GB2559627A

GB2559627A - Device

Info

Publication number: GB2559627A
Application number: GB1702405.0A
Authority: GB
Inventors: Fazlul Hoque Chowdhury Dewan; Bojan Kartheka; Kshirsagar Vallabh
Original assignee: NDM Technologies Ltd
Current assignee: NDM Technologies Ltd
Priority date: 2017-02-14
Filing date: 2017-02-14
Publication date: 2018-08-15
Also published as: GB201702405D0; WO2018150168A1

Abstract

An analyte monitoring device 2 for use in contact with the skin 4 comprises a porous membrane 6 comprising an enzyme. Preferably glucose or lactate are detected via a reaction using glucose oxidase or lactate dehydrogenase to yield hydrogen peroxide which is subsequently detected using an electrode 8. Preferably the porous membrane is polyethersulfone or nylon 6, 6 such as Biodyne A or C membrane. Preferably the membrane forms a three dimensional matrix of enzyme having a thickness of less than 300 µm and the average pore size is around 0.3 µm to 0.5 µm. Preferably use of the device is preceded by an additional step such as iontophoresis, ultrasound or microneedle microporation. Methods of use of the device to continuously monitor an analyte such as glucose are disclosed.

Description

(71) Applicant(s):

NDM Technologies Ltd (Incorporated in the United Kingdom)

Holywell Park, Ashby Road, Loughborough, LE11 3AQ, United Kingdom (72) Inventor(s):

Dewan Fazlul Hoque Chowdhury Kartheka Bojan Vallabh Kshirsagar (74) Agent and/or Address for Service:

HGF Limited

Document Handling - HGF - (Birmingham),

City Walk, LEEDS, LS11 9DX, United Kingdom (56) Documents Cited:

WO 2008/109739 A1 WO 1999/013336 A1 US 20060004271 A1

WO 2000/014535 A1 US 20100049016 A1

Biosensors & Bioelectronics (2015) 74 1047-1052 Chen et al 'PVDF-Nafion nanomembranes coated microneedles for in vivo transcutaneous implantable glucose sensing'

AU2002300167 (58) Field of Search:

INT CL A61B, C12Q

Other: WPI, EPODOC, BIOSIS, MEDLINE, Patent Fulltext (54) Title of the Invention: Device

Abstract Title: Skin contact analyte detection device comprising porous membrane layer with enzyme (57) An analyte monitoring device 2 for use in contact with the skin 4 comprises a porous membrane 6 comprising an enzyme. Preferably glucose or lactate are detected via a reaction using glucose oxidase or lactate dehydrogenase to yield hydrogen peroxide which is subsequently detected using an electrode 8. Preferably the porous membrane is polyethersulfone or nylon 6, 6 such as Biodyne A or C membrane. Preferably the membrane forms a three dimensional matrix of enzyme having a thickness of less than 300 pm and the average pore size is around 0.3 pm to 0.5 pm. Preferably use of the device is preceded by an additional step such as iontophoresis, ultrasound or microneedle microporation. Methods of use of the device to continuously monitor an analyte such as glucose are disclosed.

At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.

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Sensitivity of the Sensor with PBS Medium

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Application No. GB1702405.0

RTM

Date :24 October 2017

Intellectual

Property

Office

The following terms are registered trade marks and should be read as such wherever they occur in this document:

Biodyne

Nafion

Intellectual Property Office is an operating name of the Patent Office www.gov.uk/ipo

DEVICE

The present invention relates to a device, and in particular to a device for monitoring an analyte.

There have been many attempts to monitor the level of an analyte within the body of an animal. Some of these attempts have been quite intrusive, involving taking a sample of tissue or blood or the insertion of a sensor inside the skin of a human or animal subject. Other attempts have tried to assess the level of a particular substance in a less intrusive manner. One type of attempt has been to utilise a device placed in contact with an animal’s body, such as in contact with the skin to measure an analyte that is either passively or actively extracted via pores in the skin.

Such prior art devices can have disadvantages, such as being inaccurate, or insensitive to changes in concentration of an analyte due to poor sensitivity, poor specificity, or limits of detection that are not low enough to detect the low levels of analyte that is extracted. Furthermore, noise generated by inherent features of the devices can also mask the signal generated by the analyte being measured. A further limitation that has been observed is drift in the signal caused by a number of factors, often related to the inherent material properties, for example the drying out of a hydrogel that may be in contact with the skin acting as a diffusion pathway and conducting layer between the skin and the device that measures the analyte signal, such as an electrode measuring the current generated by hydrogen peroxide which in turn is a by-product of the reaction between an analyte and an enzyme present in the system, such as lactase dehydrogenase used to measure lactic acid levels, and glucose oxidase used to measure glucose levels.

The present invention seeks to provide a device and method for monitoring an analyte.

The present invention will now be described, by way of example, with reference to the following figures, in which:

Figure 1 is an illustration of a device of the invention.

Figure 2 shows the results of a monitoring device of the invention.

Figure 3 shows the results of a monitoring device of the invention.

Figure 4 shows the results of a monitoring device of the invention.

Figure 5 shows the results of a monitoring device of the invention.

Figure 6 shows the results of a monitoring device of the invention.

Figure 7 shows the results of a monitoring device of the invention.

Figure 8 shows the results of a monitoring device of the invention.

Figure 9 shows the results of a monitoring device of the invention.

Figure 10 shows the results of a monitoring device of the invention.

Figure 11 shows the results of a monitoring device of the invention.

Figure 12 shows the results of a prior art monitoring device.

Figure 13 shows the results of a monitoring device of the invention.

Figure 14 shows the results of a monitoring device of the invention.

Figure 15 shows the results of a monitoring device of the invention.

Figure 16 shows the results of a monitoring device of the invention.

Figure 17 shows the results of a monitoring device of the invention.

Figure 18 shows the results of a monitoring device of the invention.

Figure 19 shows the results of a monitoring device of the invention.

Figure 20 shows the results of a monitoring device of the invention.

Figure 21 shows the results of a monitoring device of the invention.

Figure 22 shows the results of a monitoring device of the invention.

Figure 23 shows the results of a monitoring device of the invention.

Figure 24 shows the results of a monitoring device of the invention.

According to the present invention, there is provided an analyte-monitoring device comprising:

a porous membrane layer comprising an enzyme; and analyte monitoring means;

wherein, when placed on the skin of a patient the membrane layer is an interface between the skin of the patient and the analyte monitoring means.

Preferably, the analyte is glucose or lactate.

Conveniently, the device does not comprise a hydrogel.

Advantageously, the device is configured to continuously monitor the concentration of the analyte in a patient.

Preferably, the enzyme is glucose oxidase or lactase dehydrogenase.

Conveniently, the enzyme has a concentration in the porous membrane layer of 0.45 to 0.60 mg/crrr² of membrane surface area for a membrane thickness of 158 to 192 pm thickness, or 0.45 mg to 0.60 mg per 0.158 pm³ to 0.192pm³ ofthe 3-dimensional porous membrane.

Advantageously, the enzyme is physically adsorbed on the porous membrane layer or is chemically bonded to the porous membrane layer.

Preferably, the porous membrane layer is a porous hydrophilic polymer layer, optionally wherein the polymer is nylon, preferably nylon 6,6, and more preferably amphoteric nylon 6,6.

Conveniently, the porous membrane layer is a porous hydrophobic, or partially hydrophobic layer, optionally wherein the polymer is chemically or physically treated nylon 6,6, or surface functionalised polyethersulfone.

Advantageously, the porous membrane layer is a 3-dimensional matrix, optionally wherein the 3-dimensional matrix is nylon 6,6, or surface functionalised polyethersulfone.

Preferably, the porous membrane layer has a thickness of less than 350 pm, more preferably the membrane has a thickness of less than 250 pm.

Conveniently, the pores ofthe porous membrane layer have an average size of from about 0.1 pm to about 2.0 pm, such as from about 0.2 pm to about 1.0 pm, preferably from about 0.3 pm to about 0.5 pm.

Advantageously, the porous membrane layer and analyte monitoring means are contiguous.

Preferably, when placed on the surface of the skin of the skin of a patient, the porous membrane layer and the surface of the skin are contiguous.

Conveniently, the patch comprises means for performing iontophoresis, ultrasound, or micro-poration using microneedles or laser light.

Advantageously, the analyte monitoring means comprises a counter electrode and a working electrode, and a reference electrode.

According to another aspect of the invention there is provided the use of a device of the invention for monitoring the concentration of an analyte in a patient.

According to another aspect of the invention, there is provided a method of monitoring the concentration of an analyte in a patient comprising the steps of:

a) applying a device of the invention to the skin of the patient such that the porous membrane layer ofthe device is hydrated and in contact with the skin;

b) receiving analyte from the skin into the porous membrane layer;

c) converting the received analyte via the enzyme comprised in the porous membrane layer; and

d) generating a signal representative of the level of received analyte via the analyte detection means.

Preferably, the method involves the step of performing iontophoresis or applying ultrasound to the skin, or poration of the skin by laser light or micro needles, in order to receive analyte.

Conveniently, the step of performing iontophoresis or applying ultrasound to the skin, or poration ofthe skin by laser light or micro needles is performed prior to application of the device to the skin.

Advantageously, the analyte is glucose or lactate, and the method comprises the step of oxidising the received glucose or lactate to produce hydrogen peroxide.

Preferably, the method comprises the step of detecting the level of hydrogen peroxide produced by the analyte detection means.

The present invention concerns devices and methods relating to monitoring analytes in an animal body, particularly a human body. The analytes are moieties which are useful in monitoring, for example in connection with the health of the subject. Of particular interest are analytes whose concentration can change with time. Example of suitable analytes include glucose, lactate, phenylephrine, drugs/medicines, and ions in the plasma. Preferred analytes include glucose and lactate resulting from lactic acid build up in muscles.

The level of glucose in a subject varies with time, and it is useful to monitor the level in the prevention and treatment of certain diseases and conditions, such as diabetes and hyperglycemia.

Some prior art monitoring systems aimed at monitoring the level of analytes such as glucose suffer from disadvantages, including lack of sensitivity, high noise levels and an inability to monitor changes in concentration in a real-time or near-real time manner.

The present invention provided a device and method to monitor the level of an analyte.

Turning to Figure 1, a device 2 according to the invention is shown. The device 2 is in the form of a patch placed on the skin 4 of a subject. The dermal patch device 2 comprises a porous membrane 6 placed onto the skin 4 of the subject. The membrane 6 is positioned such that it interfaces with the face of an electrode electrode 8. In use, an analyte present in the subject leaves the skin 4 and enters the membrane 6. The porous membrane 6 comprises an enzyme which acts upon the analyte. The enzyme is bound in some way to the membrane 6 as will be discussed in more detail below. In some embodiments, the porous membrane is maintained on the skin by the use of a peripheral adhesive such as silicone adhesive or acryclic adhesive, or by forming a vacuum seal around the device, without the use of adhesive.

The device of the present invention is preferably in the form of a patch, more preferably a dermal patch.

The enzyme acts on the analyte to facilitate a chemical reaction. The reaction produces a moiety which is detected by the electrode 8. For example, the enzyme glucose oxidase (GOx) can be used in the device of the present invention to monitor glucose in a subject. The glucose oxidase enzyme (GOx) also known as notatin is an oxido-reductase that catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-d-lactone. The resultant hydrogen peroxide is electrochemically detected by the electrode 8, which is connected to suitable electronic circuitry and power supply to enable the amperometric detection of the signal generated by the reaction of the analyte with the enzyme. The electrical signal is related to the amount of hydrogen peroxide, which itself is related to the amount of glucose.

Other enzymes can be used in a similar way to monitor the levels of other analytes. For example, lactase dehydrogenase may be used to measure lactate levels extracted from the skin resulting from the build-up of lactic acid in muscle tissue.

Various methods could be used to help the analyte to pass through the skin and into the membrane. These methods include iontophoresis, ultrasound and skin poration using physical ablation with pulsed concentrated laser beams, or microneedles. General skin abrasion has also been used to enhance the permeation and/or diffusion of analytes out of the body through the skin. These steps can be alone or in combination, and can be performed before the device is applied to the subject, and/or after the device has been applied to the subject.

Preferably, the membrane does not contain a hydrogel. The applicant has found that the presence of a hydrogel can reduce analyte sensitivity and adversely affect the signal to noise ratio of the signal. This may be caused by the presence of components in the hydrogel such as buffers and surfactants and the gel-forming material itself, all of which have been noted to contribute to the noise levels to varying degrees.

Also, in a polymer membrane such as Nafion is added then an issue is that diffusion is restricted leading to poor signal. Also, gels have been found to dehydrate over time leading to signal drift over time which must be corrected by multiple calibrations which is not ideal from a patient perspective.

The present invention contemplates using a porous membrane, such as a nylon mesh, or a 3D matrix that is a ‘solid’ system. This leads to several advantages, including a reduction in background noise and very good diffusion. Also, interfering species can be trapped in the matrix and therefore prevented from reaching the electrode thus giving clean low noise signals. In addition, given the solid nature of the membrane substrate, dehydration is very low compared to gels.

The membrane used in the invention is a porous membrane. In some applications, the membranes are preferably hydrophobic, or partially hydrophobic. In some applications, the membranes are preferably hydrophilic, or partially hydrophilic. Furthermore, in some applications the membrane is amphoteric, either on the surface alone or on the surface and throughout the matrix, or within the 3-dimensional matrix alone. Amphoteric membranes have been found to provide a higher level of consistency in absorption and adsorption of the enzyme solution, and have also shown remarkably strong precision in human studies, whereby precision is measured by applying two devices to a single patient, for example one on each arm, and measuring the closeness of the signals obtained by each device. These amphoteric properties have also yielded significantly higher accuracy of analyte levels measured using the device, for example glucose measurements, when compared with finger prick glucose measurements. Such levels of accuracy, specificity and elimination of noise and signal drift using this type of membrane has not previously been reported in literature and provides a significant advance on the current state of the art, with tangible benefits to end users of this type of system for continuous monitoring. These membranes whilst mentioned in passing in numerous patents and some published literature, for this type of monitoring, have not been investigated in any detail according to the literature, and have been widely used in laboratory settings for conducting laboratory based tests, or used in instrumentation that is designed for separation and measurement of analytes.

The devices of the invention comprise a membrane that comprises an enzyme. It has been found by the applicant that good results are achieved when the enzyme is bound in some way to the membrane. The enzyme may be bound to the membrane by physical adsorption, entrapment (for example within a bead), covalent bonding or electrostatic bonding, and so on.

A preferred method of binding the enzyme to the porous membrane is with the use of metal nanoparticles or the use of a cross-linking agent such as glutaraldehyde. Where gold nanoparticles are used the enzyme may be associated with the nanoparticles in a number of ways, such as covalent bonds and electrostatic interactions. Gold nanoparticles are used in a preferred embodiment of the invention.

Conveniently, the enzyme has a concentration in the porous membrane layer of 0.45 to 0.60 mg/cm^-2 of membrane surface area for a membrane thickness of 158 to 192 pm thickness, or 0.45 mg to 0.60 mg per 0.158 pm³ to 0.192pm³ of the 3-dimensional porous membrane. In general, the enzyme is present in the membrane in an amount of less than 0.8 mg/cm'². Preferably the concentration is within the range 0.35 to 0.70 mg/crn'². More preferably the concentration is within the range 0.45 to 0.60 mg/cm'², or this may be expressed as amount of glucose oxidase, dissolved in a suitable aqueous system such as water or buffer, per 0.158 pm³ to 0.192pm³ of the 3-dimensional porous membrane, for membrane thicknesses of 145 to 170 pm.

Examples

Binding of GOx to Membranes

Glucose Oxidase (GOx) was prepared as a dispersion surrounded by Gold nanoparticles according to methods available in the published literature. 5nm and 10nm gold nanoparticles were studied. It was found that membranes coated with gold nanoparticles where the gold particle size was 10nm provided superior results to 5nm gold particles, whereby 0.27mg Glucose Oxidase was mixed with 3ml of a nano-gold suspension (containing 5.70 x 10¹² gold particles per millilitre). Additionally, the results shown in Figure 2 indicates the following findings:

1. There is a linear increase in the glucose concentration when glucose solution is added incrementally (glucose solution addition commencement is indicated by the line between about 35 and 90 minutes). The linearity shows a significantly higher gradient (therefore sensor sensitivity level) as compared with the use of hydrogels.

2. The depletion of the signal started immediately after the last glucose dispense with a rapidly decreasing slope indicating a rapid depletion rate, as compared with hydrogels which has shown longer depletion rates. A rapid depletion rate is very important to maintain a strong dynamic relationship between rise and falls in blood glucose levels for example, and the ability of the sensor to detect it without significant time lags.

3. The noise signal is very low with respect to the lowest glucose concentration signal.

GOx only on Membrane

The results obtained for GOx only on a membrane were inconsistent throughout the studies. In most cases, they did not respond to the amount of glucose added, but instead resulted in a depleting signal. The results are shown in Figures 3, 4 and 5. This further supports the contribution of the nano-gold in anchoring the enzyme in an effective manner to allow the extracted analyte, such as glucose to be presented to the enzyme for a consistent and reproducible reaction to occur.

Glutaraldehyde-GOx on Membrane

The results are shown in Figure 6. The ascending portions of the line indicates the incremental addition of glucose solutions. The results indicate:

1. Depletion occurs immediately after glucose addition is stopped.

2. Noise is noticeable at lower glucose concentrations

Concentration of GOx: GLU on Membrane

The results shown in Figures 7 and 8 depict the effect of concentration of Glucose Oxidase and ratio of glucose oxidase to Glutaraldehyde, with respect to the response signal obtained from glucose dispensed on a membrane-based medium. When the glucose oxidase:glutaraldehyde ratio is 0.4 (first graph) the results are inconclusive without any trend observed. When the ratio is 0.8 (second graph), there is a clear linear increase (orange line) observed with addition of glucose, followed by a drop in glucose level when the addition of glucose is stopped indicating immediate depletion of glucose levels. The ratio of glucose oxidase to glutaraldehyde is preferably greater than 0.5 (2 to 1 glucose oxidase to glutaraldehyde).

The solid mesh structure of the membrane allows liquid to be held within it, such as water, buffer or some other non-interfering medium through which diffusion of the relevant analytes or enzyme reaction products can occur, as they make their way to the electrode surface. However, the liquid can be difficult to retain in the membrane for long periods of time, since the liquid is easily drawn in where the membrane is hydrophilic.

Using a hydrophobic, or partially hydrophobic membrane, or a membrane that has reduced hydrophilicity will require additional energy input to drive the diffusion medium/liquid through it. This energy may be heat, sonication, agitation etc., or a combination of these. It follows that a similarly high level of energy will be required to remove the liquid from the membrane, i.e., it will have a reduced propensity to exit the membrane in the absence of an energy source, as a result of which the membrane will retain the liquid for far longer periods, leading to more controlled diffusion, and absence of drift caused by a reduction of the liquid content of the membrane/drying out of the membrane over time. This drying out usually occurs even when there is a hermetic seal around the skin, since the skin itself can absorb some ofthe moisture/liquid.

Background Signal - Use of Hydrogel/PBS Buffer Solution

The use of prior art hydrogel/PBS systems to act as an interface between the skin surface and the working electrode of the sensor has disadvantages. The analytes extracted from the skin through Iontophoresis need to diffuse through the hydrogel system to reach the working electrode for the specific analyte (e.g. glucose) to react with the enzyme coated on the working electrodes of the sensor, for the measurement of the analyte to be measured.

A typical response to additions of glucose to a prior art hydrogel device is illustrated in Figures 9 and 10. The concentrations of analyte added and the current signal obtained are aligned in parallel at same time periods

A problem faced with the use of Hydrogel/PBS systems is a current signal well before the analyte reaction that is to be measured. The signal obtained only with Hydrogel/PBS is the artefact/noise for the biosensor system.

The current signal obtained from the hydrogel/PBS system is due the dissociation of molecules into ions (Hydrogel) and free flow of ions (PBS) influenced by the current applied on the working electrode of the sensor where Hydrogel/PBS is utilised. The ions from the interface medium contribute to the current and result in an unwanted signal.

The prior art interface between the skin surface and the sensor is replaced by a porous membrane which comprises an enzyme. The porous membrane acts as a medium for the analytes from the skin to the sensor’s working electrode. This eliminates the unwanted current signal by the ions present in the prior art systems due to the elimination of such extraneous ions and materials through interaction with the membranes 3-dimensional surface area.

The results obtained from the porous membrane as the interface/medium for the Iontophoresis extraction analyte eliminates the unwanted/background signal produced with either hydrogel or buffer solutions as an interface medium. An example of the response from a device according to the invention is shown in Figure 11.

The noise resulting from the prior art Hydrogel/PBS systems on the sensor dominates the actual signal (Analyte measurement) making the SNR very low. (SNR = 2, which is considerably low for the sensor). Experimental results from a prior art hydrogel device are shown in Figure 12.

The noise level in the membrane interface of embodiments of the present invention is below the actual signal of the analyte measured, and the SNR is relatively high when compared to other interfaces mentioned above. (SNR = 11, calculated from the experimental results). Experimental results from a device according to the invention are shown in Figure 13.

Sensitivity of the Sensor

The use of the membrane in accordance with the present invention increases the sensitivity of the signal tremendously, as depicted in Figures 14 and 15. Figure 14 shows the results from a prior art device and Figure 15 shows the results from a device according to the invention.

Membranes

The following properties of a Membrane as used in the present invention are advantageous as compared to a prior art Hydrogel/Buffer Solution:

1. Porous to allow the analyte to pass through, preferably with a porosity of less than 2 pm pore diameter - more preferably less than 1 pm pore diameter, and even more preferably 0.45 pm pore diameter - becoming a selective membrane and thus keeping other components away from the sensor working electrode.

2. Intrinsically Hydrophilic membranes entrapping the water and the other components, thus not contacting the working electrode which will result in ion formation and therefore the current, avoiding giving rise to the unwanted signal.

3. Low background Signal - since the membranes used in the invention have low or substantially no free ion, they enhance the chemical reaction without the interference of the background signals mentioned above.

So, the devices of the present invention have a sensor design approach which is to divide the chemistry and the amperometric part separately as follows:

1. The membrane section is where the chemical reactions take place.

2. The sensor section is where the amperometric measurement takes place.

This Design approach is taken to provide a real time measurement with a faster accumulation/depletion rate to give more sensitive and more accurate monitoring.

Working Principle of the Designed Bio-sensor

1.

Chemical Reaction Field

The porous membrane comprises the enzyme of the specific analyte being measured using an immobilisation technique, which may be ionic, covalent, hydrogen or electrostatic bonding.

Thus, when the specific analyte enters the porous membrane reacts with the coated enzyme on the membrane giving reaction products in the membrane.

2. Electric Field

The working electrode of the sensor has a voltage applied to it providing an electric field around the surface of the membrane, activating the reaction product, such as H2O2 from the membrane to reach the working electrode due to electro-diffusion. This scenario takes place, since moieties like H2O2 are electro-active compounds due to its dipole moment binding (molecular structural property).

Thus, a reaction product reaches the working electrode resulting in the current signal equivalent to the concentration of the reaction product which is directly proportional to the specific analyte measured.

By making the working of the bio-sensor into two sections helps with the following scenarios:

1. Low background Signal

2. SNR of the sensor is high

3. Increased Sensitivity ofthe sensor

Study on membranes Bio-dyne A and C

Three continuous cumulative experiments were done both on Bio-dyne Membrane A & C twice, on each membrane.

Cumulative	Concentration	Membrane A	Membrane C
Trial 1	1-8pM	Increment	Decrease
j Trial 2	1 - 34 pM	Increment only up : to 30 pM	Increment only up to 30 pM
Trial 3	4—26 pM	Incremental	Incremental

The results are shown in Figures 16 to 18.

Observation from Results

Characteristics Membrane A

Membrane C

Analysis

Curient Range

Concentration

Range

Trial 1	0.1-0.2	Trial 1	0.8-0.7
			(Noise)
Trial 2	0.4-0.8	Trial 2	0.1-0.4
Trial 3	0.4-0.6	Trial 3	0.4-0.5
1-30uM		4 - 30uM

Mem A shows signal strength

Mem A responds well to lower concentration

Linearity

Trial 1	Increasing	Trial 1	Noise	Mem C
	Exponential			linearity
Trial 2	Roughly	Trial 2	Linear
	linear
Trial 3	Increasing	Trial 3	Roughly
	exponential		Linear

Product Bio dyne A Membrane

Description Amphoteric Nylon 6,6

Works best for: Also, Colony/Plaque Lifts, DNA and RNA suited for: Transfers

Gene Probe Assays, DNA Fingerprinting, Nucleic Acid Dot/Slot Blots, Replica Plating, ELISAs

Advantages - High sensitivity - Low background Net charge can be controlled by changing pH - Ability to strip and reprobe

Binding Interaction Hydrophobic & Electrostatic

Detection Methods Radiolabelled Probes, Enzymeantibody Conjugates Chemiluminescent - Chromogenic

Bio dyne C Membrane

Negatively-charged Nylon 6,6 Reverse Dot Blots

Protein Immobilization, Affinity Purification, ELISAs

- Negative charge over broad pH range

- Surface carboxyl groups can be derivative

- Ability to strip and re-probe Hydrophobic & Electrostatic Radiolabelled Probes, Enzymeantibody Conjugates - Chromogenic si Fluctuating Gradient - Decreasing Increasing in most Gradient i points

Mem A has better response relative to Mem C. Mem A has better sensitivity

The membrane A performs better since its net charge can be controlled by the pH activity of the chemical reaction. The change in net charge is adaptable in Membrane A which helps in the signal strength as the current flow can be high, as most of the time membrane A remains neutral whereas Membrane C has a negative charge with it that inhibits the current, which is also observed in the experimental results.

Sensitivity and lower concentration detection is possible in Membrane A. This is because

Membrane A is a nylon based filter and does not contain any functional groups or charges, making the membrane A non-functionalised, providing a great response to analyte monitoring. While Membrane C also contains nylon material, it has charges and functional groups for derivatization.

The results are shown in Figure 19.

Noise Level with respect to Membranes

During the first 30 min of the experimental analysis, glucose was not dispensed. The signal obtained during this period is studied to see baseline noise signals in the system. The results are shown in Figure 20.

These baseline signals observed do not appear to have a pattern. Moreover, the Membrane A and Membrane C responses to these signals are not well defined. It is concluded that Membrane A and Membrane C have a baseline noise level, which is therefore an innate but variable characteristic, but importantly it is significantly less, almost an order of magnitude less than the noise levels observed with hydrogel based systems.

Substantiation of Data based on Human studies of Continuous Glucose

Measurements using the Device of this Invention

The device was used with membranes A and C in a human study in an insulin-dependent diabetic subject. The glucose measurements were taken once every 5 minutes over a period of up to 10 hours, during continuous device application to the arm of the patient. Finger prick glucose measurements were also taken at pre-defined time intervals of up to hourly frequency. Two devices were worn simultaneously, one on each arm to measure precision as well as accuracy. The results are summarised in the table below, and also shown in figures 21 to 24.

100min Decreasing exponential

No Response in one Acceptable device.

Second device had a low signal

Only one watch had Acceptable a response

Parabolic shape/Unsure whether Noise

Decreasing exponential

9- Feb using 2 Membrar

HHfl

10- Feb using 2 Membrar

H—

NA

30min

One Watch had no Response

Very Good correlation

None

Very strong correlation therefore precision

Observations

Results from these tests are shown in Figures 21 to 24, and the data shows that both accuracy and precision is enhanced, and initial noise level reduced where the device uses

Membrane A compared with using Membrane C. The properties of membrane A are therefore a preferred embodiment of the invention.

Claims

1. An analyte-monitoring device comprising:

a porous membrane layer comprising an enzyme; and analyte monitoring means;

2. The device of Claim 1, wherein the analyte is glucose or lactate.

3. The device of Claim 1 or 2, wherein the device does not comprise a hydrogel.

4. The device of any preceding claim, wherein the device is configured to continuously monitor the concentration of the analyte in a patient.

5. The device of any preceding claim, wherein the enzyme is glucose oxidase or lactase dehydrogenase.

6. The device of any preceding claim, wherein the enzyme has a concentration range in the porous membrane layer of 0.45 to 0.60 mg/crrr2 of membrane surface area for a membrane thickness of 158 to 192 pm thickness, or 0.45 mg to 0.60 mg per 0.158 pm3 to 0.192pm3 of the 3-dimensional porous membrane.

7. The device of any preceding claim, wherein the enzyme is physically adsorbed on the porous membrane layer or is chemically bonded to the porous membrane layer.

8. The device of any preceding claim, wherein the porous membrane layer is a porous hydrophilic polymer layer, optionally wherein the polymer is nylon, preferably nylon 6,6, and more preferably amphoteric nylon 6,6.

9. The device of any of Claims 1 to 7, wherein the porous membrane layer is a porous hydrophobic, or partially hydrophobic layer, optionally wherein the polymer is chemically or physically treated nylon 6,6, or surface functionalised polyethersulfone.

10. The device of any of Claims 1 to 7, wherein the porous membrane layer is a 3dimensional matrix, optionally wherein the 3-dimensional matrix is nylon 6,6, or surface functionalised polyethersulfone.

11. The device of any preceding claim, wherein the porous membrane layer has a thickness of less than 300 pm.

12. The device of any preceding claim, wherein the pores of the porous membrane layer have an average size of from about 0.1 pm to about 2.0 pm, such as from about 0.2 pm to about 1.0 pm, preferably from about 0.3 pm to about 0.5 pm.

13. The device of any preceding claim, wherein the porous membrane layer and analyte monitoring means are contiguous.

14. The device of any preceding claim, wherein when placed on the surface of the skin of the skin of a patient, the porous membrane layer and the surface of the skin are contiguous.

15. The device of any preceding claim, wherein the patch comprises means for performing iontophoresis, ultrasound, or micro-poration using microneedles or laser light.

16. The device of any preceding claim, wherein the analyte monitoring means comprises a counter electrode and a working electrode, and a reference electrode.

17. Use of a device of any preceding claim for monitoring the concentration of an analyte in a patient.

18. A method of monitoring the concentration of an analyte in a patient comprising the steps of:

a) applying a device according to any of Claims 1 to 16 to the skin of the patient such that the porous membrane layer of the device is hydrated and in contact with the skin;

b) receiving analyte from the skin into the porous membrane layer;

19. The method of Claim 18, wherein the method involves the step of performing iontophoresis or applying ultrasound to the skin, or poration of the skin by laser light or micro needles, i order to receive analyte.

20. The method of Claim 19 wherein the step of performing iontophoresis or applying ultrasound to the skin, or poration of the skin by laser light or micro needles is performed prior to application of the device to the skin.

21. The method of Claim 18, 19 or 20, wherein the analyte is glucose or lactate, comprising the step of oxidising the received glucose or lactate to produce hydrogen peroxide.

22. The method of Claim 21, comprising the step of detecting the level of hydrogen 10 peroxide produced by the analyte detection means.

GB 1702405.0

1-22

Intellectual

Property

Office

Application No: Claims searched: