HK1143052B - Transdermal analyte monitoring systems and methods for analyte detection - Google Patents

Description

Transdermal analyte monitoring system and method for detecting analytes

Technical Field

The present invention relates to the field of systems and methods for improving the non-invasive sampling of biological fluids, and more particularly to systems and methods for improving the transdermal detection and quantification of analytes.

Cross reference to related patent applications

This patent application claims priority from U.S. patent application No. s.s.n.60/893,563, filed on 7/3/2007 at the united states patent and trademark office.

Background

The impact of diabetes on the health of americans is striking. According to the American diabetes Association, about 2080 million Americans have been diagnosed with diabetes in 2006. It is estimated that the cost for diabetes in 2002 is $ 1320 billion. It is estimated that 613 americans per day died in 2006 due to complications associated with diabetes.

There is a strong need for new and improved systems and methods for the treatment and detection of diabetes. Analytical biosensors provide a system that can be used to monitor diabetes. Analytical biosensors have been used for the last decade as a means to combine the advantages of electrochemical signaling with the inherent specificity in biological interactions. For example, the use of dynamic blood glucose monitoring (CGM) to monitor diabetes has become increasingly popular.

Although analytical biosensors have recently improved, the available systems suffer from several drawbacks. For example, systems that typically use hydrogel sensors have a short shelf life and may leak sensor material onto the skin of the user. Alternatively, bacterial growth or growth of other microorganisms may contaminate or cause biofouling on the biosensor, rendering its analytical measurements unreliable. In some cases, proteins, carbohydrates, cells, or cell fragments from the user can bind to the sensor and interfere with the measurement. This binding can also contaminate the biosensor.

Membranes, films, or other physical barriers have been used on sensor electrode surfaces to prevent contaminants from reaching the electrode face. Typical thin films that have been used include: cellulose acetate, poly (o-phenylenediamine), polyphenol, polypyrrole, polycarbonate, and(i.e., tetrafluoroethylene-perfluoro-3, 6-dioxo-4-methyl-7-octenesulfonic acid copolymer available from e.i.du Pont de nemours&Co., wilmington, tera). However, these films may be difficult to prepare and may not adhere effectively to the reaction surface of the electrode.

Certain CGM systems require pre-treatment of the skin with a hydrating formulation prior to system attachment. For example, with existing biosensor systems, a skin hydrating procedure is typically applied to the target skin site for 10-40 minutes after the treatment to increase skin porosity but before the sensor is applied. The water replenishment process may result in better sensor performance (good correspondence of sensor signal to reference glucose reading) than if the sensor were not pre-treated. While it can result in improved sensor performance, the skin hydrating process also requires additional labor, materials, and time that further complicate the instrument setup process and thereby increase the cost of the system. Desirably, a system that does not require a complex or time-consuming skin pretreatment procedure.

In other CGM systems, the blood glucose sensor is calibrated using a standard reference blood glucose method, and then the sensor reports a subsequent glucose reading based on the calibrated electrical signal. In principle, the subject's blood glucose concentration should be proportional to the measured electrical signal. For sensors based on enzymatic conversion of glucose, for example, when glucose oxidase (GOx) converts glucose to hydrogen peroxide (H) using water and oxygen₂O₂) And glucurolactoneEnzymatic conversion is limited by the amount of oxygen available. When the oxygen supply is limited, such as in interstitial fluid, where the glucose concentration exceeds the oxygen concentration, the enzymatic conversion of glucose will depend on the oxygen supply, which may lead to unreliable glucose readings of the sensor and thus to an influence on the sensor performance.

Various methods for alleviating oxygen limitation have been reported. Tierney et al describe the use of reverse iontophoresis to limit glucose extraction and maintain a good balance of oxygen to glucose (M. Tierney et al, Annals of Medicine, 32 (9): 632-641 (2000)). Shults et al, in U.S. Pat. No.7,110,803, disclose the use of a glucose-limiting membrane layer having a high oxygen to glucose permeability ratio. Simpson et al, in U.S. Pat. No.7,108,778, disclose the use of auxiliary electrodes to generate oxygen for sensing chemistry. However, each of these methods requires the addition of additional elements to the CGM system, thereby increasing the cost and complexity of the system. There is a need for a simple method of increasing the amount of available oxygen to a sensor without increasing the cost and complexity of the system.

It is therefore an object of the present invention to provide an improved transdermal analyte monitoring system.

It is another object of the present invention to provide a method of reducing biological deposition and/or contamination in a transdermal analyte monitoring system.

It is a further object of the present invention to provide a method for improving the accuracy of analyte detection and/or quantification using a transdermal analyte detection system.

Disclosure of Invention

Described herein is a Transdermal Analyte Monitoring System (TAMS) with extended lifetime and improved analyte detection. Generally, a transdermal analyte detection system ("TADS") includes a sensor assembly and a display and/or computing device, wherein the sensor assembly includes: (1) a hydrophilic polymer matrix, such as a hydrogel, for receiving an analyte from the skin, and (2) a sensor body containing a plurality of electrodes. In a preferred embodiment, the TAMS comprises a semi-permeable membrane at the end of the sensor, which membrane is attached to the hydrophilic polymer matrix. This membrane is in contact with the outer surface of the subject and acts as a barrier between the patient's skin and the hydrophilic polymer matrix. The semipermeable membrane may reduce the amount of biofouling of the hydrophilic polymer matrix by forming a protective barrier on the exposed surface of the hydrophilic polymer matrix as compared to the same device lacking the semipermeable membrane. In addition, the semi-permeable membrane prevents the hydrophilic polymer matrix from leaking out of the device. The hydrophilic polymer matrix typically comprises an enzyme and may optionally comprise one or more humectants.

In preferred embodiments, the TAMS comprises one or more channels or reservoirs in the sensor assembly, which increases the amount of oxygen available to react the analyte with the enzyme and produce a detectable signal.

In another embodiment, a method for improving the detection and/or quantification of an analyte by a transdermal analyte monitoring system is provided. The method comprises the following steps: an area of the skin of the living being is treated to increase porosity and the treated area is then wiped with a substrate. The substrate may be any suitable adsorbent material such as a pad, woven or non-woven fabric, felt or gauze. Typically, the substrate comprises a wiping agent such as a solvent (e.g., water, ethanol, or propanol), a phosphate buffer, lactic acid, soap, a surfactant, or a combination thereof. This wiping step makes it unnecessary to carry out a skin moisturizing treatment. After being wiped across the skin, the transdermal analyte monitoring system was used.

In another embodiment, a test kit is provided comprising a transdermal analyte monitoring system and a substrate. The substrate may be wetted with the wiping agent. Alternatively, the reagent for wiping may be contained in the cartridge separately.

Drawings

Fig. 1 shows an exemplary wired Transdermal Analyte Monitoring System (TAMS) for continuous analyte monitoring, and the sensor is shown in an exploded view. Alternatively, communication between the sensor and the detector is achieved via a wireless link (not shown in fig. 1).

Fig. 2 shows the sensor body shown in fig. 1.

Fig. 3 is a bar graph of the percent signal remaining for an exemplary biosensor with and without a barrier membrane after 24 hours in vitro application, where "n" represents the number of trials.

Fig. 4 shows histograms of signal drift (%) and 24-hour mean absolute relative error (MARD) between sensor (nA) measurements and Blood Glucose (BG) levels for devices with different membranes, calibrated every 4 hours. The letters "PES-10K" (poly (ether sulfone)) and "RC-3K" under the bar chart refer to UF membranes, "UB" refers to covalently activated PES membranes, "0.2 PES" refers to PES membranes without coating and with 0.2 μm pores, ". NAF" refers to membranes with pores of 0.2 μmCoated film, and the asterisk indicates that the MARD was calibrated every 8 hours for this sensor.

Fig. 5 shows the signal drift (%) and 24-hour MARD histogram when multiple wetting agents are contained in the hydrogel matrix. The experiment was a 24 hour in vitro application and all used 0.2pes.

FIG. 6 shows the correlation "R" by nA and BG after 4 hours of in vitro application²"and MARD to compare histograms of biosensor systems with covalently immobilized GOx and biosensor systems without covalently immobilized GOx.

FIG. 7A is a graph of blood glucose concentration (mg/dl) versus time (minutes) from a continuous, transdermal glucose sensor and a skin preparation wipe procedure performed prior to application of the sensor. The reference blood glucose ("actual BG", finger stick glucometer reading, circled solid line) level is compared to the predicted blood glucose ("predicted BG", sensor glucose reading, solid line) level. The data shows a strong correlation between the predicted BG and the actual BG (r ═ 0.950).

FIG. 7B is a graph of blood glucose concentration (mg/dl) versus time (minutes) from a continuous, transdermal glucose sensor, and no skin preparation wiping procedure prior to application of the sensor. The reference blood glucose ("actual BG", finger stick glucometer reading, circled solid line) level is compared to the predicted blood glucose ("predicted BG", sensor glucose reading, solid line) level. The data show a very poor correlation between the predicted BG and the actual BG (r ═ 0.309).

Fig. 7C is a graph of blood glucose concentration (mg/dl) versus time (minutes) from a continuous, transdermal glucose sensor and a 40 minute skin moisturizing procedure prior to application of the sensor. The reference blood glucose ("actual BG", finger stick glucometer reading, circled solid line) level is compared to the predicted blood glucose ("predicted BG", sensor glucose reading, solid line) level. The data show a strong correlation between the predicted BG and the actual BG (r ═ 0.947).

Fig. 8A is a diagram showing the bottom of an exemplary target plate having four air channels.

FIG. 8B is a diagram showing the front of an exemplary glucose sensor target plate having three cutouts around its central aperture for hydrogel chemistry as internal air reservoirs.

FIG. 8C shows an exemplary sensor mounted on top of a target plate to provide an enclosed glucose sensor.

Fig. 9 is a schematic diagram showing how an exemplary wireless Transdermal Analyte Monitoring System (TAMS) may be used for continuous analyte monitoring. The system can be used with the glucose sensor described in fig. 8A, 8B, 8C.

Fig. 10 is a Clarke error grid of the data obtained in study 1A with 222 sensor-glucose data points collected from 10 patients.

Fig. 11 is a Clarke error grid of the data obtained in study 1B with 225 sensor-glucose data points collected from 10 patients.

Fig. 12 is a Clarke error grid of the data obtained in study 2C with 147 sensor-blood glucose data points collected from 10 patients (of which 9 completed the study). Detailed Description

I. Transdermal analyte monitoring system

Systems and methods for enhancing transdermal detection of analytes are described herein. Generally, a transdermal analyte monitoring system ("TAMS") includes a sensor assembly and a display and/or computing device, wherein the sensor assembly comprises: (1) a hydrophilic polymer matrix, such as a hydrogel, for receiving an analyte from the skin, and (2) a sensor body containing a plurality of electrodes. In a preferred embodiment, the TAMS comprises a semi-permeable membrane at the end of the sensor, which membrane is attached to the hydrophilic polymer matrix. This membrane acts as a semipermeable barrier between the patient's skin and the hydrophilic polymer matrix.

Applying the TAMS to an area on the skin of an animal; typically, the animal is a mammal, and in preferred embodiments the animal is a human.

When this system is used, the hydrogel contains an enzyme that continuously reacts with the analyte, thereby generating an electrical signal. The electrical signal is then detected using the electrode assembly. The electrical signal is correlated with a value of the analyte.

The analyte to be monitored may be any analyte of interest including, but not limited to: glucose, lactate, blood gas (e.g., carbon dioxide or oxygen), blood pH, electrolytes, ammonia, proteins, or other biological substances present in biological fluids such as blood, plasma, serum, or interstitial fluid.

An exemplary TAMS described in U.S. patent application publication No. 20060094946 to Kellogg et al is presented in fig. 1 herein. The TAMS shown in fig. 1 can be used to perform continuous monitoring of an analyte such as glucose. As shown in fig. 1, the TAMS (100) described herein includes a sensor assembly (112) comprising: a sensor body (101), a hydrogel disc (106), and a mounting plate (102), as well as other components described herein, which may be connected to a display or computing device. During operation, the sensor assembly (112) may be positioned adjacent to a permeable area (107) of the user's skin, the permeable area (107) being shown in phantom in fig. 1. The sensor assembly (112) may be connected to a display or computing device using any suitable method. Suitable methods include: wireless connection or any other electrical connection method such as a flexible cable (109) for connection. In one embodiment, the sensor assembly (112) is connected to a potentiostat (108), which potentiostat (108) may include a printed circuit board (111). The connecting cable (109) is preferably connected to the potentiostat (108) via a connector which facilitates removal and connection of the sensor assembly (112). Suitable methods of attachment include: the connection is connected with a wireless connection by a flexible cable (109).

A TAMS with wireless connectivity is presented in fig. 9. The sensor assembly (112) includes: a target plate (120), a hydrogel (106) and a sensor, and a sensor housing (125). The sensor is used in conjunction with a micro-analyzer that wirelessly transmits data to a monitor for data processing and display.

A. Sensor assembly

The sensor assembly (112) shown in fig. 1 and 2 can be incorporated into any of a variety of sensing devices. For example, the sensor assembly may be incorporated into a receiver to provide discrete and/or continuous glucose monitoring.

The sensor assembly (112) includes a sensor body (101). The sensor body includes electrodes, as shown in fig. 2, on the surface of which an analyte or a reaction product, which is an indicator of the analyte, is electrochemically detected.

A heat transducer (103) is interposed between a sensor body (101) and a mounting board (102), and the heat transducer (103) can be packaged in a sensor package (112) in a shape corresponding to the sensor body (101). Enzyme-based electrochemical sensors, such as glucose sensors, may be sensitive to temperature fluctuations. The heat transducer (103) may be used to normalize and report changes due only to the analyte or changes in an indicator of the analyte.

The sensor assembly (112) may also include a suction cup (104), the suction cup (104) may be attached to a side of the sensor body (101) facing the heat transducer (103).

The sensor assembly (112) may also include a suction ring (105), and the suction ring (105) may be attached to a side of the sensor body (101) facing away from the suction cup (104). Preferably, a cut-away portion in the center of the suction ring (105) exposes some or all of the sensor components to the sensor body (101). The suction ring (105) and suction cup (104) may have a shape corresponding to the shape of the sensor body as shown in fig. 1.

The sensor assembly (112) comprises a hydrogel disc (106), which hydrogel disc (106) can be placed in a cut-out in the centre of the suction ring (105) adjacent to the surface of the sensor body (101).

a. Sensor body

The sensor body (101) is shown in detail in fig. 2. The sensor body 101 includes a body layer (207) with conductive lines (204, 205, and 206) disposed thereon. The leads may be formed by, for example, applying a metal in desired locations on the bulk layer (207). The working electrode (201) is generally centrally located in the sensor body (101). The working electrode (201) may comprise a catalytic and/or conductive material such as pure platinum, platinum carbon, glassy carbon, carbon nanotubes, mesoporous platinum, platinum black, palladium, gold, or platinum-iridium. The working electrode (201) may be disposed on the lead (206) such that it is in electrical contact with the lead (206). The counter electrode (202) may comprise a stable conductive material and preferably carbon, and may be disposed around the perimeter of a portion of the working electrode (201), as shown in fig. 2. The counter electrode (202) may be arranged on the lead (205) such that it is in electrical contact with the lead (205). As shown in fig. 2, a reference electrode (203) containing a binary oxidation-reduction material that can provide a stable redox potential, which preferably contains silver/silver chloride, can be disposed around another portion of the working electrode (201). The electrodes (201, 202, 203) may be formed substantially along the layout of electrically conductive lines (206, 205, 204), respectively, that run in the sensing area of the device. The electrodes (201, 202, 203) may be screen printed or sprayed on the electrical leads (206, 205, 204), respectively. The wires are routed to the sensor body (101) using screen printing or other methods known in the art in a manner that allows electrical connection to external devices or components. For example, as shown in FIG. 2, these leads may form 3X plug leads (204, 205, and 206) at the ends of the extended region of the sensor body. Thus, standard connectors can be used to connect the sensor electrodes to external devices or components.

b. Hydrophilic polymer matrix

The sensor assembly comprises a hydrophilic polymer matrix. The substrate is designed to provide a structure that forms a reservoir within the sensor assembly. The hydrophilic polymer matrix may be of any suitable shape suitable for a sensor component. Typically, the hydrophilic polymer matrix is present in the shape of the sensor body. One standard form is disc-like. The shape is selected to match the shape of the sensor. Optionally, ionic groups may be incorporated into the hydrophilic polymer matrix to provide additional functions, such as bioadhesiveness. In a preferred embodiment, the matrix is a hydrogel.

Hydrogels are a class of biomaterials used in the medical and biotechnological fields such as contact lenses, biosensors, liners for artificial implants, and drug delivery devices. Transdermal analyte monitoring systems may use one or more of the hydrogel materials described below. The types of hydrogel materials that can be used in the sensor assembly include: agarose-based hydrogels, polyethylene glycol diacrylate (PEG-DA) -based hydrogels, and vinyl acetate-based hydrogels, including polyethylene glycol diacrylate/polyethyleneimine (PEGDA-PEI) and polyethylene glycol diacrylate-N-vinyl pyrrolidone (PEGDA-NVP).

Suitable polymers that can form hydrogels include, but are not limited to: synthetic or natural polymers. Examples of synthetic polymers include: polyacrylic and polymethacrylic polymers, cellulose derivatives such as hydroxypropyl cellulose, polyethylene glycol-based polymers, copolymers and block copolymers, and other water-swellable, biocompatible polymers. Examples of natural polymers include collagen, hyaluronic acid, gelatin, albumin, polysaccharides, and derivatives thereof. Natural polymers can also be those isolated from various plant materials such as flax.

Structurally, the polymerized hydrogel is a three-dimensional macromolecular configuration. They can be prepared by a variety of methods: a) synthesis from monomers (cross-linking polymerization); b) synthesis from polymer and polymerization aid (graft and crosslink polymerization); c) synthesis from polymers and non-polymeric auxiliary agents (cross-linked polymers); d) synthesis from polymers with an energy source (boosterless cross-linking polymerization) and e) synthesis from polymers (cross-linking by reactive polymer-polymer inter-coupling).

The hydrogel can have various thicknesses. Typically, the hydrogel is about 10 to about 1000 μm, more preferably about 50 to about 700 μm, and still more preferably about 200 to about 500 μm.

As shown in fig. 1, the hydrogel disc (106) may be placed in such a way that it is stacked on the mounting plate (102) to face the user. The sensor body (101) may be attached to the mounting plate (102) using a standard connector with a latch that mates with a corresponding connector interface.

i. Agarose based hydrogels

Agarose-based hydrogels may provide several advantages for continuous transdermal monitoring of analytes. For example: agarose-based hydrogels provide one or more of the following properties: good response to glucose and hydrogen peroxide due to its high water content, high enzyme loading, good biocompatibility, and excellent permeation and diffusion properties.

Agarose hydrogels can be prepared, for example, by the following methods: adding 1-20% agarose to a solution containing 0-1M sodium or potassium phosphate, 0-1M sodium chloride, 0-1M potassium chloride, 0-2M lactic acid, and surfactant (such as 0-1M agarose gel)X-100 (United states Union carbide chemistry)&Plastics technology Co Ltd,80(ICI america corporation) or sodium lauryl sulfate), as well as buffers for other biocompatible components. For example, the loading of glucose oxidase in the agarose hydrogel can be up to 0-20% (by weight) by soaking the solid hydrogel in a solution of concentrated glucose oxidase, or by mixing a concentrated glucose oxidase powder or solution with the agarose solution in its melting stage (15-65 ℃) followed by cooling at a lower temperature (40 ℃ or lower) to gel it.

PEG-based hydrogels

PEG-based hydrogels may provide several advantages for continuous transdermal monitoring of analytes. Structurally, PEG is highly hydrophilic and exhibits a high degree of solvation in aqueous solvents. The predominant solvation of the PEG molecule effectively excludes proteins from the PEG chain space, thereby protecting the surface from protein-induced biofouling. PEG-based hydrogels crosslinked by chemistry can provide an advantage in that their physical and chemical properties can be modulated by varying the molecular weight of the PEG chains and varying the initiator concentration. For example, increasing the molecular weight of the polyethylene oxide backbone can increase the mesh size. The release of bioactive molecules, such as enzymes, can be controlled by controlling the network density. Therefore, the hydrogel containing PEG with a weight average molecular weight of 8kDa will have a higher release rate of the embedded drug than the hydrogel containing PEG with a weight average molecular weight of 3.3 kDa.

Optionally, additives may be incorporated into the hydrogel to provide additional functions, such as bioadhesive properties. For example, hyaluronic acid or polyacrylic acid may be added to the PEG macromer prior to crosslinking to create a bioadhesive hydrogel. In another embodiment, the crosslinked hydrogel may be imparted with an ionic character to provide intermolecular interactions (e.g., ionic bonds) with the entrapped drug, thereby slowing the rate of release of the drug from the matrix.

PEG-based hydrogels for use in biosensors may provide one or more of the following features: (a) a biocompatible, biofouling-free surface suitable for use in long-term exposure to biological fluids without impairing sensor function, (b) as a storage mechanism for glucose oxidase, (c) as a substrate into which ionic groups can be introduced to enhance capture of glucose oxidase, (d) as a substrate whose physical and chemical properties (network density, swellability) are adjusted by changing the molecular weight of the backbone, and (e) as a substrate with bioadhesive properties by adding ionic excipients such as chitosan gluconic acid, polyacrylic acid, polyamidoamine, polyethyleneimine and hyaluronic acid.

When the hydrogel is formed from polyethylene glycol diacrylate (PEGDA) macromolecules, polymerization (such as uv polymerization) may occur in a mold containing a pre-installed scrim page (scrim page) that may provide a support substrate and handle for the hydrogel. The PEGDA macromolecules polymerized only around the rounded head of the lollipop-type sheet, and no hydrogel was formed at the tail of the scrim sheet, so that the tail could be used as a handle (see fig. 2 and 9).

Optionally, the PEGDA hydrogel comprises an acrylate-PEG-NHS (a-PEG-N) reagent (e.g., sold by Nektar) that can be used as a linker molecule to covalently attach an enzyme (such as a GOx enzyme) to the PEGDA hydrogel network.

Vinyl acetate based hydrogels

Vinyl acetate based hydrogels (such as N-vinylpyrrolidone/vinyl acetate copolymers) may exhibit properties such as transparency, tackiness, nontoxicity, flexibility, and/or hydrophobicity. Vinyl acetate based hydrogels generally have good properties of moisturizing and trapping enzymes (such as glucose oxidase), are biocompatible, and have good adhesion to the skin so that coupling between the skin and the sensor can be improved. As reported by Chuang et al, a glucose flow sensor, which uses N-vinylpyrrolidone/vinyl acetate copolymer as a hydrogel, showed good performance in tracking blood glucose levels in diabetic patients in a glucose clamping study. Chuang et al, supra in the fourth technical annual meeting of diabetes, conducted on days 28-30 of 2004, described in "ultrasound Pretreatment for Continuous Transdermal glucose monitoring" (Philadelphia, Pa.).

Modified hydrogels

1. Covalent immobilized enzyme

Optionally, the hydrogel may be modified to include an enzyme and/or a humectant. The enzyme and/or humectant may be captured by any suitable method, including: covalent bonding and non-covalent immobilization. Examples of non-covalent immobilization include, but are not limited to: ionic interactions and physical trapping. Preferably, the enzyme is covalently linked to the hydrogel, for example by using a linker molecule. In one embodiment, the glucose oxidase is covalently immobilized in a hydrogel disc, which is particularly useful in CGM systems. For example, covalent immobilization of GOx into the PEGDA network allows for improved device effectiveness by eliminating GOx diffusion (maintaining bioavailability) and/or by stabilizing enzymes (maintaining bioactivity). The PEGDA network provides a structure with about 80% water in the matrix. It acts as a water reservoir to retain important components of the solution (e.g., buffer salts and penetrants) while also providing a transport medium for diffusion of the analyte.

At a PEGDA concentration of 15 wt%, most of the GOx is physically trapped in the mesh and thus retained in the hydrogel. However, at lower PEGDA concentrations, such as near 10% (by weight), the network is larger in mesh and will not retain GOx, thus requiring covalent immobilization.

Covalent attachment of enzymes to hydrogels using linkers

The enzyme may also be coupled to the hydrogel using a linker. The linker molecule typically includes two or more functional groups capable of reacting with the functional groups on the enzyme and the functional groups on the hydrogel. For example, the linker molecule may comprise electrophilic groups that react with nucleophilic groups (such as hydroxyl, thiol, and/or amino groups) present in the enzyme and the hydrogel. These linkers mediate the binding of the enzyme to the hydrogel surface by forming bonds with different atomic numbers. The linker molecules may be monofunctional (i.e., the functional groups are the same) or multifunctional (i.e., the functional groups are different).

Suitable linker molecules include, but are not limited to: n-succinimidyl 3- (2-pyridyldithio) propionate (SPDP, 3-and 7-interatomic), long chain-SPDP (12-interatomic), succinimidyloxycarbonyl-alpha-methyl-2- (2-pyridyldithio) toluene (SMPT, 8-interatomic), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC, 11-interatomic), and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC, 11-interatomic), m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS, 9-interatomic), N- (gamma-maleimidobutyryloxy) succinimidyl ester (GMBS, 8-atom spacing), N- (gamma-maleimidobutyryloxy) sulfosuccinimidyl ester (sulfo-GMBS, 8-atom spacing), succinimidyl 6- ((iodoacetyl) amino) hexanoate (SIAX, 9-atom spacing), 6- (6- (((4-iodoacetyl) amino) hexanoyl) amino) hexanoic acid succinimidyl ester (SIAXX, 16-atom spacer), 1, 4-bis- [3 '-2' -pyridyldithio ] propionamido) butane (DPDPDPPB, 16-atom spacer), bismaleimide hexane (BMH, 14-atom spacer) and iodoacetic acid p-nitrophenyl ester (NPIA, 2-atom spacer). One of ordinary skill in the art will also recognize that: many other coupling agents of different atomic numbers may also be used.

In addition, spacer molecules such as acrylate-polyethylene glycol-N-hydroxysuccinimide (acrylate-PEG-NHS or a-PEG-N) may also be introduced into the linker to increase the distance between the terminal reactive functional groups. Some multifunctional PEGs are commercially available from hilwatt polymers (helvetville, alabama) and Texaco Chemicals (houston, Texas). polyaminoPEGs are commercially available under the trade name "Jeffamine", which includes diamino PEGs and triamino PEGs. In a preferred embodiment, the enzyme is covalently immobilized in the hydrogel using acrylate-PEG-NHS (A-PEG-N).

Covalent attachment of enzymes to hydrogels using coupling reagents

It is also possible to couple the enzyme directly onto the hydrogel without introducing a coupling reagent, by using a reagent or by activating a certain group on the surface of the hydrogel or the enzyme, which group reacts correspondingly with a functional group on the enzyme or hydrogel.

For example, carbodiimides mediate the formation of amide linkages between carboxylic acid esters and amines or mediate the formation of phosphoramidate linkages between phosphate esters and amines. Examples of carbodiimides are 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (CMC), Dicyclohexylcarbodiimide (DCC), Diisopropylcarbodiimide (DIC), and N, N' -Carbonyldiimidazole (CDI). N-ethyl-3-phenylisoxazolium-3 '-sulfonate (Wood's reagent) mediates the formation of amide bonds by condensation of carboxylic acid esters and amines. CDI can also be used to attach amino groups to hydroxyl groups.

2. Wetting agent

In another embodiment, the hydrogel is modified to include one or more humectants. Humectants are hygroscopic substances that have a strong affinity to form hydrogen bonds with water molecules. Wetting agents typically have several hydrophilic groups such as hydroxyl, amine, or carboxyl groups. The hydrogel may contain any suitable amount of humectant to ensure that the hydrogel retains the desired level of water. Suitable amounts of humectant in the hydrogel range from 0.1 to 40% by weight, with preferred amounts ranging from 5 to 15% by weight.

Preferably, the wetting agent has a negative charge as a whole. Suitable anionic wetting agents include, but are not limited to: glycerol triacetate and polyols having a negative charge. Preferred humectants that have been tested include sodium PCA (i.e., the sodium salt of 2-pyrrolidone-5-carboxylic acid) and sodium lactate.

Certain small molecule wetting agents may be used, i.e., molecules having a molecular weight of less than 1000 Da. Examples of useful small molecule wetting agents include, but are not limited to: urea, propylene glycol, sodium lactate, and sodium Pyrrolidone Carboxylate (PCA).

Certain polysaccharide-based humectants can be used. Useful polysaccharide-based humectants include, but are not limited to: hyaluronic acid (sodium salt), carrageenan and agarose.

The humectant retains water molecules which, if not present, evaporate from the open system during the application period. Water loss from the gel can cause a number of deleterious effects including: increased transport resistance, decreased bioavailability of the catalyst providing the electrical signal (e.g., GOx enzyme), and decreased interfacial contact area due to shrinkage. Any of the above effects can interfere with device performance.

The wetting agent improves the consistency of the device by reducing the loss of water. The reduction in water loss also extends the life of the device. As disclosed in example 2, some humectants have been shown to improve device performance (as indicated by reduced signal drift) compared to the control (no humectant), while other humectants such as glycerin and hydroxyethyl urea do not increase device performance (as indicated by increased signal drift) compared to the control (no humectant) and are thus not beneficial. Preferred humectants can extend the life of the device without significantly increasing the error in the reading (such as by MARD analysis) (see FIG. 5).

c. Mounting or target plate

The mounting plate (102) may have any suitable geometry. The mounting plate is attached to the sensor body (101) using standard connectors, such as a plug-in SLIM/RCPT connector that mates with a corresponding connector interface already mounted on the mounting plate (102). In wireless systems (such as shown in fig. 8A, 8B, and 8C), a target board (120) is used in place of the mounting board. Preferably, the mounting or target plate may be made of a rigid, non-conductive material with a high dielectric constant, such as plastic, which may provide a strong support for the sensor body (101) and ensure containment of the hydrogel. Suitable materials for the mounting board include materials commonly used for printed circuit boards, which provide not only a robust support for the sensor body (101), but also a printed circuit for the sensor system.

i. Air reservoirs or passageways

In one embodiment, a sensor assembly for a TAMS includes a channel or reservoir that can supply air and/or oxygen to the hydrogel or other element of the sensor assembly that requires oxygen to operate. One or more air channels and reservoirs may be provided around the hydrogel. The air channel (122) and/or the storage compartment (124) are typically in the form of a slot or opening in the mounting plate (102) of a wired system (fig. 1) or the target plate (120) of a wireless system (see fig. 8A and 8B). These channels and reservoirs not only increase the oxygen supply (enhance oxidation) but also maintain the moisture (water) of the hydrogel. The air channels or reservoirs may be formed by molding, milling, stamping, etching, or any other mechanical or chemical process.

Fig. 8A-C illustrate examples of air passages and reservoirs in a target plate (120) in a wireless TAMS. A wired system (as shown in figures 1 and 2) can be modified in a similar way to include air channels and storage compartments. Fig. 8A shows a back view of an exemplary glucose sensor target plate having four air channels (122A, B, C and D). Fig. 8B shows a front view of an exemplary glucose sensor target plate having three cutouts (124A, B and C) around its central aperture (125) for hydrogel chemistry as internal air reservoirs. Fig. 8C shows a diagram of an exemplary wireless sensor housing (126) on top of the target plate (120) for providing an enclosed glucose sensor assembly (112).

In a preferred embodiment, the TAMS comprises a sensor for transdermal detection of analytes, wherein glucose oxidase (GOx, an enzyme) uses water and oxygen to convert glucose to hydrogen peroxide (H)₂O₂) And glucurolactone. Electrochemical glucose sensors can be designed to decompose H by using platinum electrodes₂O₂And simultaneously generates a continuous current with a continuous supply of transdermal glucose flux. If an air or oxygen reservoir or channel is included in the sensor assembly, the oxygen supply to the hydrogel may be increased and the moisture level of the hydrogel may be maintained as compared to the same hydrogel without such an air reservoir or channel. This is important for GOx to convert glucose to hydrogen peroxide, which is then electrochemically oxidized and measured to determine the amount of glucose in the blood.

B. Semipermeable membranes

In one embodiment, the TAMS comprises a protective semi-permeable membrane between the hydrogel surface and the user's skin. The protective semi-permeable membrane may have different pore sizes, compositions, charges, reactivities, and thicknesses. The pores can range from large (5 μm) to ultra-filtered (3k) to undefinedAs used herein, "undefined" refers to a membrane that currently has no standard method to characterize its pore structure, such asContains ion channels ranging in size from about 1nm to about 50nm, depending on the hydration state.

For transdermal analyte monitoring systems (such as CGM), a protective semi-permeable membrane is attached to the outside of the hydrogel to improve device performance by extending its lifetime and reducing contamination of the hydrogel with microorganisms, proteins, cellular material, etc. As an interface between the hydrogel and the skin being perforated, the membrane may reduce biofouling, such as proteins, lipids, cell debris, microorganisms, or a combination thereof.

The protective semipermeable membrane may be made from a variety of polymers, copolymers, or blends thereof. Suitable polymers include: hydrophobic polymers such as Polytetrafluoroethylene (PTFE); hydrophilic polymers (such as nylon, polyethersulfone, activated PES, (3-mercaptopropyl) trimethylsilane, cellulose acetate); electropolymerized films (such as 1, 8-diaminonaphthalene and phenylenediamine); andcoated PES.(tetrafluoroethylene-perfluoro-3, 6-dioxo-4-methyl-7-octenesulfonic acid copolymer) is a biocompatible anionic fluoropolymer, which can be coated on hydrogels as a protective layer to prevent physiological contaminants and biofouling. Based on their hydrophobicity, charge selectivity and/or exclusion size,can be used as a protective layer.

A semi-permeable membrane (such as in the form of a polymer film) may be applied to the outer surface of the hydrogel layer (106). Typically, one or more protective barrier layers may be provided between the hydrogel and the user's skin during operation. The polymer film may be coated onto the hydrogel surface using any suitable method, such as by micropipette or dip coating the sensor in an aqueous or organic solution of the polymer, followed by air drying for a number of hours before use.

In another embodiment, a protective semi-permeable membrane is attached to only one side of the hydrogel. The attachment at the interface is formed by polymerizing the hydrogel in the presence of the membrane and forming an Interpenetrating Polymer Network (IPN) at the interface region. IPNs are formed when a first polymer (such as a PEGDA hydrogel) is crosslinked in the presence of another polymer network (such as a polymer membrane).

In one embodiment, the semi-permeable membrane is negatively charged (e.g., charged) at the hydrogel/skin interfaceCoated PES) to prevent loss of negatively charged components from the hydrogel into the skin. Negatively charged wetting agents (e.g., NaPCA) and osmotic agents (e.g., lactic acid) are often added to hydrogels to improve system stability.

Protective membranes are known to bind proteins or other biological agents through covalent, electrostatic, hydrophobic, and/or mechanical interactions. As shown by internal experiments (not published), a reduction in the amount of protein precipitation on the hydrogel was observed when the membrane was applied between the skin and the hydrogel (within a 12 hour period of in vitro application). Using extraction and bicinchoninic acid (BCA) protein analysis, an average protein precipitation of 32 μ g per gel dish was observed if there was no membrane between the skin and the hydrogel; whereas when a protective film was used, the average amount of protein precipitated was 14. mu.g per gel dish.

Method for improving analyte detection performance

A. Skin treatment

In a preferred method of using TAMS, the skin is pre-wiped prior to use of TAMS. The skin preparation wiping process is used to replace the standard skin moisturizing process in the prior art. The skin preparation wiping process is performed to wipe or clean the skin surface. Typically, after a skin preparation for increasing porosity, the skin preparation wiping procedure is applied to the targeted skin area by massaging, wiping with a wiping pad, rubbing, or other methods. This step typically requires a short amount of time (as compared to the longer standard refill procedure used in the prior art), such as about 1 to 30 seconds.

The wipes may be formed by immersing a paper, cotton or textile based substrate in an agent comprising water, phosphate buffer, lactic acid, soap, surfactant, or any other chemical, solvent, or mixture thereof that can be used to clean a targeted skin area after any skin pretreatment procedure, such asUltrasonic skin penetration system (SontraMedical). Preferably, the agent is an inorganic or organic solvent, such as water, ethanol, isopropanol, or mixtures thereof. An exemplary formulation of the agent is an aqueous solution containing 30-95% isopropyl alcohol and the wiper material is gauze.

a. Detection box

In one embodiment, the cartridge includes a transdermal analyte detection system and a skin preparation wipe, optionally including a wiping reagent such as phosphate buffer, lactic acid, soap, surfactant, or solvent. In one embodiment, the substrate is pre-soaked with the wiping agent. In another embodiment, the wiping reagent is provided as a separate component of the test kit.

B. Improvement of biosensor oxygen supply

In a preferred embodiment, the sensor assembly is designed to add oxygen supply to the hydrogel and/or other elements of the sensor assembly that require oxygen to operate. Air channels and reservoirs may be placed around the hydrogel. The air passages and reservoirs are typically in the form of slots or openings in the mounting plate (102) or target plate (fig. 8). These channels and reservoirs not only increase the oxygen supply (enhance oxidation) but also maintain the moisture (water) of the hydrogel. Preferably, the mounting or target plate may be made of a rigid, non-conductive material with a high dielectric constant (such as plastic), which may provide a strong support for the sensor body and ensure containment of the hydrogel. The air channels or reservoirs may be formed by molding, milling, stamping, etching, or any other mechanical or chemical process.

Method of use

The TAMS described herein can be used to monitor biological analytes, such as blood glucose concentration of a user, and/or to deliver therapeutic compounds as needed. Applying TAMS to an area on the skin of an animal; typically, the animal is a mammal, and in a preferred embodiment, the mammal is a human.

For example, a person at risk of developing diabetes or a diabetic person may use the apparatus to monitor their blood glucose concentration levels and administer insulin accordingly to those concentration levels as needed. Insulin may be administered by the user or by the device. Other analytes may also be monitored.

Continuous glucose monitoring allows measurement of blood glucose concentration independent of the amount of body fluid accumulated in the sensor. In continuous glucose monitoring, for example, one may prefer to minimize the accumulation of both glucose and hydrogen peroxide in the hydrogel so that the current measured by the electrochemical sensor reflects the flow of glucose through the region of skin permeability in real time. This advantageously allows continuous real-time transdermal glucose monitoring.

To use the transdermal analyte monitoring system described herein, first, the skin area of the user is made more permeable by any suitable means. Typical methods of increasing skin permeability include: tape stripping, rubbing, sanding, abrading, laser ablation, Radio Frequency (RF) ablation, application of chemicals, ultrasonic introduction, iontophoresis, electroporation, application of penetration enhancers. For example, a skin pretreatment procedure may apply low energy ultrasound to the skin (e.g.An ultrasonic skin penetration system) or controlled skin abrasion.

When wireless TAMS is used, typically, the target plate (120) is placed on the skin at a site for increased permeability. Then, a skin pretreatment process is performed. The method is particularly suitable for useAs a skin penetration system.

In a preferred embodiment, after the skin pretreatment step, the treated skin is cleaned by wiping or rubbing the treated area of skin for a short period of time (such as about 1 to 30 seconds), such as with a skin preparation wipe.

A sensor assembly, such as the one shown in fig. 1 (wired system) or fig. 9 (wireless system), is then attached to the permeable area of the skin (107) such that the semi-permeable membrane (not shown in fig. 1) is in contact with the permeable skin. When using wireless TAMS, typically, the hydrogel and sensor are placed in the target plate and aligned with the central aperture (125). The sensor housing (126) is then attached and attached to the target plate (120) to form the complete sensor assembly (112).

The analyte can be extracted via the treated permeable region (107) of the user's skin and pass through the semi-permeable membrane to be brought into contact with the hydrogel disc (106) of the sensor assembly (101).

For example, an analyte such as glucose may be transported by diffusion through the semi-permeable membrane and into the hydrogel disk (106), where the analyte may contact the glucose oxidase. The glucose then reacts with the glucose oxidase present in the hydrogel disc (106) to form gluconic acid and hydrogen peroxide. Next, the hydrogen peroxide is transported to the electrode surface in the sensor body (101) where it is electrochemically oxidized. The current generated in this oxidation reaction is indicative of the rate of production of hydrogen peroxide in the hydrogel, which is related to the amount of glucose flux through the skin (the flux of glucose through a fixed area of the skin). The flow of glucose through the skin is proportional to the glucose concentration in the blood of the user.

In this way, the user's blood glucose concentration can be continuously monitored by displaying the blood glucose concentration on the potentiostat (108) in a continuous, real-time manner using signals from the sensor assembly.

In principle, any sensor that measures hydrogen peroxide using the working electrode (201), the counter electrode (202), and the reference electrode (203) can be configured in the same manner. Examples of sensors are biosensors for glucose, lactate or other substances using oxidase enzymes introduced into a hydrogel (106). In continuous glucose monitoring, the electrochemical sensor is preferably operated in potentiostatic mode. In potentiostatic mode, the potential between the working electrode and the reference electrode of the three-electrode cell is maintained at a preset value. The current between the working electrode and the counter electrode is measured. The sensor is maintained in this mode as long as the required battery voltage and current do not exceed the potentiostatic current and voltage limits. In the potentiostatic mode of operation, the potential between the working electrode and the reference electrode can be selected to achieve selective electrochemical measurements for a particular analyte or analyte indicator.

Other modes of operation may be employed to study the kinetics and mechanisms of electrode reactions occurring on the surface of the working electrode, or in electroanalytical applications. For example, depending on the electrochemical cell mode of operation, when the potential of the working electrode is measured with the reference electrode, a current can flow between the working electrode and the counter electrode. Those skilled in the art will understand that: the mode of operation of the electrochemical sensor may be selected depending on the application.

Examples

Example 1: use of a protective semi-permeable membrane to form hydrogel/membrane compositions and improve TAMS performance

The following films were tested: [ a ] A]Polyethersulfone without coating (PES): symmetric membranes with pore diameters of 0.2, 1.2 and 5.0 μm, respectively; asymmetric membranes with pore diameters of 0.3, 1.0 and 2.0 μm, [ b ]]Coated PES: for each of the 6 different pore sizes listed aboveCoating test, [ c ]]PES (pore diameter 0.45 μm) activated with aldehyde function, [4 ]]Amphoteric and cationic nylon 66 (pore diameter 0.2 μm), [ d ]]Ultrafiltration membrane: regenerated Cellulose (RC) having a molecular weight cut-off of 3.5 k; PES having a molecular weight cut-off of 10k, [ e ]]Nafion 1135 membrane with ion channels of about 35 nm.

Formation of hydrogel/film composition: cutting the film into a disk shape, immersing in a buffer and placing at the bottom of a polymerization mold; placing a scrim sheet on the film; injecting a polymer solution into the mold cavity; the mold is exposed to ultraviolet light to form a polymer.

For the product composed ofFilm of PES coated using an automatic coaterThe solution was precoated with PES film. Coating parameters include machine speed, coating bar size,Solvent, and the number of coating layers, and it varies with the pore size of the PES. Coating parameters affect the thickness of the coating, its depth of immersion into the film, consistency, and lifetime. For example, when using a 20-gauge rod, 5% will be applied at a rate of 8 inches/secondWhen the solution (in 45% ethanol) is coated in a monolayer on 0.2 μm PES, the result isTo shallow surface coatings; when using 20-size rod, 20% of the total weight of the rodThe solution (in 80% ethanol) gives a deeper coating when applied in multiple layers over 5.0 μm PES.The depth of the coating was determined by dyeing the coating film with cationic methylene blue.

Where the pore size of the membrane is smaller than 3.4k PEG macromolecules (e.g., 3k cellulose is used), a smaller 0.75k PEG macromolecule is used to attach the membrane to the 3.4k PEG network. In this case, 0.75k PEG macromolecules can be used to form two interconnected IPNs when the PEG macromolecules (at 3.4k daltons) cannot penetrate the pores of the membrane (at 3k daltons). The 0.75k PEG macromolecules were first polymerized in one face of the 3k membrane; subsequently, the 3.4k PEG macromolecules polymerized at the new membrane face, which now appears as a network of 0.75k PEG.

A 24-hour in vitro study was run to investigate the effect of each membrane on the performance of the glucose sensor. Test groups without membrane or with different membranes were compared. A film that extends the lifetime of the device without increasing MARD error is identified as a preferred film. Each membrane was applied to the outer surface of a sensor assembly in a CGM device (provided by Sontra Medical) and then applied to the sonicated skin of the subject. In response, the device provides an electrical signal in nanoamperes (nA), which is calibrated to the subject's Blood Glucose (BG) using a finger stick blood glucose meter. Throughout the in vitro study (up to 24 hours), finger prick BG samples were sampled as follows: sampling at a frequency of once per hour during the time the subject is awake; or samples are taken at a frequency of once every 15 minutes around the meal time and the samples are correlated with the signal of the device. Analysis of this correlation provides information about the accuracy, consistency and duration of validity of the performance of the device.

In general, the addition of either membrane to the hydrogel extends the useful life of the glucose sensor. As shown in figure 3, in the in vitro application without membrane, only 55% of subjects had any one 24-hour response; for in vitro application with membranes, 83% of subjects had a 24-hour response (see fig. 3).

There is a difference between the membranes and the criterion for selecting the best membrane is that it has the lowest signal drift over the entire 24 hour period, but still provides a good signal correlation (measured in nA versus BG) (i.e. no significant increase in MARD error). Among various films evaluated, those having 5.0 μm poresThe coated PES membrane was determined to be optimal as it showed the lowest signal drift (about 54% over the entire 24 hour period) and acceptable MARD (see fig. 4).

Example 2: a humectant is incorporated into the hydrogel buffer to mitigate water loss and improve device performance.

A series of tests were performed with various humectants contained in the hydrogel. Two broad classes of humectants were tested. The first large class includes small molecule humectants, typically Natural Moisturizing Factors (NMF), and the second large class includes polysaccharides. The following small molecule humectants were tested: glycerol, urea, hydroxyethyl urea, propylene glycol, sodium lactate (Na lactate) and sodium pyrrolidone carboxylate (nacpa). The following polysaccharide humectants were tested: hyaluronic acid (sodium salt), carrageenan and agarose.

For small molecule wetting agents, each wetting agent is dissolved in the polymer solution prior to polymerization. A specific concentration of wetting agent in the polymer solution is also maintained in the hydrogel buffer. This prevents the concentration gradient of the humectant from changing, which can increase the loss of humectant by diffusion during rinsing and storage.

For polysaccharide humectants, the same general procedure as described above for small molecule humectants is used. However, some of the humectants evaluated (such as agarose and isolated carrageenans) require heating to obtain proper solubility and cooling to form a gel. The PEGDA concentration was reduced to 10% to increase the solubility of the polysaccharide.

Screening tests were first performed to select the best humectant to be evaluated for limited in vitro tests. Screening assays included a comparison of solubility and drying rate.

A 24 hour in vitro study was conducted to investigate the effect of each humectant on the performance of the glucose sensor. The test groups were compared without humectant or with different types of humectants. A humectant that extends the life of the device without increasing MARD error is identified as a preferred humectant. In vitro testing involved applying the device to volunteers for 24 hours and then comparing the life performance with different humectants.

Similar to example 1, the purpose of this investigation of the incorporation of wetting agents was to improve device performance. The test results are provided in fig. 5. While many humectants, including sodium lactate, carrageenan, and agarose, have shown some promise, Na PCA consistently provides the lowest signal drift (see fig. 5). In addition, when Na PCA is used withWhen the coated PES membrane was used with this, there was no water loss throughout the 24 hour in vitro study. Data collected from 27 subjects showed an actual water gain of 2%. However, typically, there is a loss of water in the 24 hour study. In the control group of the study (36 subjects without Na PCA), a mean water loss of 19% was observed.

Example 3: covalently immobilized glucose oxidase (GOx) in PEGDA hydrogel

A series of experiments were performed to establish a practical enzyme immobilization strategy. acrylate-PEG-NHS (A-PEG-N) reagent (Nektar) was chosen as a linker or immobilization reagent. Parameters of interest include the ratio of immobilized reagents to enzyme, the order of the reactions, and the incubation time.

A prepolymerization step was used for incubating the enzyme with the acrylate-PEG-NHS (A-PEG-N) immobilization reagent. 3% GOx was dissolved in polymerization buffer and an excess (7 to 1 molar ratio) of A-PEG-N was added. The molar ratio of 7 to 1 was chosen to ensure binding without interfering with the activity of the enzyme. The solution was left to incubate at 4 ℃ overnight (reaction at room temperature for 3 hours was also effective). PEGDA is typically added the next day to complete the formulation of the polymer solution, followed by uv curing.

There is evidence that covalent immobilization with 10% PEGDA polymer containing 3% GOx was successful. Without covalent immobilization, when GOx was placed in wash buffer, it filtered out of the hydrogel dish and turned the solution to a distinct yellow color (uv absorbance at 460nm ═ 0.16). The washing solution did not yellow by covalent immobilization (uv absorption at 460nm ═ 0.02).

The potentiostatic test provides evidence that the enzyme is still active after covalent immobilization, which shows no significant difference in response to glucose levels: the control system was about 700nA and the covalent immobilization system was about 650 nA.

After the covalent immobilization parameters were established, in vitro experiments were performed to determine if the consistency of the readings of the system had been improved. In vitro testing involved applying the device to volunteers for 4 hours, followed by statistical analysis and calculation of r²And MARD values, to compare device performance without covalent immobilization.

In a 4 hour comparison study of the in vitro device performance of covalently immobilized GOx versus non-covalently immobilized GOx, the immobilized GOx system was able to better track BG changes (nA vs BG correlation). The results of this study are shown in figure 6. As shown in fig. 6, this in vitro study revealed that the system exhibited a more consistent trace with covalently immobilized GOx, with r2 of 0.68 and a MARD of 12.27. In contrast, for non-covalently immobilized GOx, r²The value was 0.41 and the MARD was 20.44.

Example 4: skin preparation procedure for transdermal detection of analytes

The target plate is first applied to the skin. Then pass through a target plateIs applied to the skin area. Then will beTurned on for one second or more and automatically turned off by the embedded control algorithm of the device. Through use(Sontra Medical Corp.) after a skin pretreatment procedure to increase skin porosity, the treated skin site is wiped with a skin preparation wipe. The skin preparation wipe used in this study was a gauze pad pre-soaked in a 70%/30% isopropyl alcohol/water mixture.

Figures 7A and 7B illustrate the difference in sensor performance between when and when a skin preparation wipe procedure has been applied to a subject. In contrast to the skin preparation wipe procedure, fig. 7C shows the results of the same subject when a 40 minute hydrating procedure (i.e., a prior art procedure) was used. As shown, wiping the treated skin with the skin preparation wipe showed performance comparable to a 40 minute moisturizing procedure, and both of these methods performed better than without any skin preparation procedure. The skin preparation wipe removes and/or cleans away any material that blocks the pores, which is expected to improve the transdermal path for both analyte extraction and drug delivery.

Example 5: clinical study using three different configurations of continuous transdermal glucose sensors

The glucose biosensor comprisesElectrochemical sensor and water for use with ultrasound skin permeation systemGel, and can continuously introduce glucose into the sensor. Glucose flowing through the skin is consumed by the biosensor as it reacts with the glucose oxidase in the hydrogel. The chemical reaction produces a continuous electrical signal, which is recorded by a glucose monitor. Due to the permeability enhancement and hydrogel chemistry caused by SonoPrep, the glucose flux detected by the sensor can provide a glucose reading every minute over a time force of up to 24 hours by wireless connection. See fig. 9 for a schematic of a wireless biosensor system.

The following procedure was used in each study. These steps are schematically illustrated in fig. 9. First, the target plate is placed on the skin site of the patient. SonoPrep was then applied to the skin site for 5 to 15 seconds (step 1). SonoPrep was then removed from the target plate. The treated skin site was then wiped with a skin preparation wipe containing ethanol. Next, the hydrogel and the sensor are placed in the target plate (step 2). For each patient, a disposable glucose sensor was placed on the SonoPrep-treated skin site. The sensor housing is then placed over the hydrogel and the sensor assembly is closed (step 3). The sensors are connected to a micro-analyzer which wirelessly transmits the digitized data to a monitor for data processing and display (step 3). The glucose sensor signal was referenced to the finger stick glucometer reading in study 1A and study 1B, and to the blood glucose sampled through the fourth line in study 2C.

Table 1 describes the configuration of the sensor, the type of membrane used (if a membrane is used), and the type of humectant contained in the hydrogel (if any humectant is present) in each study. The sensors used in each study were intended to provide enhanced oxidation (such as illustrated in fig. 8A, 8B, and 8C). In addition, the hydrogel used in each study contained 3% GOx covalently immobilized in 15% PEGDA.

TABLE 1 sensor configuration, materials and duration in each study

Study number	Sensor arrangement	Duration of time	Film	Wetting agent
					1	A	12h	Is free of	Is free of
1	B	12h	Biodyne B	Is free of
					2	C	24h	5.0PES.NAF	10％Na PCA

Study 1A: study 1 with sensor configuration A

10 diabetic patients were tested using the method described above. As noted in table 1, the study was conducted for 12 hours. The sensors used in this study had no membrane on their hydrogel. In addition, the hydrogel contains no wetting agent.

222 data points obtained in this study were analyzed to support the development of a blood glucose prediction algorithm. The results are summarized in the Clarke error grid in fig. 10. As shown in fig. 10, the results show that the sensor accurately predicts blood glucose readings using a single point calibration per minute for periods as long as 12 hours after a one hour warm-up period.

The measurements of the biosensor and the reference blood glucose were compared and statistical analysis showed a MARD (mean absolute relative error) of 12.4%. 98.7% of the data falls in the A + B region of the Clarke error grid, with 89.6% in the A region. The study again demonstrated excellent correlation in data (average r 0.87) (see fig. 10). These statistics are summarized in table 2, along with statistics of other studies described in this example.

Study 1B: study 1 with sensor configuration B

The same study protocol and configuration as in study 1A was used, except that a filtration membrane (Biodyne B) was used in combination with the hydrogel. 10 diabetic patients were tested using the method described above. As noted in table 1, the study was conducted for 12 hours. The sensors used in this study had a membrane on their hydrogel (Biodyne B). In addition, the hydrogel contains no wetting agent.

225 data points were collected in this study. The results are summarized in the Clarke error grid in fig. 11. As shown in fig. 11, the results show that the sensor accurately predicts blood glucose readings using a single point calibration per minute for up to 12 hours after a one hour warm-up period.

The measurements of the biosensor and the reference blood glucose were compared and statistical analysis showed a MARD (mean absolute relative error) of 20.4%. 96.9% of the data falls in the A + B region of the Clarke error grid, with 70.7% in the A region. The correlation coefficient between the biosensor and the reference blood glucose measurement was 0.64. These statistics are summarized in table 2, along with statistics of other studies described in this example.

Study 2: study with sensor configuration C

Patients were subjected to a 24 hour clinical study during and after cardiovascular surgery. As noted in Table 1, the sensors used in this study had a membrane on their hydrogel (with a membrane)Coated 5.0 PES). In addition, the hydrogel contained a humectant (10% by weight NaPCA).

During surgery, the patient's core temperature is reduced to about 20 ℃, and the patient's heart is stopped beating with the aid of a bypass blood circulation pump. Drugs such as insulin and heparin are administered to the patient, and blood glucose sampling is performed through the fourth line and blood glucose analysis is performed using a blood glucose analyzer.

In the first part of the study, it was determined that humidity and iodine (disinfectant used to treat the skin prior to surgery) adversely affected the sensor and caused device failure. Temporary modifications are then made to the device configuration and installation procedures (e.g., to avoid the use of iodine in the skin area) to address those issues.

In the second part of the study after device modification, 10 patients participated in and nine completed the study. 147 sensor blood glucose data points were collected and analyzed using the same glucose prediction algorithm as used in study 1A.

The results are summarized in the Clarke error grid in fig. 12. As shown in fig. 12, the results show that the sensor accurately predicts blood glucose readings every minute during and after the procedure for up to 24 hours.

Comparing the measurements of the biosensor and the reference blood glucose, statistical analysis showed that the MARD (mean absolute relative error) was 11.2% and 100% of the data fell in the a + B region of the Clarke error grid, with 86.4% in the a region. This study shows that with proper device configuration and installation, the transdermal glucose monitor can provide accurate continuous glucose readings for up to 24 hours, even in a surgical ICU setting.

TABLE 2 summary of statistical analysis in clinical studies

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present claims.

Claims (15)

1. A transdermal analyte monitoring system, comprising:

a sensor component, wherein the sensor component comprises a hydrogel and a sensor body comprising a plurality of electrodes, wherein the sensor body is in fluid communication with the hydrogel, wherein the hydrogel comprises an anionic humectant and an enzyme, wherein the amount of the anionic humectant in the hydrogel ranges from 0.1% to 40% (by weight), wherein the anionic humectant is selected from the group consisting of sodium lactate and sodium pyrrolidone carboxylate (NaPCA).

2. The transdermal analyte monitoring system of claim 1, further comprising a semi-permeable membrane, wherein the semi-permeable membrane is in fluid communication with the hydrogel.

3. The transdermal analyte monitoring system of claim 2, wherein the hydrogel and the semi-permeable membrane form an interpenetrating polymer network.

4. The transdermal analyte monitoring system of claim 1, wherein the hydrogel comprises: a polymer selected from the group consisting of polyethylene glycol diacrylate (PEGDA), agarose, polyethylene glycol diacrylate/polyethyleneimine (PEGDA-PEI), polyethylene glycol diacrylate-N-vinyl pyrrolidone (PEGDA-NVP), acrylate-polyethylene glycol-N-hydroxysuccinimide (a-PEG-N), or a blend or copolymer of these polymers.

5. The transdermal analyte monitoring system of claim 1, wherein the enzyme is an oxidase.

6. The transdermal analyte monitoring system of claim 1, wherein the enzyme is covalently immobilized in the hydrogel.

7. The transdermal analyte monitoring system of claim 6, wherein the enzyme is covalently immobilized in the hydrogel with A-PEG-N.

8. The transdermal analyte monitoring system of claim 1, wherein the anionic wetting agent is sodium pyrrolidone carboxylate (NaPCA).

9. The transdermal analyte monitoring system of claim 1, wherein the sensor assembly comprises at least one channel or reservoir for supplying oxygen to the hydrogel.

10. The transdermal analyte monitoring system of claim 1, wherein the enzyme is immobilized in the hydrogel by non-covalent immobilization.

11. A cartridge, comprising:

the transdermal analyte monitoring system of any one of claims 1 to 10; and

a substrate comprising a phosphate buffer, lactic acid, soap, surfactant, or solvent.

12. A cartridge, comprising:

the transdermal analyte monitoring system of any one of claims 1 to 10;

a substrate, and

a reagent selected from the group consisting of phosphate buffers, lactic acid, soaps, surfactants, and solvents.

13. A method of increasing the sensitivity, stability or accuracy of a transdermal analyte monitoring system according to any one of claims 1 to 10 comprising increasing the amount of oxygen supplied to the hydrogel.

14. The method of claim 13, wherein the source of oxygen is air.

15. A transdermal analyte monitoring system according to any one of claims 1 to 10 for use in a method according to any one of claims 13 and 14.