WO2004063393A1

WO2004063393A1 - Measurement of analyte concentration

Info

Publication number: WO2004063393A1
Application number: PCT/GB2004/000055
Authority: WO
Inventors: Gabor Tajnafoi
Original assignee: Hypoguard Limited
Priority date: 2003-01-14
Filing date: 2004-01-13
Publication date: 2004-07-29
Also published as: EP1583841A1; GB2397375A; GB0300764D0

Abstract

A diagnostic test device that measures reflectance at two different optical wavelengths to determine the change in colour of one or more chemicals, the change in colour being dependent upon the concentration of an analyte in a fluid sample applied to the chemicals. The concentration of the analyte can be determined from the ratio of the reflectance values at the two different wavelengths. In a further embodiment the optical source(s) and the optical detector are arranged so as to be within the same optical axis.

Description

MEASUREMENT OF ANALYTE CONCENTRATION

BACKGROUND OF THE INVENTION

The present invention relates to a device for measuring changes in colour chemistry to quantitatively estimate the concentration of a constituent chemical in a solution, and a method of operating such a device.

DESCRIPTION OF THE PRIOR ART

There is an increasing demand for highly portable medical diagnostic equipment that is user-friendly permitting operation by patients without intervention from medically trained staff. Such diagnostic equipment enables patients to monitor the concentration of a chemical constituent in biological material, for example glucose concentration in blood, and to seek further medical advice in the event that the measured value exceeds a threshold or is outside of a predetermined range of values. It can also be used by medically trained staff, at the bedside, in a hospital or in a nursing home.

Currently available blood glucose reflectance technology consists of a hand held reflectance meter and a disposable, chemistry-containing strip. A blood sample is applied to a sample application area on the uppermost surface of the strip. The strip further comprises a support, for easy handling, and the sample application area comprises specialised structures which contain reagent chemistries for reacting with glucose as well as removing cellular material from the blood. Colour is generated by the reaction between the glucose and the reagent chemistries and is displayed at the lower surface of the strip, the colour being related to the glucose concentration.

Conventional systems operate by applying blood to a strip which has been placed within a meter (this is commonly referred to as in-meter dosing) . There are a number of situations, particularly in nursing homes or with patients having impaired vision, where it is preferable to apply blood to a strip external to the meter and then place the strip within a meter for measurement (this is commonly referred to as off-meter dosing) . It would therefore be desirable to have a system which can operate in both an in-meter and off-meter situation.

Conventional test meters contain a light source, and it is necessary to characterise the incident radiation and reflection (often described as feedback) within a meter. Conventionally this has been achieved by:

a) estimating the feedback at the time of production. However, this technique can not compensate for variations of feedback with time or for batch-to- batch variations in the reflective surface of test strips; b) incorporating an internal reference surface or an additional light route to determine the feedback; or c) taking a "relative" measurement between the initial unused colour of the strip and the colour of the strip after the reaction with the blood sample is fully developed. This technique is very difficult to implement with off-meter dosing.

For all meter configurations the time at which analytical reflectance measurements are made is very important in providing reliable , repeatable quantitative estimates of glucose concentration.

Conventionally such equipment receives a plastic strip that comprises a carrier that contains one or more diagnostic reagent materials. A fluid sample, which may be a biological material, is placed on one side of the carrier. The sample will then pass into the carrier and the target chemical constituent being tested for will react with the diagnostic reagent material (s). The reagent (s) will undergo a change in colouration that will vary with the concentration of the target chemical constituent present in the fluid sample. Colouration change means that more colour dye (of the same colour) will be associated with higher concentrations. Typically the device will measure the change in colouration from the side of the carrier opposite to the side that the fluid sample was applied to. The carrier will be illuminated with one or more optical wavelengths and an optical detector used to measure the magnitude of the reflections from the carrier. Typically one of the optical wavelengths is related to a reflection wavelength of the colour that is generated by the presence of the target constituent chemical. Thus, the amount of light that is detected by the optical detector will vary as the colour of the reagent changes in response to the concentration of the target constituent chemical .

The received signal is the change in colouration. This is either by comparing the initial signal to the final signal as described by US4935346 and continuations or comparing the signal to a reference signal produced by an additional reference surface. (Another photodiode may be used to produce the reference signal . )

US 5 968 760 discloses a system in which a blood sample is placed on a reagent strip and is illuminated with two different wavelengths: one of the wavelengths is used to measure the colour change of the reagent (s) in response to an analyte concentration in the sample. The second wavelength is used to measure background interference, e.g. from haemocrit, contamination of part of the reagent strip by the sample etc. The analyte concentration is determined by correcting the colour change measurement, determined using the first wavelength, with the background interference measurement that is made at the second wavelength..

Test methods are also known in which blood samples are illuminated at a number of different wavelengths and the measured reflectivities are used to determine a sample property (see, for example, EP 380 664) . However, these techniques are limited to measurement of whole blood samples and are not applicable to the measurement of a colour change in response to the application of a sample to a reagent strip.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided an apparatus for measuring analyte concentration in a fluid sample, the apparatus comprising: a first optical wavelength source and a second optical wavelength source; an optical detector and an opening, the first optical wavelength source and the second optical wavelength source being aligned, in use, to illuminate a substrate comprising one or more reagents that change colour in accordance with analyte concentration to which a fluid sample has been applied, the substrate being received within the opening, the optical detector being aligned, in use, to measure light reflected from the substrate at the first optical wavelength and the second optical wavelength, wherein the reflected light at the first optical wavelength and the reflected light at the second optical wavelength are both indicative of a colour change of the one or more reagents dependent on the analyte concentration in the fluid sample, the control means determining a ratio of the reflectance at the first optical wavelength and the reflectance at the second optical wavelength and determining the analyte concentration in accordance with the determined reflectance ratio.

On one embodiment of the present invention, the first optical wavelength source, the second optical wavelength source and the optical detector are all substantially parallel .

Alternatively, the first optical wavelength source and the second optical wavelength source may be substantially parallel with a rotational offset between the optical detector and the first optical wavelength source and the second optical wavelength source. The rotational offset between the optical detector and the first optical wavelength source and the second optical wavelength source may be between 55° and 70°. Preferably the rotational offset between the optical detector and the first optical wavelength source and the second optical wavelength source is substantially 65°.

The first optical wavelength source may have a wavelength of substantially 700 nm and the second optical wavelength source may have a wavelength of substantially 575 nm.

According to a second aspect of the present invention there is provided method of measuring an analyte concentration in a fluid sample, the method comprising the steps of: i) applying the fluid sample to an area of a substrate comprising one or more reagents that change colour in accordance with analyte concentration; ii) measuring the reflectance of the area of the substrate at a first optical wavelength, the reflectance being indicative of a colour change of the one or more reagents dependent on the analyte concentration in the fluid sample; iii) measuring the reflectance of the area of the substrate at a second optical wavelength, the reflectance being indicative of a colour change of the one or more reagents dependent on the analyte concentration in the fluid sample; and iv) determining the analyte concentration by calculating a ratio of the reflectance value at the first optical wavelength to the reflectance value at the second optical wavelength and determining the analyte concentration in accordance with the ratio.

Step iv) may alternatively comprise the step of determining the analyte concentration by comparing the ratio with the contents of a database.

Preferably the method comprises the additional step of v) ) outputting an indication of the analyte concentration to a user.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention and its method of operation will be described by way of illustration only and with respect to the accompanying drawings, in which

Figure 1 shows a schematic depiction of a device according to the present invention. Figure 2 is a graphical depiction comparing the performance of a device according to the present invention with the performance of a conventional device;

Figure 3 is a graphical depiction illustrating the effect of the rotational offset of the optical detector upon the performance of a device according to the present invention and a conventional device; and Figure 4 is a graphical depiction of the local linearity of the reflection measured by a device according to the present invention when a fluid sample is applied to a carrier;

Figure 5 is a graphical depiction of the change in reflection measured by a device according to the present invention when a fluid sample is applied to a carrier;

Figure 6 is a graphical depiction of the linear relationship between two parameters of a device according to the present invention; Figure 7 is a further graphical depiction comparing the performance of a device according to the present invention with the performance of a conventional device; and

Figure 8 shows a schematic depiction of a device according to a second embodiment of the present invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Figure 1 shows a schematic depiction of a device 100 according to the present invention. The device 100 comprises receiving means 20, optical source 30, optical detector 40 and control means 50. The receiving means 20 comprises an opening 22 that is positioned and aligned so as to be illuminated by the light that is generated from the optical source 30. Preferably the optical source 30 comprises two light emitting diodes 31, 32 that each emit light at one of the desired optical wavelengths. Both the optical source 30 and the optical detector 40 are in communication with the control means 50. The optical detector 40 is provided with an aperture 42 that reduces the amount of spurious off-axis light that might otherwise be detected by the optical detector, causing inaccuracies in the measurements being made by the device . Alternatively, a lightguide may be positioned such that one end of the lightguide is aligned with light reflected from the carrier and the other end is aligned with the detector.

Such devices tend to be capable of use in two different modes, commonly referred to as in-meter dosage and off- meter dosage. In-meter dosage involves the user adding the fluid containing the target constituent chemical to the carrier whilst the carrier is held within the device. In off-meter dosage the fluid is added to the carrier whilst the carrier is outside of the device. The carrier will subsequently be inserted into the device for a measurement to be taken.

When operating a meter according to the present invention in the in-dosage application, the user inserts the strip into the device, causing the meter to turn on. When the timer elapses the reflectance will be measured at the two different wavelengths. (If the light source comprises two LEDs, then it may not be possible to measure the reflectance values at both wavelengths at the same time, and thus the reflectance values can be calculated from a sequence of measurements (this assumes that when the reaction is complete any change in colour will be very slight and can be calculated using a linear approximation) .

For the off-meter dosage application the user applies a fluid sample to the carrier outside the meter. The strip is then inserted into the meter and the meter turned on. In order to make an accurate measurement it is necessary to determine whether the fluid sample has been applied to the carrier for a sufficient period of time in order for the colour change to be complete. This can be determined using the dynamic characteristics of the chemistry- produced colour changes . A preferred method by which this determination can be made is described in W099/18426) . The reference used is the colour when the reaction is complete, and thus it is not necessary to know the original carrier colour. The ratio method has been found to support the dynamic endpoint detection in the cases investigated so far.

Once the optical source illuminates the carrier then the control means 50 will prompt the optical detector to begin measuring the light that is reflected from the carrier. The measurement data from the optical detector will be transmitted to the control means for subsequent processing. As a an alternative to the methods described above the control means may cause the carrier to be illuminated for a set period of time, or until the measurement data received by the control means meets one or more pre-determined criteria, such as, for example, passing a threshold value, maintaining a threshold value for a given period of time, two signals attaining a ratio, etc. The control means 50 analyses the received measurement data, preferably by calculating the ratio of the reflectances for the two optical wavelengths and then determining the concentration of the target chemical constituent, either by direct calculation or by reference to a look-up table. If a ratio of reflectance values is to be calculated then it may be calculated using slightly modified reflection values that are calculated based upon the electrical amplifier's bias (which can be determined during production) .

The device then provides an indication to the user of the concentration of the target constituent chemical in the biological material via visual indicators 60 and/or audible indicators 62, for example a numerical indication of the target constituent chemical concentration. Informed users may then use the data provided by the diagnostic device to manage their condition through diet, exercise or medication, or seek the advice of a medical professional .

In an alternative the optical source 30 may comprise a white light source, or another wideband source that comprises the optical wavelengths of interest, as opposed to a discrete optical source for each wavelength of interest. Similarly, the optical detector 40 may be a wide band detector from which the wavelengths of interest can be extracted, or a discrete optical detector can be provided of the wavelengths of interest, with each detector having a band pass filter centred upon the respective wavelength of interest.

One particular application of a device according to the present invention is the measurement of glucose in blood, as diabetes is becoming an increasing problem as Western populations age and become more prosperous. Theoretical analysis and experimentation have shown that when used with standard reagents for measuring blood/glucose levels it is possible to measure glucose concentrations with a greater repeatability than is possible with conventional measuring devices.

Known measuring devices tend to use a 635 nm optical source to determine glucose concentrations by measuring changes in reflectance at that wavelength. Measuring devices that utilise a further optical source, for example 700 nm, use the second wavelength to correct for other factors, for example haemocrit and oxygenation behaviour within the diagnostic reagents. In the present invention both wavelengths are used to generate reflectance values and the glucose concentration is derived using a ratio of the two reflectance values .

Figure 2 is a graphical comparison between the performance of a conventional diagnostic device using a single wavelength (indicated by triangles in the graph) and the performance of a device according to the present invention using one fixed wavelength and one variable wavelength. (indicated by squares in the graph) . The vertical axis is a coefficient of variation (CV) value, which is a relative representation of measurement standard deviation; the lower the CV value, the more repeatable the measurement technique. The data in Figure 2 for the conventional diagnostic device was generated by taking a number of measurements from approximately 300 nm to 100 nm and calculating a CV value based on those measurements. For the device according to the present invention measurements were again taken across the 400 nm to 1000 nm range. The CV values for each wavelength were calculated using the ratio of reflectance measured at each wavelength with the reflectance measured at 576 nm (the singularity that would be present at 576 nm has been removed from the graph) .

Figure 2 shows that the CV values calculated for the device according to the present invention are lower than those calculated for the conventional device across the entire wavelength range, indicating that the device according to the present invention provides more repeatable measurements than conventional devices. An ideal meter would have low and constant CV values across the concentration range that is of interest.

The calculated CV values and hence the performance of the meter has been observed to be dependent upon both the wavelengths used and the angle that there is between the optical detector and the optical source. Figure 3 shows the variation of CV values with the offset between the optical source and the optical for a conventional device measuring reflectance at 700 nm and a device according to the present invention (measuring reflectance at both 570 nm and 700 nm) . Figure 3 shows that the optimum offset angle appears to be between 55 °& 70° and also that the CV values for the device according to the present invention are lower than the CV values for the conventional device . Experimentation has shown that an offset angle of approximately 65° is of significant benefit.

Figure 4 shows a graphical depiction of the variation of reflected light as the blood sample is applied to the test strip. The various traces show the local linearity with respect to time of a number of different samples (each of which has a different glucose concentration) . The local linearity characteristics are generally similar, with an initially negative trend for the first 2-3 seconds after the sample is applied, with a minima value of approximately -5 to -10% local linearity, followed by a rapid rise to a positive maxima at approximately 4-5 seconds after sample application. The magnitude of the maxima is greater than that of the minima, with peak values of 15-30% local linearity. By approximately several seconds after sample application the local linearity values are effectively zero (excluding noise effects) . Figure 5 shows the corresponding measurements of reflection for the same samples that are shown in Figure 4.

It is been found that the measuring process for the on- meter dosing application can be initiated by detecting the local linearity minima and then measuring the reflection values at both wavelengths The glucose concentration can be determined by calculating the ratio of the two wavelength values and then making a further calculation or accessing a look-up table. Other techniques may also be used to initiate the measurement, for example, selecting a different point from the local linearity curve or detecting one or more conditions based upon the reflection values, such as detecting a steady state value for a given period of time, measuring a relative decrease from the initial value, initiating a timer upon detecting such a condition, etc.

It will be readily understood that if different diagnostic reagents were to be used to detect glucose concentrations then different wavelengths may be needed so that the device operated in a regional of minimal CV values (that is, the most repeatable measurement of glucose concentration. In the example discussed above a combination of 576 nm & 700nm was used with the DAOS product, made by Dojindo Ltd of Kumamoto, Japan (which comprises 4-Aminoantipiryne and N-Ethyl-N- (2-hydroxy-3- sulfopropyl) -3, 5-dimethoxyaniline) . If the reagent set used in the QuickTek meter-system (which is 4- Aminoantipiryne and N-Ethyl-N- (2-hydroxy-3-sulfopropyl) - 3,5-dimethylaniline) , a product of Hypoguard Limited, Woodbridge, Suffolk, UK, is used then experimentation has shown that the best pair of wavelengths is 570 nm and 870 nm (Figure 7 shows the comparison of CV values between a variable single wavelength and a combination of 870 nm and a variable single wavelength) . If the reagent is the o- Toluidine reagent (available from Sigma-Aldrich Corp., St. Louis, MO, USA, product no. 6356) , then the best pair of wavelengths is 660 nm & 740 nm. The two wavelength values for a particular reagent system can be determined by performing a number of scans using a single wavelength and a range of wavelengths, changing the single wavelength for each different scan. This creates a matrix of CV values for different wavelength combinations and an appropriate choice may be made from within the matrix.

Conventional meters that detect colour changes assume that the optimum wavelength or wavelengths to be used depend on the colour of the signal producing dye and are not be affected by the arrangement of the various optical elements .

If the amount of reflected light in an optical arrangement is denoted by reflection = A*a + B*β [l] where A and B are geometry associated constants, β is associated with the concentration to be measured

(generally linked to the colour of the surface) and a is associated with the rest of the reflected light (generally describes the roughness and reflectivity of the surface) .

Conventionally, a relative measurement is used, that is a ratio between the inspected signal and a reference signal is determined. A reason for doing this is to take account of the intensity of the light being used. If the reflection from final coloured surface is compared with the reflection from the surface once the colour has changed, then the geometry related constants A and B will be the same, however a and β will be associated with the developed colour stage. The reflectance ratio, r, used to predict the concentration can therefore be described as:

where the geometric parameter becomes C, B≠O otherwise there would be no way to predict concentration) .

Manufactured meters will have slightly different optical arrangements due to mechanical tolerances. Let r₀ denote the reflection values produced by a reference meter (and subsequent production meters should have similar values to this) and r denote the reflection produced by a subsequent production meter. It is possible to identify the meter differences with the difference between the C constant, which describes the geometrical arrangement, with C describing the geometrical arrangement of the production meter and C₀ describing the reference meter. This can be defined by the following equations:

Let f r - r_Q denote the function which describes the relationship between them. As it is intend to use the meters to measure colour variations it is valid to assume; r₀(β) = f(r(β)) [4]

Assuming /is linear (i.e . f(r) = a * r + b ) , then r₀(β) = f(r(β))= a *r(β) + b [5] and thus

C. '8 + β C*3 ₊ β _{+ b}

C₀ *a + β C*a + β Differentiate both sides by β and re-arrange to give C * + β a = [7] C_Q *a + β which can be applied to [6] to derive a b = (C₀-C)* [8]

C_Q *a + β

If both a(C) and Jb(C) are linear in C then b(a) will be linear in a, giving

C= a *(C₀ * + β)~ β

191 a and

which gives b(a) = -* (! - a) [11] where the gradient of the linear function is

b'(«) = -- [12] a

Figure 6 shows the relationship between the values of the a parameter and the b parameter. The data points were derived from measurements taken with a reference meter and with meters having different geometries. The linearity shown in Figure 6 indicates clearly that the geometric parameter, C, plays an important part in understanding the relationship between the parts of the reflected light and the colour of the surface being measured and that the optical geometry of a meter is very significant in the operation of the meter. The next factor to be considered in relation to the performance of a meter is the wavelength that is used within the meter. Generally a meter operates by determining a relationship between the concentration (s) to be measured and the received signal (s) , (normally a reflectance ratio is the received signal) . This relationship is often referred to as the calibration curve. (and is often described using the K/S transformation to linearize the relationship) . Let the mathematical representation of the relationship be denoted by r . r i→ c or T(r) ~ c , where r denotes the reflection ratio and c denotes the concentration of constituent to be measured.

In order to determine the precision of concentration measurements the Coefficient of Variation (CV) values of concentration (cCV) will be compared with the CV values calculated from the underlying reflected signal (rCV) . Coefficient of Variation is a relative representation of the standard deviation and is the most suitable representation of the precision of measurements. The cCV is obviously affected by the mathematical transformations that were used to calculate it . When compared to the underlying rCV it depends only on this transformation, T, and nothing else. Mathematically this can be shown by:

The T transformation, which is dependent upon the design of the meter, will determine the performance of the meter. For optimum meter performance the meter will provide accurate repeatable measurements (that is, the ratio of rCV to cCV is constant) across the range of concentration values that are of interest . The performance characterisation used to estimate the calibration curve is thus very important, may be described by

T(r₀) = g *r₀ ^h [14] where g and h are constants, the 0 index refers to a reference instrument that generally satisfies that condition that cCV/rCV=constant . Another meter's transformation function (as/is linear) can be written as

T(r) = g*(a *r + b)^h [15]

This causes this meter's performance to vary through the concentration range and the effect can be quantitatised by applying [13] to give

cCV _ r* h *g* (a *r + b)^{h l} *a _ r *h *a _ h

[16] rCV g*(a *r + b)^h a *r + b 1+- a*r

This constraint can be used to select the wavelength (s) incorporated into the meter design once the geometrical arrangement of the optics has been designed.

Experimentation has shown that the conventional arrangement of the optical elements is not the most effective. Figure 8 shows a schematic depiction of a second embodiment of the present invention. The device 200 comprises receiving means 220, optical source 230, optical detector 240 and control means 250. The receiving means 220 comprises an opening 222 that is illuminated by the optical source 230 that preferably comprises two light emitting diodes 231, 232 that each emit light at different optical wavelengths. In an alternative embodiment, the light source 230 may comprise a single LED (or other monochromatic light source) .

The LEDs 231, 232 and the optical detector 240 are substantially parallel to each other, such that the two LEDs both illuminate the opening 222 in the receiving means and that the detector 240 is aligned with the opening so as to receive light reflected from the sample that is received within the opening. Both the optical source 230 and the optical detector 240 are in communication with the control means 250. It will be understood that the positioning of the LEDs relative to the detector may be varied, for example by positioning both LEDs to one side of the detector.

The second embodiment relies upon the insight of the inventor that the light reflected f-rom the sample is dependent upon two factors. The first factor is the colour of the sample, which is in turn dependent upon the analyte concentration in the fluid. Light will be absorbed by the dye materials within the fluid from the light source 230 and then emitted. The light emitted in this fashion is indicated schematically by distribution 280. The second factor is reflections that are dependent upon Snell's law and the geometry and roughness of the surface of the carrier comprising the sample. The light reflected in this fashion is indicated schematically by distribution 270. It has been determined experimentally that the light that is reflected due to surface effects is generally of a larger magnitude than the light that is absorbed and emitted by the dye materials and thus conventional meters (that is those using a similar optical geometry to the arrangement shown in Figure 1) are positioned so as to measure the largest signal i.e. the combination of reflected light and emitted light. However, the emitted light is the only light that is of interest and the reflected light is effectively noise. Therefore, the embodiment shown in Figure 8 enables the emitted light to be detected whilst reducing the deleterious effects of the reflected light.

Similarly, the use of the invention to detect other analytes will require the use of different wavelengths to enable the repeatable operation of the device. References to optical wavelengths should be understood to include the visible spectrum as well as the ultra-violet and infra-red spectral regions near to the visible spectrum. Although a meter according to the present invention may be a portable device for use by non-medical personnel , the present invention may also be provided as a larger device, for example a bench-top machine, that is intended for use by medical personnel, for example within a doctor's surgery or a hospital .

Claims

1. An apparatus for measuring analyte concentration in a fluid sample, the apparatus comprising: a first optical wavelength source and a second optical wavelength source; an optical detector and an opening, the first optical wavelength source and the second optical wavelength source being aligned, in use, to illuminate a substrate comprising one or more reagents that change colour in accordance with analyte concentration to which a fluid sample has been applied, the substrate being received within the opening, the optical detector being aligned, in use, to measure light reflected from the substrate at the first optical wavelength and the second optical wavelength, wherein the reflected light at the first optical wavelength and the reflected light at the second optical wavelength are both indicative of a colour change of the one or more reagents dependent on the analyte concentration in the fluid sample, the control means determining a ratio of the reflectance at the first optical wavelength and the reflectance at the second optical wavelength and determining the analyte concentration in accordance with the determined reflectance ratio.

2. An apparatus according to claim 1 wherein the first optical wavelength source, the second optical wavelength source and the optical detector are all substantially parallel .

3. An apparatus according to claim 1 wherein the first optical wavelength source and the second optical wavelength source are substantially parallel and there is a rotational offset between the optical detector and the first optical wavelength source and the second optical wavelength source.

4. An apparatus according to claim 3 wherein the rotational offset between the optical detector and the first optical wavelength source and the second optical wavelength source is between 55° and 70°.

5. An apparatus according to claim 4 wherein the rotational offset between the optical detector and the first optical wavelength source and the second optical wavelength source is substantially 65°.

6. An apparatus according to any preceding claim in which the first optical wavelength source has a wavelength of substantially 700 nm and the second optical wavelength source has a wavelength of substantially 575 nm.

7. A method of measuring an analyte concentration in a fluid sample, the method comprising the steps of; (i) applying the fluid sample to an area of a substrate comprising one or more reagents that change colour in accordance with analyte concentration;

(ii) measuring the reflectance of the area of the substrate at a first optical wavelength, the reflectance being indicative of a colour change of the one or more reagents dependent on the analyte concentration in the fluid sample;

(iii) measuring the reflectance of the area of the substrate at a second optical wavelength, the reflectance being indicative of a colour change of the one or more reagents dependent on the analyte concentration in the fluid sample; and

(iv) determining the analyte concentration by calculating a ratio of the reflectance value at the first optical wavelength to the reflectance value at the second optical wavelength and determining the analyte concentration in accordance with the ratio.

8. A method according to claim 7, wherein step (iv) alternatively comprises the step of determining the analyte concentration by comparing the ratio with the contents of a database.

9. A method according to claims 7 or 8, the method comprising the additional step of

(v) outputting an indication of the analyte concentration to a user.