimate mechanical properties. Retained activity, mechanical stability, and operational stability can be customized according to a diagnostic or continuous sensing application by changing the protein and cross-linking process parameters. A more detailed description of a specific embodiment of the invention is described below in connection with Figure 2b. Figure 2b illustrates in a flow diagram, a process according to an embodiment of the present invention for producing a protein pellet 18. The embodiment illustrated in Figure 2b is a process for formulating and immobilizing a sensor matrix protein. In preferred embodiments, the method may be employed to yield protein pellets that are mechanically and chemically stable over time and that can be inserted, in their hardened state, into sensing devices. In the embodiment illustrated in Figure 2b, initially, a protein mixture or suspension is obtained or formulated, as shown at 22. In the example embodiment, the protein mixture that is obtained or formulated is one that is free of polymeric additives and concentrated using ultrafiltration or other chromatographic techniques. In an example embodiment, the protein mixture comprises a GOx and HSA mixture. The GOx concentration may vary for different embodiments of the invention. For example, the GOx concentration may be within the range of approximately 350 mg/ml (67,000 U/ml) to approximately 700 mg/ml (150,000 U/ml). In such embodiments, the HSA concentration may vary between about 23-32.5% (w/v), depending on the GOx concentration. Or, collagen or other structural proteins could be used instead of or in addition to HSA. For embodiments employing enzymes other than GOx, concentrations other than those discussed herein may be utilized. For example, depending on the enzyme employed, concentrations ranging from approximately 10% weight per weight to 70% weight per weight may be suitable. The concentration may be varied not only depending on the particular enzyme being employed, but also depending on the desired properties of the resulting protein matrix. For example, a certain concentration may be utilized if the protein matrix is to be used in a diagnostic capacity while a different concentration may be utilized if certain structural properties are desired. Those skilled in the art will understand that the concentration utilized may be varied through experimentation to determine which concentration (and of which enzyme or protein) may yield the desired result. Although GOx is employed in the embodiment described herein, other proteins and/or enzymes may also be used or may be used in place of GOx, including, but not limited to, hexose oxidase, lactate oxidase, and the like. Other proteins and/or enzymes may also be used, as will be evident to those skilled in the art. Moreover, although HSA is employed in the example embodiment, other structural protein, such as collagen or the like, could be used instead of or in addition to HSA. Next, a cross-linking reagent is added to the protein mixture as shown at 24. In one embodiment, an amine cross-linking reagent, such as, but not limited to, glutaraldehyde, is added to the protein mixture. The addition of a cross- linking reagent to the protein mixture creates a protein paste. The concentration of the cross- linking reagent to be added may vary according to the concentration of the protein mixture. In an example embodiment, the GOx concentration may be about 67,000 U/ml and the HSA concentration may be about 25% (w/v), while the final glutaraldehyde concentration in the enzyme matrix may be approximately 1.25%. Although glutaraldehyde is utilized in the embodiment described above, other cross-linking reagents may also be used or may be used in place of glutaraldehyde, including, but not limited to, an amine reactive, homofunctional, cross- linking reagent such as Disuccinimidyl Suberate (DSS). Another example is l-Ethyl-3 (3- Dimethylaminopropyl) Carbodumide (EDC), which is a zero-length cross-linker. EDC forms an amide bond between carboxylic acid and amine groups. Other suitable cross-linkers also may be used, as will be evident to those skilled in the art. Once a cross-linking reagent is added to the protein mixture, the combined mixture can be injected into or otherwise applied to a mold. This may be done, for example, by drawing the combined mixture into a syringe and injecting the mixture into a mold. In some embodiments, the combined mixture may be highly reactive, and thus, may harden quickly. Therefor, the combined mixture should be injected into, or otherwise applied to, the mold quickly before the mixture hardens. The shape or form of the mold may be dependent on the desired size and shape of the resulting protein pellet. For example, the mold could be configured to produce a protein pellet that is precisely the desired shape and size. However, the mold may also be configured to yield a protein matrix that still needs to be cut, machined, or otherwise formed to the desired shape and size. Figure 3 shows a perspective view of one example of a mold that can be utilized in accordance with an embodiment of the invention. The mold may be formed to have a chemically inert surface. One way to insure that the mold has a chemically inert surface is to use materials such as, but not limited to, teflon, delrin, and the like. The mold in the illustrated embodiment is comprised of two opposing blocks 30 and 36. In the illustrated orientation, the bottom block 30 has intersecting channels 32 on its top surface 34. The intersecting channels 32 are each formed by semicylindrical recesses in the top surface 34 of the bottom block 30. A top block 36 has a flat bottom surface. In the illustrated embodiment, the top surface 34 of the bottom block 30 and the bottom surface of the top block 36 are pressed or screwed together. The protein mixture may be injected into the mold through an opening or hole 38 that passes all the way through the top block 36. Other embodiments may employ molds of varying configurations. Variations that may be employed in other embodiments are illustrated in Figures 4a-4d. As is illustrated in Figures 4a-4d, the recesses (or channels) in the mold may be configured differently. For example, the recesses could be parallel lines, a spiral, or multiple groups of intersecting lines. Also, although the embodiment described herein employs recesses (or channels) that are semicylindrical, other suitable shapes or channel cross-section configurations could be employed. The cross-section configuration may be varied according to the desired application. For example, the channel cross-sections could be rectangular, cylindrical, tubular, spherical, or any other desired or suitable configuration. Other suitable application specific mold configurations will be apparent to those skilled in the art. Once the combined mixture has been placed in the mold, the mixture is incubated in the mold. In one example embodiment, the mixture is incubated at room temperature for approximately two hours. Following the incubation period, the mixture may be cross-linked, but it may not yet be fully hardened. In other embodiments, the incubation may be carried out for other suitable time periods and/or may be carried out at an elevated temperature. The primary form may be imparted to the protein matrix during this initial molding operation. In other words, the mold may define the ultimate shape of the protein matrix. Following incubation, the protein matrix then may be placed in an atmosphere containing a cross-linking agent in order to maintain the compositional integrity and achieve ultimate mechanical properties. In the example embodiment, after incubation, the mixture is exposed to a vaporized cross-linking reagent. However, in other embodiments, other non-liquid-based environments may be utilized. The non-liquid cross-linking process further hardens and solidifies the mixture. Those skilled in the art will understand that suitable non-liquid environments that range from very high pressure to very low pressure may be suitable. The environment's temperature could also be varied according to experimentation. In the example embodiment, the non-liquid cross- linking process may be done by opening the mold and exposing the mixture to a vapor phase in a reactor for a suitable time period to further harden and solidify the mixture. In an example embodiment in which the mold is configured as illustrated in figure 3, the two blocks of the mold may be separated after which the mixture may still reside in the recesses 32 of the block 30. The block 30, with the mixture still in the recesses 32, may then be placed into a chamber in a vapor phase reactor. In one example embodiment, the mixture, while still in the recesses 32 of the opened mold, is exposed to vapor phase for approximately 16 hours. In other embodiments, other suitable exposure time periods may be employed. The vapor phase cross-linking reagent may comprise, for example, approximately 12.5% (w/w) glutaraldehyde in a water vapor. In other embodiments, other suitable vapor phase cross-linking reagents may be employed. The inclusion of a water vapor in the vapor phase may be desirable in contexts in which the protein mixture should remain hydrated. For example, enzymes may cease to be active if the reactor is dry. In an example embodiment, after the mixture has been exposed to the vaporized cross- linking reagent for a suitable period of time, the mixture may be removed from the mold and introduced to or placed in a cross-linking solution to further cross-link the hardened mixture. The mixture may, at this point in the process, resemble the configuration of the recesses in whatever type of mold was employed in the process. For example, if a mold as shown in figure 3 was employed, then at this point in the process, the hardened mixture will resemble semi- cylindrical rope-like structures. In the example embodiment in figure 2, after further cross- linking the GOx-HSA matrix with vapor glutaraldehyde, the hardened ropes may be exposed to a 2.5% buffered glutaraldehyde solution. The glutaraldehyde in the solution may react with remaining or residual amine groups to further cross-link the GOx-HSA matrix. Other embodiments might employ glutaraldehyde solutions with suitable concentrations varying, as required, according to the proteins or enzymes utilized. Further embodiments might use cross- linking solutions with cross-linking reagents other than glutaraldehyde, such as, but not limited to, EDC and DCC, and the like. In an example embodiment, the hardened mixture is submerged in the cross-linking solution for approximately one hour. In other embodiments, other suitable time periods may be employed. Moreover, those skilled in the art will understand that this part of the process could be omitted in other embodiments. In an example embodiment, after the hardened mixture has been introduced to or placed in the cross-linking solution, then the hardened mixture is washed. In an embodiment where the hardened mixture was not placed in a cross-linking solution at all, then the hardened mixture may be washed after the vapor phase. The hardened mixture may be washed in a variety of washing solutions, including but not limited to, phosphate buffered saline (PBS), sterile de-ionized water, or any other suitable washing solution. Moreover, the hardened mixture may be washed a single time or it may be washed more than one time. In an example embodiment, the hardened mixture is washed five times with sterile deionized water, phosphate buffered saline, and or glycine buffer. The purpose of washing the hardened mixture is to remove any residual glutaraldehyde. In other embodiments, methods other than washing could be employed to remove residual glutaraldehyde, or to remove residual amounts of a different cross-linking reagent that might have been employed, without deviating from the spirit or scope of the invention. In other embodiments, the washing process or the removal of residual amount of cross- linking reagents by other means, could be omitted entirely. In the example embodiment, after the hardened mixture has been washed, it may then be cut, machined, or otherwise formed into pieces that are the desired shape and size. The hardened mixture may be easily cut at this point in the process. As discussed above, in an example embodiment, the desired form for the sensor matrix protein is a semi-cylindrical pellet structure. The example mold illustrated in figure 3 yields rope-like structures that may be cut into pellet-sized pieces. The primary structure or form for the protein matrix is imparted during the initial molding operation. Accordingly, the cutting or machining process could take place, for example, before the vapor phase, or at any other point in the process after the mixture has been incubated in the mold. In other embodiments, a mold may be employed which forms the mixture to the desired shape and size (i.e., a semi-cylindrical pellet structure). Of course, if an embodiment employs a mold that forms the mixture to the desired shape and size, then the mixture would not need to be cut, machined, or otherwise formed as a part of the process. In that case, cutting, machining, or otherwise forming the mixture may be omitted from the process. Moreover, in the example embodiment, the resulting pellet is sized to be a line-to-line fit for a sensor in which it may be placed. In another embodiment, the mixture may be molded such that it is slightly oversized to insure a snug fit within a device into which it might be placed, such as, but not limited to, a sensing device. It is an advantage of an embodiment of the invention that the concentration of GOx is maximized so as to extend the life of a sensor. In other embodiments of the invention, the GOx concentration is increased to further extend the life of a sensor. For example, in another embodiment, the same procedure described above and illustrated in Figure 2 may be utilized. However, the concentrations of GOx, HSA, and glutaraldehyde may be varied. For example, an embodiment might employ a GOx concentration of approximately 100,000 U/ml with a HSA concentration of approximately 32.5% (w/v). In such an embodiment, the GOx-HSA mixture may be immobilized with approximately 100 μl of a 12.5% (w/w) glutaraldehyde solution. Such an embodiment may yield a protein pellet that remains physically and chemically stable for approximately one year. In another embodiment, the same procedure described above and illustrated in Figure 2 may be utilized employing a GOx concentration of approximately 135,000 U/ml with an HSA concentration of approximately 23% (w/v). In such an embodiment, the GOx-HSA mixture may be immobilized with approximately 100 μl of a 25% (w/w) glutaraldehyde solution per 1 ml GOx-HSA mixture. This embodiment may yield a protein pellet that can remain physically and chemically stable for more than one year. In another embodiment, the same procedure described above and illustrated in Figure 2 may be utilized employing a GOx concentration of approximately 150,000 U/ml with an HSA concentration of approximately 32.5% (w/v). In such an embodiment, the GOx-HSA mixture may be immobilized with approximately 100 μl of a 12.5 % (w/w) glutaraldehyde solution per 1 ml GOx-HSA mixture. Those skilled in the art will of course understand that the concentrations of GOx, HSA, and glutaraldehyde incorporated in these embodiments could be varied without diverting from the scope or spirit of the invention. Another embodiment of the invention involves further enhancing the operational activity of the protein matrix by utilizing additives in the protein mixture. For example, one embodiment involves adding another substance, such as a volume of silicone particles, to the protein mixture to possibly enhance operational stability. The addition of the silicone particles to a protein mixture may result in the sensing device maintaining its sensitivity for a longer time. It is believed that the silicone particles prevents oxygen depletion significant enough to increase the rate of product specific enzyme deactivation. The example embodiment illustrated in Figure 2 and described above remains the same in this embodiment, with the only difference being the addition of another substance, such as silicone particles, to the protein mixture. In the embodiment utilizing silicone particles, the silicone particles added may be approximately 1% (w/v) to 5% (w/v) by volume so as to not change the response of a sensor in which the resultant protein pellet could ultimately be utilized. However, in other embodiments, either more or less silicone could be added to the protein mixture. In this embodiment, the total volume of the silicone particles added is preferably less than 20% of the volume of the SMP pellet. Those skilled in the art will understand that the volume of silicone added and the precise size of the silicone particles can be varied through experimentation without straying from the scope or spirit of the invention. Figures 8a and 8b show glucose calibration curves for sensors employing protein matrices with no additive (8a) and with a silicone additive (8b). The tightness of the calibration bands indicates the stability of the sensor. When a sensor loses its activity, then the calibration curve may tend to drift, as illustrated in Figure 8a. The drift is not as pronounced in Figure 8b when the protein matrix includes a silicone additive. The embodiments described above can be utilized to prepare any protein matrix. It has been found a protein matrix formulated by the above embodiments may be particularly well suited for use in both short term and long term biosensors. The immobilization process can produce a protein matrix that is physically and chemically stable over time. The final protein matrix can be formed with the advantageous qualities of being low swelling, non-leaching, and having high activity and high mechanical strength properties. These properties are essential to the creation of long lived and stable sensors. Although the embodiments described above were described, by way of example, with reference to formulating and immobilizing glucose oxidase, other embodiments may formulate proteins and/or enzymes other than glucose oxidase. For example, in other embodiments, proteins and/or enzymes such as hexose oxidase, lactose oxidase, and the like, could also be formulated and immobilized. Those skilled in the art will appreciate and understand that the process is applicable to a wide variety of protein and/or enzymes. In addition, while the embodiments described above are described, by way of example, in terms of forming or molding a protein pellet that may be used in a sensing device, those skilled in the art will appreciate and understand that embodiments of the process could be utilized to form or mold biological tissue replacements, arteries, heart valves, and the like. This may be done, for example, by immobilizing blood plasma proteins in a mold shaped as an artery, heart valve, and the like. The immobilization process should yield an artificial artery, heart valve, and the like, that is mechanically and chemically stable. The embodiments disclosed herein are to be considered in all respects as illustrative and not restrictive of the invention. The scope of the invention is indicated by the appended claims, rather than the foregoing description. All changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

What is claimed is:
1. A method for formulating a protein comprising: combining a protein mixture with a cross-linking reagent; placing the protein mixture into a mold after combining the protein mixture with a cross-linking reagent; hardening the protein mixture; and removing the hardened protein mixture from the mold.
2. A method for formulating a protein according to claim 1, wherein hardening the protein mixture comprises exposing the protein mixture to a non-liquid cross-linking process.
3. A method for formulating a protein according to claim 2, wherein hardening the protein mixture further comprises incubating the protein mixture prior to exposing the protein mixture to the non-liquid cross-linking process.
4. A method for formulating a protein according to claim 2, wherein hardening the protein mixture further comprises immersing the protein mixture in a cross-linking solution after exposing the protein solution to the non-liquid cross-linking process.
5. A method for formulating a protein according to claim 2, wherein exposing the protein mixture to a non-liquid cross-linking process comprises exposing the protein mixture to a vapor phase that is approximately 12.5% (w/w) glutaraldehyde for approximately 16 hours.
6. A method for formulating a protein according to claim 3, wherein incubating the protein mixture comprises maintaining the protein mixture at approximately room temperature for approximately two hours.
7. A method for formulating a protein according to claim 4, wherein immersing the protein mixture in a cross-linking solution comprises submerging the protein mixture in a buffered solution that is approximately 2.5% (w/w) glutaraldehyde for approximately one hour.
8. A method for formulating a protein according to claim 1, wherein the protein mixture comprises glucose oxidase and human serum albumin.
9. A method for formulating a protein according to claim 1, wherein the cross- linking reagent is glutaraldehyde.
10. A method for formulating a protein according to claim 8, wherein the cross- linking reagent is glutaraldehyde.
11. A method for formulating a protein according to claim 1 , wherein the cross- linking reagent is selected from a group consisting of glutaraldehyde, disuccinimidyl suberate (DSS), and l-Ethyl-3 (3-Dimethylaminoρroρyl) Carbodumide (EDC).
12. A method for formulating a protein according to claim 8, wherein the glucose oxidase has a concentration that is between approximately 67,000 U/ml and 150,000 U/ml.
13. A method for formulating a protein according to claim 9, wherein the human serum albumin has a concentration that is between approximately 23% (w/v) and 32.5% (w/v).
14. A method for formulating a protein according to claim 12, wherein the human serum albumin has a concentration that is between approximately 23% (w/v) and 32.5% (w/v).
15. A method for formulating a protein according to claim 1, wherein the mold is configured to form the protein mixture into at least one elongated rope-like structure.
16. A method for formulating a protein according to claim 15, wherein the method further comprises cutting the at least one elongated rope-like structure into pieces.
17. A method for formulating a protein according to claim 15, wherein the elongated rope-like structure is semi-cylindrical.
18. A method for formulating a protein according to claim 1, wherein the mold comprises: a block with a surface wherein the surface has at least one recess in it; and wherein the protein mixture is placed in the recess when it is placed into the mold.
19. A method for formulating a protein according to claim 18, wherein the recess comprises at least one channel.
20. A method for formulating a protein according to claim 19, wherein the recess is semi-cylindrical.
21. A method for formulating a protein according to claim 19, wherein the recess comprises multiple intersecting channels.
22. A method for formulating a protein according to claim 1 , wherein the method further comprises adding silicone to the protein mixture.
23. A method for formulating a protein according to claim 22, wherein adding silicone to the protein solution comprises: obtaining silicone particles; and adding the silicone particles to the protein mixture.
24. A method for formulating a protein according to claim 23, wherein the volume of the silicone particles is less than 20% of the volume of the protein mixture.
25. A method for formulating a protein according to claim 23, wherein mixing the silicone particles into the protein mixture occurs prior to the protein mixture being placed into the mold.
26. A method for formulating a protein according to claim 1, wherein the method further comprises washing the protein mixture after the completion of hardening the protein mixture.
27. A method for formulating a protein according to claim 1, wherein the method further comprises cutting the protein mixture into at least two pieces after removing the protein mixture from the mold.
28. A sensor comprising: a sensor body; and an active protein disposed within the sensor body, the active protein comprising gluscose oxidase, human serum albumin, and a cross- linking reagent.
29. A sensor according to claim 28, wherein the active protein is hardened before it is disposed within the sensor body.
30. A sensor according to claim 28, wherein the cross-linking reagent is selected from a group consisting of glutaraldehyde, disuccinimidyl suberate (DSS), and l-Ethyl-3 (3- Dimethylaminopropyl) Carbodumide (EDC).
31. A protein matrix formed according to the method of claim 1.
PCT/US2002/030942 2001-10-23 2002-09-27 Method for formulating and immobilizing a protein matrix and a protein matrix for use in a sensor WO2003035891A2 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU2002327774A AU2002327774A1 (en) 2001-10-23 2002-09-27 Method for formulating and immobilizing a protein matrix and a protein matrix for use in a sensor
DE60226759T DE60226759D1 (en) 2001-10-23 2002-09-27 Method for producing a sensor
EP02763782A EP1437937B1 (en) 2001-10-23 2002-09-27 Method for producing a sensor
CA002457545A CA2457545A1 (en) 2001-10-23 2002-09-27 Method for formulating and immobilizing a protein matrix and a protein matrix for use in a sensor
JP2003538391A JP2005507007A (en) 2001-10-23 2002-09-27 Protein matrix formulation and immobilization method and protein matrix for use in sensors

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
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Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7409238B2 (en) 1998-08-20 2008-08-05 Becton, Dickinson And Company Micro-invasive method for painless detection of analytes in extracellular space
WO2011041715A2 (en) 2009-10-01 2011-04-07 Medtronic Minimed, Inc. Analyte sensor apparatuses having interference rejection membranes and methods for making and using them
WO2011063259A2 (en) 2009-11-20 2011-05-26 Medtronic Minimed, Inc. Multi-conductor lead configurations useful with medical device systems and methods for making and using them
WO2011084651A1 (en) 2009-12-21 2011-07-14 Medtronic Minimed, Inc. Analyte sensors comprising blended membrane compositions and methods for making and using them
WO2011091061A1 (en) 2010-01-19 2011-07-28 Medtronic Minimed, Inc. Insertion device for a combined sensor and infusion sets
WO2011115949A1 (en) 2010-03-16 2011-09-22 Medtronic Minimed, Inc. Glucose sensor
WO2011163303A2 (en) 2010-06-23 2011-12-29 Medtronic Minimed, Inc. Sensor systems having multiple probes and electrode arrays
WO2012154548A1 (en) 2011-05-06 2012-11-15 Medtronic Minimed, Inc. Method and apparatus for continuous analyte monitoring
US8394463B1 (en) 2009-01-23 2013-03-12 Medtronic Minimed, Inc. Crosslinking compounds at negative pressures and materials made by such methods
WO2013177573A2 (en) 2012-05-25 2013-11-28 Medtronic Minimed, Inc. Foldover sensors and methods for making and using them
WO2014008297A1 (en) 2012-07-03 2014-01-09 Medtronic Minimed, Inc. Analyte sensors and production thereof
WO2014089276A1 (en) 2012-12-06 2014-06-12 Medtronic Minimed, Inc. Microarray electrodes useful with analyte sensors and methods for making and using them
WO2014116293A1 (en) 2013-01-22 2014-07-31 Medtronic Minimed, Inc. Muting glucose sensor oxygen response and reducing electrode edge growth with pulsed current plating
WO2015069692A2 (en) 2013-11-07 2015-05-14 Medtronic Minimed, Inc. Enzyme matrices for use with ethylene oxide sterilization
WO2017189764A1 (en) 2016-04-28 2017-11-02 Medtronic Minimed, Inc. In-situ chemistry stack for continuous glucose sensors
WO2017195035A1 (en) 2016-05-10 2017-11-16 Interface Biologics, Inc. Implantable glucose sensors having a biostable surface
WO2017214173A1 (en) 2016-06-06 2017-12-14 Medtronic Minimed, Inc. Polycarbonate urea/urethane polymers for use with analyte sensors
US9968742B2 (en) 2007-08-29 2018-05-15 Medtronic Minimed, Inc. Combined sensor and infusion set using separated sites
WO2018170363A1 (en) 2017-03-17 2018-09-20 Medtronic Minimed, Inc. Metal pillar device structures and methods for making and using them in electrochemical and/or electrocatalytic applications
WO2019005687A1 (en) 2017-06-30 2019-01-03 Medtronic Minimed, Inc. Sensor initialization methods for faster body sensor response
CN110023745A (en) * 2016-12-09 2019-07-16 东北大学 The durable biosensor and droplet deposition fixing means based on enzyme
WO2019147578A1 (en) 2018-01-23 2019-08-01 Medtronic Minimed, Inc. Implantable polymer surfaces exhibiting reduced in vivo inflammatory responses
WO2019156934A1 (en) 2018-02-07 2019-08-15 Medtronic Minimed, Inc. Multilayer electrochemical analyte sensors and methods for making and using them
WO2019157043A1 (en) 2018-02-08 2019-08-15 Medtronic Minimed, Inc. Glucose sensor electrode design
WO2019157106A2 (en) 2018-02-08 2019-08-15 Medtronic Minimed, Inc. Methods for controlling physical vapor deposition metal film adhesion to substrates and surfaces
WO2019222499A1 (en) 2018-05-16 2019-11-21 Medtronic Minimed, Inc. Thermally stable glucose limiting membrane for glucose sensors
WO2021021538A1 (en) 2019-07-26 2021-02-04 Medtronic Minimed, Inc. Methods to improve oxygen delivery to implantable sensors
WO2021021867A1 (en) 2019-08-01 2021-02-04 Medtronic Minimed, Inc. Micro-pillar working electrodes design to reduce backflow of hydrogen peroxide in glucose sensor
WO2022026542A1 (en) 2020-07-31 2022-02-03 Medtronic Minimed, Inc. Sensor identification and integrity check design
WO2022093574A1 (en) 2020-10-29 2022-05-05 Medtronic Minimed, Inc. Glucose biosensors comprising direct electron transfer enzymes and methods of making and using them
WO2022164981A1 (en) 2021-01-29 2022-08-04 Medtronic Minimed, Inc. Interference rejection membranes useful with analyte sensors
EP4071251A1 (en) 2021-04-09 2022-10-12 Medtronic MiniMed, Inc. Hexamethyldisiloxane membranes for analyte sensors
EP4134665A1 (en) 2021-08-13 2023-02-15 Medtronic MiniMed, Inc. Dry electrochemical impedance spectroscopy metrology for conductive chemical layers
EP4162874A1 (en) 2021-10-08 2023-04-12 Medtronic MiniMed, Inc. Immunosuppressant releasing coatings
EP4174188A1 (en) 2021-10-14 2023-05-03 Medtronic Minimed, Inc. Sensors for 3-hydroxybutyrate detection
EP4190908A1 (en) 2021-12-02 2023-06-07 Medtronic Minimed, Inc. Ketone limiting membrane and dual layer membrane approach for ketone sensing
EP4310193A1 (en) 2022-07-20 2024-01-24 Medtronic Minimed, Inc. Acrylate hydrogel membrane for dual function of diffusion limiting membrane as well as attenuation to the foreign body response

Families Citing this family (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6338790B1 (en) 1998-10-08 2002-01-15 Therasense, Inc. Small volume in vitro analyte sensor with diffusible or non-leachable redox mediator
US8226814B2 (en) * 2001-05-11 2012-07-24 Abbott Diabetes Care Inc. Transition metal complexes with pyridyl-imidazole ligands
US6676816B2 (en) * 2001-05-11 2004-01-13 Therasense, Inc. Transition metal complexes with (pyridyl)imidazole ligands and sensors using said complexes
US8070934B2 (en) 2001-05-11 2011-12-06 Abbott Diabetes Care Inc. Transition metal complexes with (pyridyl)imidazole ligands
US6932894B2 (en) 2001-05-15 2005-08-23 Therasense, Inc. Biosensor membranes composed of polymers containing heterocyclic nitrogens
US7052591B2 (en) 2001-09-21 2006-05-30 Therasense, Inc. Electrodeposition of redox polymers and co-electrodeposition of enzymes by coordinative crosslinking
US7192766B2 (en) * 2001-10-23 2007-03-20 Medtronic Minimed, Inc. Sensor containing molded solidified protein
US7299082B2 (en) 2003-10-31 2007-11-20 Abbott Diabetes Care, Inc. Method of calibrating an analyte-measurement device, and associated methods, devices and systems
US8165651B2 (en) * 2004-02-09 2012-04-24 Abbott Diabetes Care Inc. Analyte sensor, and associated system and method employing a catalytic agent
US7699964B2 (en) 2004-02-09 2010-04-20 Abbott Diabetes Care Inc. Membrane suitable for use in an analyte sensor, analyte sensor, and associated method
US7545272B2 (en) 2005-02-08 2009-06-09 Therasense, Inc. RF tag on test strips, test strip vials and boxes
US7885698B2 (en) 2006-02-28 2011-02-08 Abbott Diabetes Care Inc. Method and system for providing continuous calibration of implantable analyte sensors
US11730407B2 (en) 2008-03-28 2023-08-22 Dexcom, Inc. Polymer membranes for continuous analyte sensors
US8583204B2 (en) 2008-03-28 2013-11-12 Dexcom, Inc. Polymer membranes for continuous analyte sensors
US8858501B2 (en) * 2008-04-11 2014-10-14 Medtronic Minimed, Inc. Reservoir barrier layer systems and methods
US8206353B2 (en) * 2008-04-11 2012-06-26 Medtronic Minimed, Inc. Reservoir barrier layer systems and methods
US9295776B2 (en) * 2008-04-11 2016-03-29 Medtronic Minimed, Inc. Reservoir plunger head systems and methods
CN102292053A (en) 2008-09-29 2011-12-21 卡迪尔克阀门技术公司 Heart valve
WO2010040009A1 (en) 2008-10-01 2010-04-08 Cardiaq Valve Technologies, Inc. Delivery system for vascular implant
EP4119098A1 (en) 2009-04-15 2023-01-18 Edwards Lifesciences CardiAQ LLC Vascular implant and delivery system
US20120148850A1 (en) * 2009-11-24 2012-06-14 Yongwoo Lee Sorption reinforced catalytic coating system and method for the degradation of threat agents
US8579964B2 (en) 2010-05-05 2013-11-12 Neovasc Inc. Transcatheter mitral valve prosthesis
US9308087B2 (en) 2011-04-28 2016-04-12 Neovasc Tiara Inc. Sequentially deployed transcatheter mitral valve prosthesis
US9554897B2 (en) 2011-04-28 2017-01-31 Neovasc Tiara Inc. Methods and apparatus for engaging a valve prosthesis with tissue
US9345573B2 (en) 2012-05-30 2016-05-24 Neovasc Tiara Inc. Methods and apparatus for loading a prosthesis onto a delivery system
US10583002B2 (en) 2013-03-11 2020-03-10 Neovasc Tiara Inc. Prosthetic valve with anti-pivoting mechanism
US9681951B2 (en) 2013-03-14 2017-06-20 Edwards Lifesciences Cardiaq Llc Prosthesis with outer skirt and anchors
US9572665B2 (en) 2013-04-04 2017-02-21 Neovasc Tiara Inc. Methods and apparatus for delivering a prosthetic valve to a beating heart
CA2933166C (en) 2013-12-31 2020-10-27 Abbott Diabetes Care Inc. Self-powered analyte sensor and devices using the same
US10569071B2 (en) 2015-08-31 2020-02-25 Ethicon Llc Medicant eluting adjuncts and methods of using medicant eluting adjuncts
US10245034B2 (en) * 2015-08-31 2019-04-02 Ethicon Llc Inducing tissue adhesions using surgical adjuncts and medicants
US11298059B2 (en) 2016-05-13 2022-04-12 PercuSense, Inc. Analyte sensor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6171586B1 (en) * 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
US6267958B1 (en) * 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation

Family Cites Families (80)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US426638A (en) * 1890-04-29 Belt rest
US424696A (en) * 1890-04-01 Fan-blower
CH564031A5 (en) * 1968-03-29 1975-07-15 Anvar
US4240438A (en) 1978-10-02 1980-12-23 Wisconsin Alumni Research Foundation Method for monitoring blood glucose levels and elements
US4486408A (en) 1981-04-07 1984-12-04 Kiel Johnathan L Insoluble crosslinked cytotoxic oxidase-peroxidase system
US4478946A (en) 1981-07-02 1984-10-23 South African Inventions Development Corporation Carrier bound immunosorbent
US4568335A (en) 1981-08-28 1986-02-04 Markwell Medical Institute, Inc. Device for the controlled infusion of medications
US4628928A (en) 1982-08-09 1986-12-16 Medtronic, Inc. Robotic implantable medical device and/or component restoration system
US4771772A (en) 1982-08-09 1988-09-20 Medtronic, Inc. Robotic implantable medical device and/or component restoration system
US4479796A (en) 1982-11-15 1984-10-30 Medtronic, Inc. Self-regenerating drug administration device
US4650547A (en) 1983-05-19 1987-03-17 The Regents Of The University Of California Method and membrane applicable to implantable sensor
US4484987A (en) 1983-05-19 1984-11-27 The Regents Of The University Of California Method and membrane applicable to implantable sensor
JPS60260611A (en) 1984-06-08 1985-12-23 Mitsubishi Petrochem Co Ltd Production of high-molecular weight cresol novolak resin
US4890620A (en) 1985-09-20 1990-01-02 The Regents Of The University Of California Two-dimensional diffusion glucose substrate sensing electrode
US4994167A (en) 1986-04-15 1991-02-19 Markwell Medical Institute, Inc. Biological fluid measuring device
US4757022A (en) 1986-04-15 1988-07-12 Markwell Medical Institute, Inc. Biological fluid measuring device
US4703756A (en) 1986-05-06 1987-11-03 The Regents Of The University Of California Complete glucose monitoring system with an implantable, telemetered sensor module
US4891104A (en) * 1987-04-24 1990-01-02 Smithkline Diagnostics, Inc. Enzymatic electrode and electrode module and method of use
FR2617763B1 (en) * 1987-07-07 1989-12-01 Essilor Int METHOD OF MANUFACTURING CONTACT LENS IN A NATURAL PROTEIN POLYMER, BY MOLDING BEFORE CROSS-LINKING
JP2672561B2 (en) * 1988-01-29 1997-11-05 テルモ株式会社 Membrane cover sensor
US5266688A (en) 1988-06-21 1993-11-30 Chiron Corporation Polynucleotide sequence for production of glucose oxidase in recombinant systems
US5094951A (en) 1988-06-21 1992-03-10 Chiron Corporation Production of glucose oxidase in recombinant systems
US4911168A (en) 1989-01-13 1990-03-27 Pacesetter Infusion, Ltd. Method of screening and selecting intraperitoneal medication infusion pump candidates
US5270446A (en) 1989-04-04 1993-12-14 Suntory Limited Decolorized crosslinked products and method for decolorization of crosslinked products
US5431160A (en) * 1989-07-19 1995-07-11 University Of New Mexico Miniature implantable refillable glucose sensor and material therefor
US5985129A (en) 1989-12-14 1999-11-16 The Regents Of The University Of California Method for increasing the service life of an implantable sensor
US5593852A (en) 1993-12-02 1997-01-14 Heller; Adam Subcutaneous glucose electrode
US5773270A (en) 1991-03-12 1998-06-30 Chiron Diagnostics Corporation Three-layered membrane for use in an electrochemical sensor system
US5328460A (en) 1991-06-21 1994-07-12 Pacesetter Infusion, Ltd. Implantable medication infusion pump including self-contained acoustic fault detection apparatus
GB9311784D0 (en) 1993-06-08 1993-07-28 Univ Alberta Vascular bioartificial organ
US5497772A (en) 1993-11-19 1996-03-12 Alfred E. Mann Foundation For Scientific Research Glucose monitoring system
US5791344A (en) 1993-11-19 1998-08-11 Alfred E. Mann Foundation For Scientific Research Patient monitoring system
US5494562A (en) 1994-06-27 1996-02-27 Ciba Corning Diagnostics Corp. Electrochemical sensors
US5667983A (en) 1994-10-24 1997-09-16 Chiron Diagnostics Corporation Reagents with enhanced performance in clinical diagnostic systems
US5741319A (en) 1995-01-27 1998-04-21 Medtronic, Inc. Biocompatible medical lead
US5995860A (en) 1995-07-06 1999-11-30 Thomas Jefferson University Implantable sensor and system for measurement and control of blood constituent levels
US5741211A (en) 1995-10-26 1998-04-21 Medtronic, Inc. System and method for continuous monitoring of diabetes-related blood constituents
US6002954A (en) 1995-11-22 1999-12-14 The Regents Of The University Of California Detection of biological molecules using boronate-based chemical amplification and optical sensors
DE69633573T2 (en) 1995-11-22 2005-10-06 Medtronic MiniMed, Inc., Northridge DETECTION OF BIOLOGICAL MOLECULES USING CHEMICAL AMPLIFICATION AND OPTICAL SENSOR
SE9504233D0 (en) 1995-11-27 1995-11-27 Pacesetter Ab Implantable medical device
US5834232A (en) * 1996-05-01 1998-11-10 Zymogenetics, Inc. Cross-linked gelatin gels and methods of making them
AU3596597A (en) 1996-07-08 1998-02-02 Animas Corporation Implantable sensor and system for in vivo measurement and control of fluid constituent levels
JP2943700B2 (en) 1996-07-10 1999-08-30 日本電気株式会社 Biosensor
US5696314A (en) 1996-07-12 1997-12-09 Chiron Diagnostics Corporation Multilayer enzyme electrode membranes and methods of making same
US5707502A (en) 1996-07-12 1998-01-13 Chiron Diagnostics Corporation Sensors for measuring analyte concentrations and methods of making same
US5804048A (en) 1996-08-15 1998-09-08 Via Medical Corporation Electrode assembly for assaying glucose
US5932175A (en) 1996-09-25 1999-08-03 Via Medical Corporation Sensor apparatus for use in measuring a parameter of a fluid sample
JP2000507457A (en) 1996-11-14 2000-06-20 ラジオメーター・メディカル・アクティーゼルスカブ Enzyme sensor
US5964993A (en) * 1996-12-19 1999-10-12 Implanted Biosystems Inc. Glucose sensor
EP0958495B1 (en) 1997-02-06 2002-11-13 Therasense, Inc. Small volume in vitro analyte sensor
US6001067A (en) 1997-03-04 1999-12-14 Shults; Mark C. Device and method for determining analyte levels
US5919216A (en) 1997-06-16 1999-07-06 Medtronic, Inc. System and method for enhancement of glucose production by stimulation of pancreatic beta cells
US6093167A (en) 1997-06-16 2000-07-25 Medtronic, Inc. System for pancreatic stimulation and glucose measurement
US6125291A (en) 1998-10-30 2000-09-26 Medtronic, Inc. Light barrier for medical electrical lead oxygen sensor
US6134459A (en) 1998-10-30 2000-10-17 Medtronic, Inc. Light focusing apparatus for medical electrical lead oxygen sensor
US6248080B1 (en) 1997-09-03 2001-06-19 Medtronic, Inc. Intracranial monitoring and therapy delivery control device, system and method
US6144866A (en) 1998-10-30 2000-11-07 Medtronic, Inc. Multiple sensor assembly for medical electric lead
US6198952B1 (en) 1998-10-30 2001-03-06 Medtronic, Inc. Multiple lens oxygen sensor for medical electrical lead
US6125290A (en) 1998-10-30 2000-09-26 Medtronic, Inc. Tissue overgrowth detector for implantable medical device
US6259937B1 (en) * 1997-09-12 2001-07-10 Alfred E. Mann Foundation Implantable substrate sensor
WO1999017095A1 (en) 1997-09-30 1999-04-08 M-Biotech, Inc. Biosensor
US5941906A (en) 1997-10-15 1999-08-24 Medtronic, Inc. Implantable, modular tissue stimulator
US6027479A (en) 1998-02-27 2000-02-22 Via Medical Corporation Medical apparatus incorporating pressurized supply of storage liquid
US6103033A (en) 1998-03-04 2000-08-15 Therasense, Inc. Process for producing an electrochemical biosensor
US5992211A (en) 1998-04-23 1999-11-30 Medtronic, Inc. Calibrated medical sensing catheter system
US6175752B1 (en) 1998-04-30 2001-01-16 Therasense, Inc. Analyte monitoring device and methods of use
US5934232A (en) 1998-06-12 1999-08-10 General Motors Corporation Engine valve lift mechanism
WO1999066964A1 (en) 1998-06-23 1999-12-29 Surgical Sealants, Incorporated Carbodiimide cross-linked albumin for bioadhesives, surgical sealants and implantable devices
US6280587B1 (en) 1998-07-02 2001-08-28 Nec Corporation Enzyme electrode and a biosensor and a measuring apparatus therewith
US6251260B1 (en) 1998-08-24 2001-06-26 Therasense, Inc. Potentiometric sensors for analytic determination
US6159240A (en) 1998-08-31 2000-12-12 Medtronic, Inc. Rigid annuloplasty device that becomes compliant after implantation
US6201980B1 (en) 1998-10-05 2001-03-13 The Regents Of The University Of California Implantable medical sensor system
US6163723A (en) 1998-10-22 2000-12-19 Medtronic, Inc. Circuit and method for implantable dual sensor medical electrical lead
US6261280B1 (en) 1999-03-22 2001-07-17 Medtronic, Inc Method of obtaining a measure of blood glucose
USD426638S (en) 1999-05-06 2000-06-13 Therasense, Inc. Glucose sensor buttons
USD424696S (en) 1999-05-06 2000-05-09 Therasense, Inc. Glucose sensor
US6368274B1 (en) 1999-07-01 2002-04-09 Medtronic Minimed, Inc. Reusable analyte sensor site and method of using the same
US6343225B1 (en) * 1999-09-14 2002-01-29 Implanted Biosystems, Inc. Implantable glucose sensor
EP1354031A2 (en) 2000-07-31 2003-10-22 Maxygen, Inc. Nucleotide incorporating enzymes
US7192766B2 (en) * 2001-10-23 2007-03-20 Medtronic Minimed, Inc. Sensor containing molded solidified protein

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6267958B1 (en) * 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
US6171586B1 (en) * 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1437937A2 *

Cited By (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7409238B2 (en) 1998-08-20 2008-08-05 Becton, Dickinson And Company Micro-invasive method for painless detection of analytes in extracellular space
US8965470B2 (en) 1998-08-20 2015-02-24 Becton, Dickinson And Company Micro-invasive method for painless detection of analytes in extracellular space
US9968742B2 (en) 2007-08-29 2018-05-15 Medtronic Minimed, Inc. Combined sensor and infusion set using separated sites
US8394463B1 (en) 2009-01-23 2013-03-12 Medtronic Minimed, Inc. Crosslinking compounds at negative pressures and materials made by such methods
WO2011041715A2 (en) 2009-10-01 2011-04-07 Medtronic Minimed, Inc. Analyte sensor apparatuses having interference rejection membranes and methods for making and using them
WO2011063259A2 (en) 2009-11-20 2011-05-26 Medtronic Minimed, Inc. Multi-conductor lead configurations useful with medical device systems and methods for making and using them
WO2011084651A1 (en) 2009-12-21 2011-07-14 Medtronic Minimed, Inc. Analyte sensors comprising blended membrane compositions and methods for making and using them
WO2011091061A1 (en) 2010-01-19 2011-07-28 Medtronic Minimed, Inc. Insertion device for a combined sensor and infusion sets
US10448872B2 (en) 2010-03-16 2019-10-22 Medtronic Minimed, Inc. Analyte sensor apparatuses having improved electrode configurations and methods for making and using them
WO2011115949A1 (en) 2010-03-16 2011-09-22 Medtronic Minimed, Inc. Glucose sensor
WO2011163303A2 (en) 2010-06-23 2011-12-29 Medtronic Minimed, Inc. Sensor systems having multiple probes and electrode arrays
WO2012154548A1 (en) 2011-05-06 2012-11-15 Medtronic Minimed, Inc. Method and apparatus for continuous analyte monitoring
US9008744B2 (en) 2011-05-06 2015-04-14 Medtronic Minimed, Inc. Method and apparatus for continuous analyte monitoring
US11020028B2 (en) 2012-05-25 2021-06-01 Medtronic Minimed, Inc. Foldover sensors and methods for making and using them
US9493807B2 (en) 2012-05-25 2016-11-15 Medtronic Minimed, Inc. Foldover sensors and methods for making and using them
WO2013177573A2 (en) 2012-05-25 2013-11-28 Medtronic Minimed, Inc. Foldover sensors and methods for making and using them
WO2014008297A1 (en) 2012-07-03 2014-01-09 Medtronic Minimed, Inc. Analyte sensors and production thereof
WO2014089276A1 (en) 2012-12-06 2014-06-12 Medtronic Minimed, Inc. Microarray electrodes useful with analyte sensors and methods for making and using them
US10772540B2 (en) 2012-12-06 2020-09-15 Medtronic Minimed, Inc. Microarray electrodes useful with analyte sensors and methods for making and using them
US10194840B2 (en) 2012-12-06 2019-02-05 Medtronic Minimed, Inc. Microarray electrodes useful with analyte sensors and methods for making and using them
WO2014116293A1 (en) 2013-01-22 2014-07-31 Medtronic Minimed, Inc. Muting glucose sensor oxygen response and reducing electrode edge growth with pulsed current plating
US10426383B2 (en) 2013-01-22 2019-10-01 Medtronic Minimed, Inc. Muting glucose sensor oxygen response and reducing electrode edge growth with pulsed current plating
US11266332B2 (en) 2013-01-22 2022-03-08 Medtronic Minimed, Inc. Muting glucose sensor oxygen response and reducing electrode edge growth with pulsed current plating
WO2015069692A2 (en) 2013-11-07 2015-05-14 Medtronic Minimed, Inc. Enzyme matrices for use with ethylene oxide sterilization
WO2017189764A1 (en) 2016-04-28 2017-11-02 Medtronic Minimed, Inc. In-situ chemistry stack for continuous glucose sensors
WO2017195035A1 (en) 2016-05-10 2017-11-16 Interface Biologics, Inc. Implantable glucose sensors having a biostable surface
WO2017214173A1 (en) 2016-06-06 2017-12-14 Medtronic Minimed, Inc. Polycarbonate urea/urethane polymers for use with analyte sensors
CN110023745A (en) * 2016-12-09 2019-07-16 东北大学 The durable biosensor and droplet deposition fixing means based on enzyme
CN110023745B (en) * 2016-12-09 2022-08-23 东北大学 Robust enzyme-based biosensor and droplet deposition immobilization method
EP3552005A4 (en) * 2016-12-09 2020-08-12 Northeastern University Durable enzyme-based biosensor and process for drop deposition immobilization
WO2018170363A1 (en) 2017-03-17 2018-09-20 Medtronic Minimed, Inc. Metal pillar device structures and methods for making and using them in electrochemical and/or electrocatalytic applications
WO2019005687A1 (en) 2017-06-30 2019-01-03 Medtronic Minimed, Inc. Sensor initialization methods for faster body sensor response
WO2019147578A1 (en) 2018-01-23 2019-08-01 Medtronic Minimed, Inc. Implantable polymer surfaces exhibiting reduced in vivo inflammatory responses
WO2019156934A1 (en) 2018-02-07 2019-08-15 Medtronic Minimed, Inc. Multilayer electrochemical analyte sensors and methods for making and using them
WO2019157043A1 (en) 2018-02-08 2019-08-15 Medtronic Minimed, Inc. Glucose sensor electrode design
WO2019157106A2 (en) 2018-02-08 2019-08-15 Medtronic Minimed, Inc. Methods for controlling physical vapor deposition metal film adhesion to substrates and surfaces
WO2019222499A1 (en) 2018-05-16 2019-11-21 Medtronic Minimed, Inc. Thermally stable glucose limiting membrane for glucose sensors
WO2021021538A1 (en) 2019-07-26 2021-02-04 Medtronic Minimed, Inc. Methods to improve oxygen delivery to implantable sensors
WO2021021867A1 (en) 2019-08-01 2021-02-04 Medtronic Minimed, Inc. Micro-pillar working electrodes design to reduce backflow of hydrogen peroxide in glucose sensor
WO2022026542A1 (en) 2020-07-31 2022-02-03 Medtronic Minimed, Inc. Sensor identification and integrity check design
WO2022093574A1 (en) 2020-10-29 2022-05-05 Medtronic Minimed, Inc. Glucose biosensors comprising direct electron transfer enzymes and methods of making and using them
WO2022164981A1 (en) 2021-01-29 2022-08-04 Medtronic Minimed, Inc. Interference rejection membranes useful with analyte sensors
EP4071251A1 (en) 2021-04-09 2022-10-12 Medtronic MiniMed, Inc. Hexamethyldisiloxane membranes for analyte sensors
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EP4310193A1 (en) 2022-07-20 2024-01-24 Medtronic Minimed, Inc. Acrylate hydrogel membrane for dual function of diffusion limiting membrane as well as attenuation to the foreign body response

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EP1437937A2 (en) 2004-07-21
US8187851B2 (en) 2012-05-29
EP1437937B1 (en) 2008-05-21
WO2003035891A3 (en) 2003-11-20
US20050006807A1 (en) 2005-01-13
DE60226759D1 (en) 2008-07-03
US7435569B2 (en) 2008-10-14
US20050214741A1 (en) 2005-09-29
US20030077772A1 (en) 2003-04-24
US20090081753A1 (en) 2009-03-26
JP2005507007A (en) 2005-03-10
EP1437937A4 (en) 2005-11-30
US7192766B2 (en) 2007-03-20
US8137927B2 (en) 2012-03-20
ATE396278T1 (en) 2008-06-15
CA2457545A1 (en) 2003-05-01
AU2002327774A1 (en) 2003-05-06

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