WO2000078207A2

WO2000078207A2 - Analyte detection

Info

Publication number: WO2000078207A2
Application number: PCT/GB2000/002358
Authority: WO
Inventors: Robert Peter Chilcott; Caroline Anne Rowland
Original assignee: The Secretary Of State For Defence
Priority date: 1999-06-22
Filing date: 2000-06-15
Publication date: 2000-12-28
Also published as: WO2000078207A3; AU5547800A

Abstract

A method and apparatus for non-invasive detection of analytes in blood is described. The method employs electroporation of skin to obtain a sample of interstitial fluid for analysis.

Description

ANALYTE DETECTION

This invention relates to the detection of analytes More particularly, the present invention relates to the non-invasive detection of analytes in body fluids.

It is often necessary to analyse body fluids for the qualitative or quantitative detection of analyte. The body fluid most often analysed is blood which is obtained invasively, generally by venepuncture. Obtaining samples in this manner causes problems with the handling of the blood and with the disposal of contaminated equipment Additionally, where a subject has to regularly provide samples the inconvenience and pain associated with the taking of samples is both undesirable and burdensome This is a particular burden on subjects who rely on self monitoring for diagnosis of treatment, such as insulin- dependent diabetics or some epileptics who monitor their treatment following a fit. Problems also arise when blood must regularly be taken from infants, especially neonates, where blood volume is very small.

The present invention will now be described with particular reference to its preferred application to the monitoring of blood sugar levels. It is not. however, intended that the invention be limited to such use as π finds equal utility in other applications such as the detection of electrolytes, alcohol, anti-epileptic drugs, anti-asthmatic drugs such as theophyl ne. lithium, anti-depressants, digoxin. anti-cancer drugs such as fluorouracil, hormones such as vasopressin and especially anabolic steroids, proteins or the like

It is common practice for insulin-dependent diabetics to regulate their own treatment by monitoring their blood sugar

els and to adjust their insulin dosage accordingly Capillary blood is generally used for assessing blood sugar levels, and is obtained from a "finger-prick" using a lancet or a proprietary blood sampling device. The blood obtained is placed on a reactive stπp and either allowed to react and the reaction assessed by eye or by a proprietary blood glucose monitoring deuce

This is an inconvenient procedure for subjects and results in the production of sampling equipment contaminated by blood and in the subject developing callouses on their fingers which makes it difficult to obtain subsequent samples

Recently, non-invasive methods and apparatus for implementing these methods have been produced. For example US 5730714 descπbes a method and a device which uses iontophoresis and EP 0889703 descπbes a method and a device which uses radiation for analyte detection

The present inventors ha\ e found that reverse electroporation of the skin can be used to obtain a sample of interstitial fluid, that is the fluid which bathes the cells of the upper skin layer (epidermis) The interstitial fluid bathing the epidermis is in dynamic equilibrium with the systemic circulation and hence detection of analyte within the interstitial fluid is indicative of its presence in the systemic circulation and hence the quantification of the analyte in the fluid can be used to deduce systemic concentration Iontophoresis employs a constant low voltage current which may be uncomfortable or at least inconvenient for the subject, whereas electroporation employs a rapid, high v oltage pulse, the pulse length of which may be sufficiently short for a pain response not to register Use of electroporation is known for deli ery to cells, but it has not previously been used for extraction of fluid from cells since extraction of solutes at a cellular or tissue level was previously thought j not to yield sufficient quantities tor subsequent detection and quantification

The advantages of using electroporation rather than iontophoresis to obtain the sample include the fact that electroporation is applied for only a short period of time (seconds to minutes) whereas iontophoresis has to be applied for long time periods (20 minutes or more), and the fact that electroporation is less irritating to the subject since it is reversible within a short (30 minute) time span

Additionally, there is a minimal risk of infection from the sample w hen obtained by electroporation since bacteπa, viruses or other micro-organisms cannot be removed from the body by electroporation

Accordingly, the invention provides apparatus for electroporating the skin in order to obtain a body fluid sample, the apparatus compπsing means for applying an electπcal charge to the skin, control means for controlling the charge applied to the skin and collecting means for collecting the body fluid obtained

Preferably, the apparatus for electroporating the skin is associated w ith means for detecting the presence of analyte Ideally, the two are w ithin a single piece of apparatus

Preferably the means for applying an electrical charge to the skin tor example an electrode, includes means for controlling the voltage delivered to the skin The electrodes may be commercially av ailable electrodes which minimise the depth of the electric field and thus decrease the risk of stimulating the nerv es in the skin The selection of suitable propπetary electrodes is well within the scope of the person skilled in the an Advantageously, the control means comprises a microprocessor. Such a microprocessor may be commercially available. Preferably the control means comprises signal processing means which convert a detector input signal into units of concentration of analyte in addition to controlling the magnitude and frequency of voltage application to the means for applying a charge to the skin and hence to the skin.

Control of the voltage may include control of the time for which the voltage is applied and the magnitude of the \ oltage Such means may include the incorporation of an appropnate micro-processing device which will automatically adjust the electrical parameters.

Preferably, the apparatus is capable of being miniaturised, for example to resemble a wπstwatch. Where the apparatus is in the form of a wπstwatch, the power unit may be associated with the strap rather than the main body of the watch. Additionally, in such an embodiment a general indication of analyte concentration, for example "low^", "high" or "normal" may be given rather than an absolute value. In the case of glucose "normal" may equate to 3 - 10 mmol.l^"' . "low" to <3 mmol.l ' and "high" to > 10 mmol.l^{' 1}.

The apparatus may be linked to telemetry apparatus to allow remote monitoring of the subject, for example in an intensive care unit, a containment suite or a special care baby unit. Such remote monitoring may also prove advantageous in v etennary medicine. Ideally, the apparatus is configured to allow automatic sampling at predetermined time intervals. Optionally, the apparatus is linked to an alarm unit to indicate the presence of an abnormal level of analvte. Λ further advantage of the apparatus of the present invention resides in the fact that the polarity of the charge applied to the skin may be varied in dependence on the analyte to be

detected

Preferably, the analyte to be detected is selected from glucose, electrolytes, alcohol, anti- epileptic drugs, anti-asthmatic drugs such as theophyl ne, lithium, anti-depressants, digoxin. anti-cancer drugs such as fluorouracil. hormones such as vasopressin and especially anabolic steroids and proteins

Most preferably, the analyte is glucose.

In a second aspect, the present invention relates to the extraction of chemicals dissolved in the systemic circulation More particularly, in the second aspect the present invention relates to the non-invasi e extraction of solutes dissolved in the interstitial fluid of the skin

Accordingly, the present invention further provides a non-invasive method of detecting an analyte in a body fluid, the method including the steps of electroporating the skin to obtain a sample of interstitial fluid and analysing the fluid for the presence of analyte.

λn advantage of the method is that it is independent of the presence or nature of a charge on the analvte to be detected Optionally, the skin may be bathed in an osmotically active solution to promote movement of solute from the interstitial tissue The solute may be ionic, such as sodium chloride, or preferably non-ionic, such as glycerol However, the use of such a solution may be inappropriate where an electrolyte is to be detected by electrochemical means.

Alternatively, an osmotically active colloid may be used. The advantage of using a colloid is that it requires replacement less frequently than a solution since it can be washed and reused and would not interfere with the electrochemical detection of solutes.

Preferably, the voltage is applied to the skin as a pulse, preferably for a time peπod of between 1 ms and 5 minutes. Most preferably, the pulse is of the order of seconds. More than one pulse may be applied

The voltage pulses may be applied at a frequency of 0 01 - 1000 Hz, more preferably at 0.1 - 700 Hz.

Detection of the analyte may employ known methods, for example the detection of glucose may employ a glucose electrode.

Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings of which

Figure 1 is a graph showing the extraction of glucose from excised human skin by reverse electroporation under isotonic conditions w ith glucose deficiency (condition isotonic A). Figure 2 is a graph show ing the extraction of glucose from excised human skin bv reverse electroporation under isotonic conditions with 4mM glucose added (condition isotonic B).

Figure 3 is a graph showing the extraction of glucose from excised human skin by reverse electroporation under isotonic conditions with 4mM glucose added (condition isotonic B) using an alternative pulsing regime to that used for Figure 2.

Figure 4 is a graph showing the extraction of glucose from excised human skin by reverse electroporation under hypeπonic conditions, not being in ionic equilibrium (condition hypertonic A).

Figure 5 is a graph showing the extraction of glucose from excised human skin by reverse electroporation under hypertonic conditions in ionic equihbπum (condition hypertonic B),

Figure 6 is a graph compaπng glucose penetration through a membrane at various intervals after pulsing for isotonic B and hypertonic B conditions, and

Figure 7 shows schematically electroporation electrodes suitable for use in the present invention

The precise compositions of solutions associated with the various hypertonic and isotonic conditions referred to abov e are given in examples 1 to 4

Electroporation of excised skin

Skin preparation Epidermal membranes were prepared bv heat-stripping Briefly, subcutaneous fat was removed from human breast or abdominal skin obtained from reduction surgery that was free of overt pathology The skin was fullv immersed in hot

water (60°C) for 45 seconds and the epidermal la er was remov ed bv blunt dissection It

was then wrapped in aluminium toil and stored at — 25°C until required Skin absorption experiments were performed in static. Franz-type glass diffusion cells with an area available for diffusion of 2 54cm^" Coiled silv er wire was positioned in receptor chambers ia sampling arms and were soldered in situ to the positive output of a signal generator (Digitimer D330) Coils of silver wire placed into the donor chamber were connected to the negative output Magnetic stirrers were placed in the receptor chambers of all cells Human epidermal membranes were then placed in between donor and receptor chambers on top of a metal gauze support Cells were left heated overnight with receptor chambers filled with 5ml D-PBS and cotton wool soaked in saline was placed in donor chambers to prevent dehydration of the skin Negative electrodes, soldered to twin round screened cable, were suspended in donor chamber fluid using BluTac. Electrodes were then connected to the positive and negative channels of a multichannel stimulator

Variation between skin samples in diffusion cells was minimised by using a minimum number of skin sources Groups of 4 or 6 diffusion cells were placed onto an aluminium

heating block maintained at a constant temperature of 35°C (giving a membrane

temperature of 30°C) by a circulating water bath (Haake. Germany) Each heating block

was situated on a 6 position magnetic stirrer plate (Whatman. Kent. UK)

Prior to skin absorption experiments, tπtiated water was used to test the structural integrity of the epidermal membranes of each cell Receptor chambers were filled with 5ml of 0 9% saline and the v olume was adjusted so that the meniscus in the sampling arm was

level with the membrane to av oid hvdrostatic effects From each cell, 20ul zero hour standards were removed and added to 5ml of scintillation fluid in scintillation vials.

Tritiated water (Η₂O, 2ml, lμCi.ml^{" 1}) was added to the donor chamber of each cell and

left for 20 min. A 20μl sample was then removed from the receptor chamber as for

standards. The specific activity of each sample was counted in a RackBeta 1214 scintillation counter. Epidermal membranes allowing more than 20% of the applied dose to penetrate during this time were judged to be damaged. Saline and tritiated water were removed from donor and receptor chambers of structurally viable cells.

Skin Penetration Experiments: An appropriate fluid (see examples) was added to donor

chamber and zero hour receptor chamber samples (50μl) were taken. Each sample volume

was replaced immediately in each cell. ^I4C L-Glucose ( lOμl, 0.03μmol, 2μCi) was added

to each receptor chamber and zero hour donor chamber samples were taken. Tritiated

water ( lOμl) was then added to each donor chamber. Cells were left for 1 hour until a

further sample was taken. Sampling arms and donor chambers were occluded throughout the experiment to prevent evaporation. Electricity was then applied across the skin.

Control cells had no electrical stimulation. Samples of donor and receptor chamber fluid were taken from all cells immediately before and after pulsing and at regular intervals thereafter, and were placed in 5ml scintillation fluid. To determine specific activity of

samples, triplicate standards were made by placing lOμl of Η;O or ¹⁴C glucose into a

known volume of D-PBS. Samples (50μl) were then taken from each solution and placed

in 5ml scintillation fluid.

Liquid Scintillation Counting: Samples were counted using a Liquid scintillation counter (Wallac 1409 DSA) using a programme set up for dual measurement of Η and ^l C . All data obtained were computer analysed.

The maximum current that could be obtained by the pulsing apparatus limited the voltage applied to cells and therefore lower voltages than desired (<100V) were utilised.

Example 1

Extraction of glucose from excised human skin by reverse electroporation under isotonic conditions with glucose deficiency , hereafter known as isotonic A conditions.

Skin surface was bathed in glucose deficient Dulbecco's Medium. Pulses of 30s, 60s and 300s duration were applied to the skin, via the silver electrodes, at a pulsewidth of 10ms and frequency of 10Hz at 1 hour intervals. The results are shown in Figure 1. for control (A) and electroporated ( ♦ ) excised human skin membranes in vitro All points are

average ± standard deviation of n=6 diffusion cells. Arrows indicate time and duration of electroporation.

These data clearly indicate that reverse electroporation can extract significant quantities of glucose from human skin Glucose was detectable within five minutes of the first pulse (duration 30 seconds). Larger quantities of glucose were extracted using a longer duration (but the same pulse-width and frequency). For a pulse duration of up to 60 seconds, the permeab sation of the skin appeared to be reversible. No glucose penetrated through control (non-electroporated) skin Example 2

Extraction of glucose from excised human skin by reverse electroporation under isotonic conditions with 4mM glucose added, hereafter known as isotonic B conditions

Skin surface was bathed in Dulbecco's Medium with 4mM glucose added Pulses of 30s, 60s and 300s duration were applied to the skin, via the silver electrodes, at a pulsewidth of 10ms and frequency of 10Hz at 1 hour intervals The results are shown in Figure 2. for

control (■) and electroporated ( ♦ ) excised human skin membranes in vit. o All points are

average ± standard deviation of n=4 diffusion cells Arrows indicate time and duration of electroporation.

These data clearly indicate that reverse electroporation can extract significant quantities of glucose from human skin Glucose was detectable within fi e minutes of the first pulse (duration 30 seconds) Larger quantities of glucose were extracted using a longer duration (but the same pulse-width and frequency) For a pulse duration of up to 60 seconds, the permeabhsation of the skin appeared to be reversible No glucose penetrated through control (non-electroporated) skin

Further experiments were performed using isotonic B conditions Pulses of 60s duration were applied to the skin, v ia the silver electrodes, at a pulsewidth of 10ms and frequency

of 10Hz The results are shown in Figure 3. for control (■) and electroporated ( ♦ ) excised

human skin membranes in vitw All points are average ± standard deviation of n=6 diffusion cells Arrows indicate time and duration of electroporation These data clearly indicate that reverse electroporation can extract significant quantities of glucose from human skin.

Example 3

Extraction of Glucose from excised human skin by reverse electroporation under hypertonic conditions with hypertonic solutions not in ionic equilibrium, hereafter known as hypertonic A conditions.

Skin was bathed in a hyper-tonic solution ( 10 x Phosphate buffered saline, pH 7.4). Pulses of 30s, 60s and 300s duration were applied to the skin, via silver electrodes, at 1 hour

intervals and the results can be seen in Figure 4 (control (▲) and electroporated (♦ )

excised human skin membranes). All points are average ± standard deviation of n=6 diffusion cells. Arrows indicate time and duration of electroporation.

These data clearly indicate that reverse electroporation can extract glucose from human skin. Glucose was detectable within five minutes of the first pulse (duration 30 seconds). Larger quantities of glucose were extracted using a longer duration (but the same pulsewidth and frequency). For a pulse duration of up to 60 seconds, the permeablisation of the skin appeared to be reversible. No glucose penetrated through control (non- electroporated) skin. Use of hypertonic solution led to a significant reduction in the amount of glucose extracted in comparison with isotonic conditions (Figures 1 and 2). This may have been due to an increased conductivity of the whole system resulting in decreased voltage across the skin membrane. Example 4

Extraction of Glucose from excised human skin by reverse electroporation under hypertonic conditions with hypertonic solutions in ionic equilibrium, hereafter known as hypeπonic B conditions

Skin was bathed in a hypertonic solution of approximately 17% glycerol in Dulbecco's medium. A pulse of 60s duration was applied to the skin, via silver electrodes, after 1 hour at a pulsewidth of 10ms and frequency of 10Hz. The results can be seen in Figure 5 (control (X) and electroporated (A) excised human skin membranes) All points are average ± standard deviation of n=6 diffusion cells Arrows indicate time and duration of electroporation.

Discussion

Figure 6 compares the glucose penetration through the membrane at various time intervals after the application of the pulse for isotonic B and hypertonic B conditions These data were derived from Figures 3 and 5 These data clearly indicate that reverse electroporation tan extract glucose from human skin using hypeπonic conditions The initial rate of glucose mo ement as slower using

hypertonic B conditions than using isotonic B conditions. 17±9μg and 44± 17μg of glucose

were collected respectively from the hypertonic and isotonic conditions in the first 30 minutes after pulsing How ev er. the amount of glucose moved ov er times in excess of 2 hours was greater under hypertonic B conditions than under isotonic A conditions Furthermore, the glucose movement was significant for at least 5 hours after application of the pulse in the case of the hypertonic B conditions, compared to a figure of about 2.5

hours for the isotonic B conditions The peak rates of flow of glucose were 139±26 and

372±50 μg/hr/cm' for the isotonic B and hypertonic B conditions respectively

The decrease in glucose flux to control rates following electroporation also suggests that increases in skin permeability induced by electroporation are transient and that skin damage is not caused by electroporation

These data show that electroporation can be used to extract glucose across human epidermal membranes. The use of a transdermal osmotic gradient created by a non-ionic material was found to greatly increase the efficiency of glucose extraction. Furthermore, the maximum current that could be obtained from the pulsing apparatus (about 800mA) limited the voltage applied to cells and therefore lower voltages than desired (i.e. <100V) were used Hence, the invention in suit shows excellent extraction of glucose using voltages that are lower than the ideal level

The amount of tritiated water passing through the skin was monitored in the electroporation experiments Flux rates immediately before and after pulsing were calculated allowing the assessment of changes in membrane permeability w ith respect to changes in membrane integrity Flux rates increased immediately after pulsing in all experiments but returned to levels comparable to control levels after one hour This implies that increases in skin permeability were re ersible. This suggests that the skin is not damaged by electroporation Fuπhermore. it was found that in general the total amount of Η₂O moved through the membrane was reduced under hypertonic conditions when compared to isotonic conditions. This suggests that the direction of water flow was from receptor to donor chamber ( i.e. towards the hypertonic or glucose-free solution), thus reducing the ^"Η;O movement from donor to receptor and enhancing glucose movement.

This is consistent with the data presented in the examples above.

Example 5

Measurement of Blood Glucose Extraction In Vivo

Extraction of blood glucose from the skin of volunteers is achievable by the following

protocol:

Two electrodes, each contained within plastic tubes filled with saline as shown in Figure 7 are attached to the forearm of a volunteer. The electrodes are connected to a D330 MuIstiStim apparatus. A pulse of electricity [ lOOv. l OHz. 1 ms pulsewidth, 30 second duration] is applied and the saline in the plastic tubes is analysed by a standard colourimetric assay or a glucose electrode to determine the concentration of glucose

extracted.

Claims

1. Apparatus for electroporating the skin in order to obtain a body fluid sample, the apparatus comprising means for applying an electrical charge to the skin, control means for controlling the charge applied to the skin and collecting means for collecting the body fluid obtained.

2. Apparatus according to claim 1, in which the apparatus is associated with means for detecting the presence of analyte

3. Apparatus according to claim 1 or claim 2, in which the means for applying an electπcal charge to the skin includes means for controlling the voltage delivered to the skin.

4 Apparatus according to any one of claims 1 to 3. in which the control means comprises a microprocessor.

5. Apparatus according to any preceding claim, in which both the time for which the voltage is applied and the magnitude of the voltage are controlled.

6 Apparatus according to any preceding claim, in which the apparatus is in the form of a wπstwatch

7 Apparatus according to any of claims 1 to 6. in which the apparatus is linked to telemetry apparatus to allow remote monitoring of a subject

8 Apparatus according to any one preceding claim in which the apparatus includes an alarm unit to indicate the presence of an abnormal level of analyte.

9 A non-invasive method of detecting an analyte in a body fluid, the method including the steps of electroporating the skin to obtain a sample of interstitial fluid and analysing the fluid for the presence of analyte

10. A method according to claim 9, in which the skin is bathed in an osmotically active solution to promote movement of solute from the interstitial tissue.

11. A method according to claim 10 in which the osmotically active solution comprises an ionic solute.

12 A method according to claim 1 1 in which the ionic solute comprises sodium chloride.

13. A method according to claim 10 in which the osmotically active solution compnses a non-ionic solute

14 A method according to claim 13 in which the non-ionic solute comprises glycerol.

15. A method according to claim 9. in which the skin is bathed in an osmotically active colloid to promote movement of solute from the interstitial tissue

16. A method according to any one of claims 9 to 15, wherein the electroporating step comprises application of a pulsed voltage to the skin.

17. A method according to claim 16 in which the or each pulse width is between 1 ms and 5 minutes.

18. A method according to either of claims 16 or 17, in which the pulsed voltage is applied at a frequency in the range of 0.01 - 1000 Hz.

19. A method according to claim 18, in which the pulsed voltage is applied at a frequency in the range of 0.1 - 700 Hz.

20. Apparatus substantially as hereinbefore described with reference to Figure 5 of the accompanying drawings.

21. A non-invasive method of detecting an analyte in a body fluid by electroporation substantially as hereinbefore described.