WO2015097190A2

WO2015097190A2 - Device and method for characterisation of biological samples

Info

Publication number: WO2015097190A2
Application number: PCT/EP2014/079080
Authority: WO
Inventors: Dewan Fazlul Hoque Chowdhury
Original assignee: Dermal Diagnostics Limited
Priority date: 2013-12-23
Filing date: 2014-12-22
Publication date: 2015-07-02
Also published as: GB2531956A; GB2521627A; WO2015097190A3; GB201322953D0; GB201522844D0

Abstract

An apparatus for measuring the concentration of an analyte in a sample extracted from the skin (10) of a patient comprises a collection chamber (3) adjacent to the skin; a radiation source (7) configured to irradiate the sample in the collection chamber (3); a reflector (4) configured to reflect incident radiation back through the sample; and a sensor (11) configured to measure the radiation emitted from the sample. The reflector (4) may shield the skin (10) from the incident radiation and have a convoluted or perforated shape to permit sufficient diffusion of the extracted analyte to all parts of the surface of the reflector from its edges. The reflector (4) may be a reflective surface of reverse iontophoresis electrodes (9) used to extract the sample from the skin. In a further apparatus the reflector (4) is implanted beneath an outer layer of the skin (10) so that instead of being extracted, the sample can be measured in vivo. An implanted reflector (4) may be formed from an assemblage of reflective particles. A mask (5) may be provided to prevent the incident radiation bypassing the reflector (4).

Description

DEVICE AND METHOD FOR CHARACTERISATION OF BIOLOGICAL SAMPLES

Background

Numerous attempts have been documented for the non-invasive measurement of analytes in biological subjects such as humans and animals. This has been driven by the clinical need to attain finer control over chronic conditions such as diabetes, as well as for monitoring the absorption of drugs into the blood circulation. Noninvasive means are desirable as they can lead to reduced costs, reduced time, and enhanced compliance by minimizing the disturbance caused to the subject.

A wide range of examples are cited in literature to extract analyte from the skin, including sonophoresis, abrasion techniques, and reverse iontophoresis, followed by measurement of the analyte outside the skin. Measurement techniques include acoustic e.g., sonophoretic methods; optical and spectroscopic techniques such as near-infrared, and far-infrared spectroscopy, radiowave impedance (whereby non- ionic solutes such as glucose attenuates the amplitude of the radiowaves), skin impedance spectroscopy, polarimetry (or optical rotation of polarised light), and Raman spectroscopy, photo-acoustic methods, optical coherent tomography, amongst others that measure the analyte directly in the skin. Mid-infra red spectroscopy has also been applied in the measurement of glucose concentrations in samples in-vitro.

Table 1 summarises some of the main techniques and the parameters used to measure and quantify analytes.

Table 1

Technique* Definition* Parameters used

(Bazaev et. al.,)

Near Infra-red Absorption or emission data in Wavelength 700nm- Spectroscopy the 0.7 to 2.5 μιη region of the 2500nm

spectrum are compared to

known data for glucose. Raman Laser light is used to induce Wavelength 785nm, Spectroscopy emission from transitions near 532nm

the level excited.

Photoacoustic Laser excitation of fluids is Wavelength 1550nm- Spectroscopy used to generate an acoustic 1850nm or 2100nm- response and a spectrum as 2300nm; 532nm, 1064nm the laser is tuned.

Scatter Changes The scattering of light can be Interferometer with low used to indicate a change in coherence radiation source the material being examined, and interferometric e.g., Optical Coherent photodetector. Reflected Tomography. radiation is superimposed on reference fiber optic bundle radiation and resulting interferometric signal detected using photodiode.

Polarization The presence of glucose in a Wavelength 523nm, Changes fluid is known to cause a 635nm, 632.8nm

polarization preference in the

light transmitted.

Mid-Infrared Absorption or emission data in Wavelength 2500nm- Spectroscopy the 2.5 μιη - 25 μιη region are 25,000nm

examined and used to

quantitate glucose in a fluid.

*http://photonicssociety.org/newsletters/apr98/overview.htm

Bazaev et. al, Biomedical Engineering, Volume 45, No. 6, March 2012, pp. 229-233.

Patent literature contains numerous examples of methods of enhancing the sig strength generated from these types of non-invasive measurement techniques:

WO 2013/173237 (Al) - describes a focusing element for focusing an incident light from a laser light source, and an optical element for collecting a signal from the sample with a reflected light sensor situated on the inner housing of the spectrophotometer. US 2013/266258 (Al) - describes a means of converting an optical input to a differently shaped optical output, using stacked waveguides.

WO 2012/173686 (Al) - describes an apparatus for stabilizing an optical, thermal, and mechanical interface between a spectroscopic and/or imaging system and biological sample, using a window retainer which does not obstruct light travelling back from the sample to the imaging or spectroscopic system.

US 2011/194183 (Al) - describes an optical window for re-directing scattered radiation. It further describes an aperture of the optical window fabricated from a reflective material such that light emerging from the sample outside the area of the aperture is redirected back to the sample.

WO 2010/141258 (Al) - describes an apparatus for emitting optical radiation onto a sample and for collecting in-elastically scattered radiation from a sample, and comprises an off-axis reflector and filter to transmit the in-elastically scattered radiation; the reflective optics are used to both deliver the excitation beam and collect the scattered radiation.

WO 2007/127909 (A2) - describes the use of optical fiber bundles positioned to receive the scattered light collected by the optics.

All of the above inventions relate to the use of optical radiation for the purpose of detecting scattered light from the sample containing the analyte, which is generally described as being human skin. It follows that the pathway between incident radiation and the sample containing the analyte of interest must be optically transparent to allow the radiation to reach its target. Furthermore the incident light source is essentially unidirectional towards the sample containing the analyte, i.e., the skin, on the assumption the surface being irradiated, e.g., the skin is a substantially thick three-dimensional substrate. Reverse iontophoresis is widely documented in literature to be able to effectively extract a number of charged and uncharged analytes from the interstitial fluid of the skin. The major benefit is that the sensing process is unaffected by the numerous other molecules that constitute the chemical and biological environment within the skin, from which the analytes are essentially filtered out to the surface of the skin, and whilst there is still a mixture of analytes that is extracted, the process of detection or sensing is significantly simplified in that the volume or quantity of interfering species is reduced, in particular macromolecules such as proteins present in the skin that would interfere with the sensing are generally not extracted from the skin using reverse iontophoresis due to the size of the protein molecules. Optical methods involving the exposure of the skin to a light source, followed by measurement of the scattered signal suffer from a number of impediments: noise generated from the complex skin composition

differences in light absorption due to skin colour and skin thickness minimal distance of penetration by the light source

heating of exposed tissue leading to burns and tissue damage

presence of moisture/sweat on the skin leading to variability

overlapping spectral signals from tissue composition

Acoustic methods of measuring the desired analyte in vivo suffer from analogous problems.

The above has lead to efforts to refine the signals generated, using physical means such as improved optics, and mathematical means such as complex algorithms, but with limited success. Indeed several commercial entities have ceased to operate due to lack of precision and accuracy of their optical non-invasive systems, initially developed for glucose monitoring. An improved non-invasive method would therefore provide a tool for monitoring chronic conditions, with meaningful clinical outcomes. Summary

In a first embodiment of this invention a reflector is implanted just below the surface of the skin. A radiation source located outside the body is configured to irradiate the sample volume of skin between the reflector and the surface; the reflector is configured to receive incident radiation that has passed through the sample and reflect it back through the sample; and a sensor located outside the body is configured to measure features of the radiation emitted from the sample from which information about the concentration of the analyte can be derived. (In this specification, the term "radiation" is used to encompass acoustic waves as well as electromagnetic and other forms of radiation.)

Providing a reflective surface behind the sample to be illuminated causes the radiation to pass twice through the sample and enhances the extent of irradiation of the analyte, with a concurrent increase in scattered light returning to the collection/detector optics and increased signal strength. It also permits the radiation source and the sensor to be conveniently packaged together on the same side of the sample.

The implantation depth of the reflector should be sufficient to permit the build-up of micro-circulation of capillaries between the reflective surface and the outer surface of the skin, creating a fixed focal point where optical, acoustic or impedance or other physical techniques may be applied to measure the signal generated by the sample. Furthermore enhanced (micro) vascular growth may be achieved by application of one or more appropriate growth factors to the skin in that region as is known in the current state of the art, or as is taught by Yuan Liu et al., (BioMed Research International, Volume 2013 (2013), Article ID 561410). The growth factors may be applied in a separate process, either before or after implantation of the reflector, or the growth factors may be embedded in or coated on the surface of the reflector so as to be delivered to the sample region of the skin when the reflector is implanted. Alternatively the implantation depth of the reflector may be sufficient to ensure a (rich) supply of interstitial fluid which would form the sample volume to be measured. This differs from existing implanted sensors in two fundamental ways: implanted sensors, for example those used for glucose measurement, using glucose oxidase, or fluorescent technologies, suffer from bio-fouling, i.e., the build-up of cells and micro- vasculature around the implanted device which leads to drifting of the signals that are generated, thus requiring multiple calibrations using blood glucose values determined by finger-prick in order to re-calibrate the system. In this invention however the build-up of cellular and micro-vascular network over the reflector is preferred to ensure a sufficiently large and representative quantity of the analyte within the sample volume.

Secondly, current implanted sensors are required to be removed and replaced after a period of time, which varies from 5 to 14 days for glucose oxidase based sensors and is up to 6 months for optical based sensors. The implant described in this invention may be retained in the skin on a permanent or long term basis as it is an entirely inert material that does not react chemically, and provides a physical surface from which to reflect optical or acoustic waves. This has important ramifications in that optical or acoustic measurements taken from different locations can lead to wide variations in the accuracy of the data generated, thus the ability to select and maintain a specific area that would reduce that variability is beneficial. The type of material that would be used for such implant would have the general characteristics of being a solid, non-porous material with smooth surfaces. This can be created from metals, ceramics and plastics/polymers, or a composite thereof, which are biocompatible, and suitable for long term implantation, such as materials used in bone graft surgery, hip replacements etc.

In a variant of this embodiment of the invention, the implanted reflector comprises an assemblage of reflective particles instead of a single reflective body. The particles are preferably implanted to form a layer parallel to the outer surface of the skin. An advantage of this is that the reflector can be implanted using minimally invasive techniques such as the use of microneedles to inject the reflector particles into the skin at the desired depth. Alternatively, the particles can simply be suspended in a buffer solution, saline solution or water for injection using known techniques for fluids.

The particles may be uniform in shape and size, or irregular and polydispersed. The material, size and shape of the particles and their density and distribution within the skin are such that the irradiated light waves or sonic waves are at least partially reflected by them towards the detector. The wavelength of the incident light or sonic wave may vary over a broad spectrum, as shown in Table 1 above. Near- and mid- infrared wavelengths in particular provide detectable signals from reflected light.

The reflector surface area should be maximized. The smaller the size of the reflective particles, the larger the surface area (for a given volume of material). Tiny particles, for example nano- or micro-particles will offer a very large surface area. A further objective is to occlude proteins that are present in the underlying tissue and interstitial fluid, therefore the particle must be at least equal to the size of such proteins, the larger of which have a theoretical diameter of around 5nm. It follows therefore that a spherical particle of at least 5nm diameter would be adequate in shielding components of the interstitial fluid from the radiation. The particles may however be disc- or rod-shaped or irregular.

The maximum particle size would be dictated by what can be physically embedded in the skin, while providing an optimal surface area to volume ratio. The maximum diameter is expected to be less than 1000 μιη and the particles are preferably below 100 μιη in diameter.

If the assemblage of particles is non-uniform in size, the particle size should be measured as the mean of the individual particle diameters. If the particles are not spherical, the diameter of each particle should be measured as the maximum Feret diameter.

A problem may arise if the particles are too small because they will be absorbed into the blood stream and carried away from the sample volume. Another problem if the particles are not absorbed and merely lodge within the tissue under the upper layer of the skin, is that they are prone to phagocytosis or attack by other inflammatory and immunological mediators that may result in their encapsulation and possible removal from the tissue. In either case, the effectiveness of the particulate reflector is reduced.

In order to prevent this the particles can be encapsulated in a material that does not impede the transmission of the radiation to and from the reflective surface of the particle. An example would be silicone though other polymers or glass or ceramic materials could be used. Medical grade polymers such as methacrylates would also be suitable, for example poly methyl methacrylate (PMMA). In such case the particles would be suspended in the encapsulating material and then injected as small micro-doses just under the skin. The encapsulating material would not be absorbed or itself be phagocytosed or attack the body as it would be neutral and not reactive and the micro-dose would be too large to undergo any form of attack by the body's defence mechanism. The encapsulation material may also be described as an 'anchoring' material whose function is to prevent the particle from being engulfed by cells in the body, i.e., the material need not entirely encapsulate the reflector particles and may instead merely attach to them to anchor them in a relatively fixed position within the skin. It is not essential that the engulfment or biodegradation should be 100% eliminated permanently: for many uses of the invention it will be sufficient to prolong it by weeks, months or even years.

In a second embodiment of the invention the sample to be analysed is first extracted by reverse iontophoresis from the skin of the patient into a collection chamber adjacent to the skin; a radiation source is configured to irradiate the sample in the collection chamber; a reflector is configured to receive incident radiation that has passed through the sample and reflect it back through the sample; and a sensor is configured to measure features of the radiation emitted from the sample from which information about the concentration of the analyte can be derived. The reflector at least in part comprises a reflective surface of the reverse iontophoresis electrodes. Extraction is followed by analysis external to the skin using optical means, acoustic or other means known in the current state of the art. This provides a means of noninvasive sensing, either continuously or intermittently, using a detection means that may be permanently interfaced to the analyte collection chamber, or may be an independent unit that is intermittently exposed over the analyte that has been extracted into the collection chamber to measure the concentration of analyte.

In either embodiment of the invention, the sensor that detects the radiation emitted from the sample may transmit measurement signals to a remote location for analysis and presentation to the user. The apparatus may incorporate a control module interfaced to the patch, containing a power source and programmable micro-chip to determine the sequence of the analyte extraction mechanism, as well as any input or output communications.

Previous reverse iontophoretic systems discuss means of determining the concentration of an analyte such as glucose by extracting the glucose and then depleting the extracted glucose by reaction with an enzyme to determine the amount extracted (e.g., the former Glucowatch^® developed by Cygnus, Inc). However, optical methods would not necessarily deplete any or all of the extracted analyte as part of the detection process. Optical sensors would likely lead to a cumulative build-up of analyte extracted into the collection chamber, other than where the collection chamber is replaced after each extraction/measurement; the latter is possible, but not necessary. UK patent application GB 2502287 A, entitled "Cumulative measurement of an analyte", teaches a means of detecting the concentration of analyte based on cumulative build-up of substantially most of the extracted analyte; this principle may be applied here.

A patch, as described in patent application GB 2461355 A entitled "Patches for reverse iontophoresis", may be used to extract and collect the analyte from the skin, containing a skin attachment means, such as an adhesive or mechanical attachment means such as peripheral vacuum seal, or pressure applied using a belt of some form around the patch, a chamber containing an analyte diffusion or conducting medium, and a means of inducing withdrawal or extraction of the analyte from the skin. Techniques to extract analyte from the skin may include active methods and passive methods. Active methods are defined herein as methods that are continually or intermittently applied to enable sample extraction. These may include reverse iontophoresis (whereby electrodes would be present in electrical communication with the sample collection chamber, via a conductive medium), and thermal inducement of forced perspiration. Passive methods are defined here as methods whereby the skin is 'treated' at the outset to remove its barrier properties sufficiently to cause the analyte to flow out of the skin. These methods may include skin poration using microneedles or laser skin poration; skin ablation or abrasion using mechanical, physical or chemical means, or a combination of these methods, to continuously extract glucose and/or interstitial fluid, or analytes from the skin containing the analyte of interest. The latter of these methods would rely on the skin intervention method to lead to the continuous and passive diffusion of the analyte out of the skin thus not requiring an electrically conductive medium in which to collect the analyte, and instead any medium, liquid or gelatinous, such as polymeric, hydrogel, glycerine based, oil based, or water based (depending on the hydrophilicity/lipophilicity of the analyte), for example could be employed to allow the analyte to either diffuse evenly throughout the medium, or diffuse into a region from which the analyte is to be measured, such that a representative concentration of the analyte can be determined using the non-invasive measurement means.

The outer surface of the patch contains an optically and/or acoustically transparent window for the transmission of the light or sound radiation, and collection of returned radiation to detect and provide a qualitative or quantitative indication of the concentration of the extracted analyte. The analyte may be an innate/internal component, e.g., physiological component of the subject, or an externally introduced component such as a drug or other foreign molecule or entity. The sensing method may also be electromagnetic, acoustic, impedance based, or other non -invasive method that is known in the current state of the art, including the use of optical fibers. The transparent window may be composed of glass, ceramic, polymer or other material known in the prior art, or an absorbent material that is softer in nature, for acoustic transmission. The key difference with this method of sensing in the second embodiment of the invention is that the analyte has been removed from its source (inside the skin) to a region that is away from a large number of interfering substances, and may be directly analysed with minimal interference. This provides the following benefits: no/minimal noise generated from the complex skin composition

not affected by skin colour and skin thickness differences

minimal distance of penetration by the light source is no longer an issue as the optical window may be in the region of tens of microns.

heating of exposed tissue leading to potential burns and tissue damage will no longer occur (with appropriate patch design)

presence of moisture/sweat on the skin leading to potential variability in data generated will be reduced or eliminated

overlapping spectral signals from skin tissue composition are reduced to those analytes that are drawn out of the skin in addition to the analyte of interest.

The signal representing the concentration of the analyte in the sample should therefore be cleaner and (depending on the efficiency of extraction of the analyte) also stronger in this second embodiment of the invention. Nevertheless, methods used in the prior art and in the first embodiment can also be employed here to improve the signal further.

In this second embodiment of the invention the reflector protects the skin from potential injury caused by the incident energy source. A mask may be provided to prevent the radiation bypassing the reflector to contact neighbouring areas of the skin surface. It is preferable to retain the analyte collection chamber directly above the area of the skin from which the analyte is extracted, to maximize the concentration of the analyte, and prevent it being diluted. However if the reflective substrate covers/occludes the skin, then the degree and efficiency of analyte extraction will be compromised, for example if extraction occurs only in the periphery of the analyte collection chamber. It would therefore be preferable to suspend the reflective substrate above the skin within the medium where the analyte is collected, e.g., buffer solution or gel, such that there is a distance between the skin surface and the rear surface of the reflective substrate, sufficient to allow the analyte to travel from the skin to the medium in the analyte collection chamber, and to diffuse throughout the chamber.

There is also a region above the reflective substrate, between it and the optical window or optically/acoustically transparent film, sufficient to allow the representative concentration of the analyte extracted to be determined from the reflected radiation. The analyte may diffuse to this region between the reflective surface and optically transparent window via the periphery of the reflective film or through perforations within the film. In the event that perforations are created within the film, a mask of optically or acoustically opaque regions may be coated or applied to corresponding regions on the optically/acoustically transparent window to minimize or prevent the direct exposure of the skin to the source of radiation or other type of energy, where that source may be damaging to the skin.

The electrodes that are used to draw the analyte from the skin by reverse iontophoresis may be positioned facing away from the skin to prevent any possibility of direct contact between the skin and the electrodes. A peripheral region around the electrode will contain exposed area of skin, from where the iontophoretic current will drive the analyte out of the skin. The electrode may be adhered to the skin or it may be suspended above the skin to allow the conductive medium to flow below as well as around the electrode substrate. However it is also well known that the current density reduces according to distance from the electrode. It would therefore be preferable to have a convoluted electrode, e.g., in a zig-zag manner such that the distance around the periphery is greater than the circumference of an otherwise disc shaped electrode, thus increasing the area of higher current density in proximity with the skin. A similar effect is achieved by creating apertures in the electrode, the aim being that no point on the surface of the electrode should be too far from the nearest edge (including the edge of one of the apertures). Preferably the maximum distance of any point on the surface from the nearest edge is less than one quarter of the square root of the area of the surface.

The term characterisation is used here to define qualitative or quantitative analysis of molecules and chemical entities within the skin or extracted from the skin, including the determination of the concentration of said analyte. Qualitative analysis may involve merely determining the relative levels of two or more analytes. Characterisation may also involve the determination of structural properties of the analyte.

It will be understood to the person skilled in the art that the reflector described above is a substrate that is able to enhance the signal generated by the sample. There may be one or more reflectors or there may be a single reflector with perforations. The reflector may be planar, or it may be three-dimensional with curved or angular surfaces, for example in the form of spherical beads, or particles, of the requisite surface properties. Furthermore given that electrodes used in iontophoresis are generally metallic, either silver, silver/silver chloride, platinum, etc., the electrode itself could also serve as the reflector on its own or in conjunction with additional reflector(s) (given that the electrodes will generally lack a smooth surface, i.e., the surface is generally rough in order to increase the active electrode surface area), where the electrode is used to induce withdrawal of the analyte from the skin.

Furthermore whilst glucose has been used as a prime example of an analyte to be measured, it will also be appreciated that the technique will also apply to other analytes such as sodium, potassium, lithium, lactate, urea, and drugs. Whilst the analyte may be extracted adjacent to the skin, it will be also appreciated that for purposes of practicality the sample may be characterised away from the immediate vicinity of the area where the sample has been extracted, and this region is broadly defined as the 'collection chamber'. The term 'adjacent' to the skin is used to define a region in proximity to the region where the sample is extracted from the skin.

Drawings Figure 1 - Cross section schematic showing the patch consisting of a skin attachment means 1, optically transparent window 2, analyte collection chamber 3, and reflective substrate 4 in contact with the skin 10.

Figure 2 - Cross section schematic similar to Fig. 1 but showing a reflective substrate 4A anchored within the adhesive layer 1 so as to be spaced a small distance from the surface of the skin 10, and conductive medium 8 shown around the underside and above the reflective substrate 4A.

Figure 3 - Cross section schematic similar to Fig. 1 but showing the reflective substrate 4B in a concave configuration, which may help to redirect the incident radiation back towards a focus at the sensor.

Figure 4 - Exploded diagram schematically depicting an optical light source 7 transmitting radiation or acoustic waves through the optically transparent window 2 and a detector 11 for sensing the radiation received through the window 2 from the sample. The detected radiation may be radiation from the source 7 that has been reflected directly from the reflector 4, in which case changes in the radiation due to its passage through the sample, such as the absorption or scattering of certain frequencies, will leave a signature characteristic of the presence and concentration of the analyte. Alternatively, the detected radiation may be that scattered or re- emitted by the analyte itself, which will have a recognizable characteristic.

In Fig. 4 the detector 11 is shown schematically as concentrically surrounding the source 7 but the positions could be exchanged, or the source 7 and detector 11 could simply be placed side by side or in any other convenient arrangement.

In Fig. 4 the reflector is shown to be perforated by apertures 6, through which the sample containing the analyte can diffuse from the skin below, thus ensuring that no part of the upper surface of the reflector 4 is so far from an edge that it cannot be reached by a representative concentration of the analyte. Preferably the maximum distance of any point on the surface from the nearest point on the edge is less than one quarter of the square root of the area of the surface. For example, if the surface area of the reflector is 4cm², the square root of the surface area is 2cm so no point on the surface should be further than ¼ x 2cm = 0.5cm from an edge. This will be true if the reflector is a 4cm x 1cm rectangle. However, if the reflector is a 2cm x 2cm square then the central point will be 1cm from the nearest edge and one or more apertures should be provided to permit adequate diffusion of the analyte to all parts of the surface. In order that the incident radiation from the source 7 should not pass through the apertures 6 and damage the skin below, the transparent window 2 is provided with a mask containing light opaque regions 5 aligned with the positions of the apertures 6 in the reflector 4.

Figure 5 - Cross section schematic showing electrodes/thermal device/analyte extraction mechanism 9 to induce the extraction of the analyte from the skin 10, positioned within the analyte collection chamber 3.

Figure 6 - Plan view of a convoluted electrode 9, which also serves as the reflector 4. By providing a long edge and a narrow width of the reflector 4, this is an alternative way of ensuring that no part of the upper surface of the reflector 4 is so far from an edge that it cannot be reached by a representative concentration of the analyte diffusing around the edges of the reflector 4 under the influence of the electrode 9.

Figure 7 - Cross section schematic showing the reflective substrate 4 implanted below the surface of the skin 10 according to an alternative embodiment of the invention. The sample 12 to be analysed in accordance with the invention is the volume of the skin located between the reflector 4 and the outer surface, as indicated by stippling. A mask 5 prevents incident radiation from the source (not shown) from bypassing the edges of the reflector 4.

Figure 8 - Cross section schematic similar to Figure 7 showing a reflective substrate 4 comprising a layer of reflective particles implanted below the surface of the skin 10. A mask 5 (not shown) could be used with this embodiment as it is in Figure 7.

Figure 9 - Schematic illustrations of various forms that the reflective particles of Figure 8 can take. Particle 20 is a simple reflective sphere, which is of a suitable size and suitable material for implantation and retention in the skin. Particle 21 comprises a smaller reflective sphere, which is either too small to be retained in the body or is not of a biocompatible material. The small sphere is therefore encapsulated in a biocompatible material that is transparent to the incident radiation, whereby the overall particle is suitable for implantation while the reflective function of the core is unaffected. Particle 22 is similar to particle 21 except that both the reflective core and the material that encapsulates it are irregular in shape. Particles 23 are similar again, except that they are not fully encapsulated by the transparent material. It will therefore not protect them against attack by the body but its size and shape may nevertheless anchor the particles against transport within the body. Particle 24 is not encapsulated at all but is purely anchored at one point by the transparent material, which may form a network 25 linking multiple particles 22,23,24 together.

The interlinking material provides protection of the particle assemblage by virtue of a steric effect, i.e., although individual particles are not entirely covered and possibly small enough to be engulfed or absorbed into the bloodstream, the mesh-like network provides steric hindrance which makes it equivalent to a larger particle size. It should be noted that the inter-linking anchoring material does not necessarily need to be transparent, and it may even be formed of the same material as the particles and be an integral part of the particle, produced by chemical lithography or laser etching of a planar substrate for example. Another method by which the meshlike structure can be achieved is to disperse a biocompatible bio-resorbable material such as a carbohydrate in particulate form, as a solid or semi-solid, within the anchoring material, for example a polymer such as silicone or methacrylate, and embed this in the skin as a layer, which then will lead to the solid, bio-resorbable parts dissolving and being absorbed into the tissue and blood stream, whilst the polymer and reflector particles embedded within will remain within the skin tissue in a mesh-like format, with voids in the regions where the bioresorbable material was present, from which it has dissolved away. This type of structure acts to provide not just anchorage but also enhanced permeability for the interstitial fluid around the reflector particles, providing a more representative sampling volume.

Claims

1. Apparatus for characterisation of an analyte in a sample extracted from the skin of a patient, the apparatus comprising:

means for extracting the sample from the skin (10) of the patient into a collection chamber (3) adjacent to the skin;

a radiation source (7) configured to irradiate the sample in the collection chamber (3);

a reflector (4) configured to receive incident radiation that has passed through the sample and reflect it back through the sample; and

a sensor (10) configured to measure features of the radiation emitted from the sample from which characterisation information about the analyte can be derived;

characterized in that the means for extracting the sample from the skin (10) of the patient comprises reverse iontophoresis electrodes (9); and the reflector (4) at least in part comprises a reflective surface of the reverse iontophoresis electrodes (9).

2. Apparatus according to claim 1, wherein the reverse iontophoresis electrodes (9) are configured to face away from the skin (10).

3. Apparatus according to claim 1 or claim 2, wherein the reflector (4) is at least partially in the form of a convoluted strip.

4. Apparatus according to any of claims 1 to 3, wherein the reflector (4) is perforated by one or more apertures (6).

5. Apparatus according to any preceding claim, wherein the reflector (4) is in the form of a surface bounded by an edge, and wherein form of the reflector (4) is such that the maximum distance of any point on the surface from the nearest point on the edge is less than one quarter of the square root of the area of the surface.

6. Apparatus according to any preceding claim, further comprising means for attaching the collection chamber (3) to the skin (10) such that the reflector (4) is spaced from the surface of the skin (10).

7. Apparatus according to any preceding claim, wherein the collection chamber (3) and reflector (4) are contained within a patch designed to be adhered or strapped to a patient's body.

8. Apparatus according to any preceding claim, wherein the radiation source (7) and sensor (11) are contained within a housing designed to be adhered or strapped to a patient's body.

9. Apparatus for characterisation of an analyte in a sample volume (12) of the surface layer of the skin (10) of a patient, the apparatus comprising:

a radiation source (7) located outside the body and configured to irradiate the sample (12);

a reflector (4) implanted beneath the surface layer of the skin (10) to define the sample volume (12), the reflector (4) being configured to receive incident radiation that has passed through the sample (12) and reflect it back through the sample; and

a sensor (11) located outside the body and configured to measure features of the radiation emitted from the sample (12) from which characterisation information about the analyte can be derived;

wherein the reflector (4) comprises a growth factor either embedded in the reflector or applied to its surface to promote capillary growth in the sample volume (12).

10. Apparatus for characterisation of an analyte in a sample volume (12) of the surface layer of the skin (10) of a patient, the apparatus comprising:

a radiation source (7) located outside the body and configured to irradiate the sample (12); a reflector (4) implanted beneath the surface layer of the skin (10) to define the sample volume (12), the reflector (4) being configured to receive incident radiation that has passed through the sample (12) and reflect it back through the sample; and

characterized in that the reflector (4) comprises an assemblage of reflective particles.

11. Apparatus according to claim 10, wherein the particles are greater than 5μιη in diameter.

12. Apparatus according to claim 10, wherein the particles are smaller than 100 μιη in diameter.

13. Apparatus according to any of claims 10 to 12, wherein each particle is attached to an anchor.

14. Apparatus according to claim 13, wherein the anchor is formed from a material that is substantially transparent to the radiation from the source.

15. Apparatus according to any of claims 10 to 12, wherein each particle is encapsulated in a material that is substantially transparent to the radiation from the source.

16. Apparatus according to claim 14 or claim 15, wherein the transparent material is a silicone, a glass, a ceramic or a polymer.

17. Apparatus according to claim 16, wherein the transparent material is a methacrylate polymer.

18. Apparatus according to any of claims 10 to 17, wherein the reflector (4) comprises a growth factor either embedded in the reflector or applied to its surface to promote capillary growth in the sample volume (12).

19. Apparatus according to any preceding claim, further comprising a mask (5) to prevent radiation from the source bypassing the reflector (4).

20. Apparatus according to any preceding claim, wherein the analyte is glucose.

21. Apparatus according to any preceding claim, wherein the features of the radiation measured by the sensor (11) include Raman scattered radiation.

22. A method of characterisation of an analyte in a sample, wherein the method comprises the steps of:

extracting the sample from the skin (10) of a patient into a collection chamber (3) adjacent to the skin;

irradiating the sample in the collection chamber (3);

using a reflector (4) to receive radiation that has passed through the sample and reflect it back through the sample; and

measuring features of the radiation emitted from the sample from which characterisation information about the analyte can be derived.

characterized in that step of extracting the sample uses electrodes (9) to carry out reverse iontophoresis; and the reflector (4) at least in part comprises a reflective surface of the reverse iontophoresis electrodes (9).

23. A method according to claim 22, further comprising a preliminary step of attaching the collection chamber (3) to the skin (10) such that the reflector (4) is spaced from the surface of the skin (10).

24. A method of characterisation of an analyte in a sample volume (12) of the surface layer of the skin (10) of a patient, the method comprising: applying a growth factor to the skin (10) to promote capillary growth in the sample volume (12);

irradiating the sample (12);

using a reflector (4) that has been previously implanted beneath the sample volume (12) of the surface layer of skin (10) to receive radiation that has passed through the sample and reflect it back through the sample; and

measuring features of the radiation emitted from the sample (12) from which characterisation information about the analyte can be derived.

25. A method according to claim 24, further comprising embedding the growth factor in the reflector (4) or applying the growth factor to the surface of the reflector (4), whereby the step of applying the growth factor to the skin is performed by the implantation of the reflector (4) beneath the sample volume.

26. A method of characterisation of an analyte in a sample volume (12) of the surface layer of the skin (10) of a patient, the method comprising:

irradiating the sample (12);

measuring features of the radiation emitted from the sample (12) from which characterisation information about the analyte can be derived;

characterized in that the reflector comprises an assemblage of reflective particles.

27. A method according to any of claims 22 to 26, further comprising using a mask (5) to prevent incident radiation bypassing the reflector (4) .

28. A method according to any of claims 22 to 27, wherein the analyte is glucose.