GB2521627A

GB2521627A - Device and method for characterisation of biological samples

Info

Publication number: GB2521627A
Application number: GB1322953.9A
Authority: GB
Inventors: Dewan Fazlul Hoque Chowdhury
Original assignee: DERMAL DIAGNOSTICS Ltd
Current assignee: DERMAL DIAGNOSTICS LIMITED
Priority date: 2013-12-23
Filing date: 2013-12-23
Publication date: 2015-07-01
Also published as: GB201522844D0; WO2015097190A2; GB201322953D0; WO2015097190A3; GB2531956A

Abstract

An apparatus for measuring the concentration of an analyte in a sample extracted from the skin 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 10 configured to measure the radiation emitted from the sample. The reflector 4 may shield the skin 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. In a further apparatus the reflector 4 can be implanted beneath an outer layer of the skin so that instead of being extracted, the sample can be measured in vivo. Possibilities for the type of radiation technique used include sonophoretic/acoustic methods, optical and spectroscopic techniques such as near-infrared and far-infrared spectroscopy, radiowave impedance, skin impedance spectroscopy, polarimetry, Raman spectroscopy, photo acoustic methods and optical coherent tomography. Skin extraction methods include sonophoresis, abrasion techniques and reverse iontophoresis.

Description

DEVICE AND METHOD FOR CHARACTERISATION OF BIOLOGICAL SAMPLES

Background

Numerous attempts have been documented for the non-invasive measurement of s 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 bthod circulation. Non-invasive means are desirable as they can lead to reduced costs, reduced time, and enhanced compliance by minimizing the disturbance caused to the subjecL A wide range of examples are cited in literature to extract analyte from the skin, including sonophoresis, abrasion techniques, and reverse iontophoresis, foflowed by measurement of the analyte outside the skin. Measurement techniques indude acoustic e.g., sonophoretic methods; optical and spectroscopic techniques such as is 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 quanti!' 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 im region of the 2SOOnm spectrum are compared to known data for glucose. ________________________ Raman Laser light is used to induce Wavelength 7BSnm, Spectroscopy emission from transitions near 532nm the level excited.

Photoacoustic Laser excitation of fluids is Wavelength l5SOnm- Spectroscopy used to generate an acoustic lSSOnm or 2lOOnm-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.Bnm polarization preference in the light transmitted.

Mid-Infrared Absorption or emission data in Wavelength 2500nm-Spectroscopy the 2.5 tm -25 m region are 25,000nm examined and used to quantitate glucose in a fluid.

thttp://photonicssociety.org/newsletters/apr98/overview.htm Bazaev et. al., Biomedical Engineering, Volume 45, No. 6, March 2012, pp. 229-233.

s Patent literature contains numerous examples of methods of enhancing the signal strength generated from these types of non-invasive measurement techniques: WO 2013/1732 37 [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 io 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, s 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 io 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 is 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 s 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 io 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 is -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 viva 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 s 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 1radiation" is used to encompass acoustic waves as well io 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 is 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 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). 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 s preferred to ensure a sufficiently large and representative quantity of the analyte within the sample vo'ume.

Secondly, current imp'anted sensors are required to be removed and replaced after a period of time, which varies from 5 tol4 days for glucose oxidase based sensors io 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 is 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 ofbeing a solid, non-porous material with smooth surfaces. This can be created from metals, ceramics and plastics/polymers, or a composite thereof, which are bio-compatible, and suitable for long term implantation, such as materials used in bone graft surgery, hip replacements etc. In a second embodiment of the invention the sample to be analysed is first extracted 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.

Extraction may use one of a number of techniques) including reverse iontophoresis, 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 non-invasive sensing, either continuously or intermittently, using a detection means that maybe 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 io 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 is 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 GE 24613SS 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 s 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 io 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 is 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 transparentwindow 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 is 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 s 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 io 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 is to minimize or prevent the direct exposure of the skin to the source of radiation or other type of energy, where that source maybe damaging to the skin.

In a further embodiment of this invention where electrodes are used to draw the analyte from the skin by reverse iontophoresis, the electrodes may by positioned facing away from the skin, screened by the reflector element, 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 maybe 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 ii-maximum distance of any point on the surface from the nearest edge is much less than the half the square root of the area of the surface.

The term characterisation is used here to define qualitative or quantitative analysis s 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 is 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 s 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 iso as to be spaced a small distance from the surface of the skin 10, and conductive medium B shown around the io 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 ii 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 anayte. 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.

io Figure 6-Plan view ofa 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.

Claims

CLAIMS1. An 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 of the patient into a collection s chamber adjacent to the skin; a radiation source configured to irradiate the sample in the collection chamber; a reflector configured to receive incident radiation that has passed through the sample and reflect it back through the sample; and io a sensor configured to measure features of the radiation emitted from the sample from which characterisation information about the analyte can be derived.
2. An apparatus according to claim 1, wherein in use the radiation source is configured to direct the radiation towards the skin but the reflector is configured to is shield the surface of the skin from the radiation.
3. An apparatus according to any of preceding claim, wherein the means for extracting the sample from the skin of the patient comprises reverse iontophoresis electrodes.
4. An apparatus according to claim 3, wherein the reverse iontophoresis electrodes are configured to face away from the skin.
5. An apparatus according to claim 3 or claim 4, wherein the reflector at least in part comprises a reflective surface of the reverse iontophoresis electrodes.
6. An apparatus according to any of claims ito 5, wherein the reflector is at least partially in the form of a convoluted strip.
7. An apparatus according to any of claims ito 5, wherein the reflector is perforated by one or more apertures.
8. An apparatus according to any preceding daim, wherein the reflector is in the form of a surface bounded by an edge, and wherein form of the reflector is such that the maximum distance of any point on the surface from the nearest edge is much less than the square root of the area of the surface.
9. An apparatus according to any preceding daim, further comprising means for attaching the collection chamber to the skin such that the reflector is spaced from the surface of the skin.io
10. An apparatus according to any preceding claim, wherein the collection chamber and reflector are contained within a patch designed to be adhered or strapped to a patient's body.
11. An apparatus according to any preceding daim, wherein the radiation is source and sensor are contained within a housing designed to be adhered or strapped to a patient's body.
12. An apparatus for characterisation of an analyte in a sample volume of the surface layer of the skin of a patient, the apparatus comprising: a radiation source located outside the body and configured to irradiate the sample; a reflector implanted beneath the surface layer of the skin to define the sample volume, the reflector being 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 and configured to measure features of the radiation emitted from the samp'e from which characterisation information about the analyte can be derived.
13. An apparatus according to any preceding daim, further comprising a mask to prevent radiation from the source bypassing the reflector.
14. An apparatus according to any preceding claim, wherein the analyte is glucose.
15. An apparatus according to any preceding claim, wherein the features of the s radiation measured by the sensor include Raman scattered radiation.
16. A method of characterisation of an analyte in a sample, wherein the method comprises the steps of: extracting the sample from the skin of the patient into a collection chamber io adjacent to the skin; irradiating the sample in the collection chamber; using a reflector 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 is characterisation information about the analyte can be derived.
17. A method according to claim 16, which uses reverse iontophoresis to extract the sample from the skin of the patient
18. A method according to claim 16 or claim 17, further comprising a preliminary step of attaching the collection chamber to the skin such that the reflector is spaced from the surface of the skin.
19. A method of characterisation of an analyte in a sample volume of the surface layer of the skin of a patient, the method comprising: irradiating the sample; using a reflector that has been previously implanted beneath the sample volume of the surface layer of skin 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.
20. A method according to claim 19, further comprising the step of applying growth factors to the skin to promote capillary growth in the sample volume.
21. A method according to any of claims 16 to 20, wherein the analyte is s glucose.
22. An apparatus substantially as described herein with reference to any of the drawings.