Composition comprising up-converting phosphors for detecting an analyte

Abstract

The present invention relates to a detector matrix for detecting at least one analyte in a sample, preferably a sample of a body fluid, comprising at least one enzyme active in the presence of said at least one analyte and at least one indicator reagent changing at least one optical property dependent on the activity of said enzyme, wherein said detector matrix further comprises up-converting phosphor particles, preferably UV-emitting up-converting phosphor particles. The invention further relates to a test element and a test device for detecting at least one analyte in a sample comprising the detector matrix of the invention, as well as to a method for the manufacture of a detector matrix, a method for the manufacture of a test element, and to a method for detecting an analyte in a sample, comprising contacting a detector matrix according to the invention with a sample suspected to comprise said analyte.

Claims

1. A covering layer matrix, comprising: a polymeric film former and up-converting phosphor particles; wherein the covering layer matrix is adapted for use as a reflecting layer in a two- or more layer test element; a strongly light-scattering agent; wherein the covering layer matrix lacks an indicator reagent; and wherein the covering layer matrix is configured to cover a detector matrix having the indicator reagent distributed within the detector matrix.

2. The covering layer matrix of claim 1, wherein the covering layer matrix is a matrix adapted for physically contacting an analyte detector matrix.

3. The covering layer matrix of claim 2, wherein the covering layer matrix is a matrix adapted for forming the second layer of the two- or more-layer test element.

4. The covering layer matrix of claim 1, wherein the up-converting phosphor particles are UV-emitting up-converting phosphor particles.

5. The covering layer matrix of claim 1, wherein the polymeric film former is or comprises polymer particles insoluble in a carrier liquid.

6. The covering layer matrix of claim 5, wherein the polymeric film former is selected from the group consisting of polyvinyl esters, polyvinyl acetates, polyacryl esters, polymethacrylic acids, polyvinylamides, polyamides and polystyrene.

7. The covering layer matrix of claim 1, wherein the covering layer matrix further comprises a filling agent.

8. The covering layer matrix of claim 7, wherein the filling agent is SiO.sub.2, a silicate, or an aluminum-silicate.

9. The covering layer matrix of claim 1, wherein the strongly light-scattering agent has a refractive index of at least 2.

10. The covering layer matrix of claim 1, wherein the strongly light-scattering agent is a metal oxide, including mixed transition metal oxides.

11. The covering layer matrix of claim 1, wherein the strongly light-scattering agent is TiO.sub.2.

12. The covering layer matrix of claim 11, wherein the strongly light-scattering agent has an average particle diameter of 0.2 μm to 0.8 μm.

13. The covering layer matrix of claim 1, wherein the covering layer matrix is adapted for use as an illumination layer.

14. The covering layer matrix of claim 1, wherein the up-converting phosphor particles are coated with one or more inert and/or hydrophilic coatings.

15. A two- or more layer test element comprising a covering layer matrix according to claim 1 and an analyte detector matrix.

16. The test element of claim 15, wherein the analyte detector matrix is the first layer and the covering layer matrix comprising the up-converting phosphor particles is the second layer.

17. The test element of claim 15, wherein the test element is adapted for a measurement setup in which excitation irradiation is supplied from a sample application side of the test element, and wherein detection of emission is performed on the opposite side of the test element.

18. The test element of claim 15, wherein the test element is a test strip, a test tape, or a test disc.

19. The test element according to claim 15, wherein the test element is a glucose test element.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) Further optional features and embodiments of the invention will be disclosed in more detail in the subsequent description of preferred embodiments, preferably in conjunction with the dependent claims. Therein, the respective optional features may be realized in an isolated fashion as well as in any arbitrary feasible combination, as the skilled person will realize. The scope of the invention is not restricted by the preferred embodiments. The embodiments are schematically depicted in the Figures. Therein, identical reference numbers in these Figures refer to identical or functionally comparable elements.

(2) In the Figures:

(3) FIG. 1 shows a schematic cross-sectional view of a test element for use in the test system according to FIG. 1.

(4) FIG. 2 shows a cross-sectional view of an exemplary embodiment of a test system and a test device according to the present invention.

(5) FIG. 3 shows spectra of the IR laser, of UCP excitation and emission and of the re-emission of a standard test strip according to the prior art comprising a PMo dye (Phosphor molybdic acid) in the presence and in the absence of glucose. 168: UCP excitation, from the documentation of the manufacturer (Honeywell Product Data Sheet 53101 Lumilux® Green UC2); 166: IR-laser spectrum; 162: UCP emission, measured from the test strip; 160: light reflected from a conventional teststrip in the presence of glucose; 164: light reflected from a conventional teststrip in the absence of glucose. Y-Axis: RE.sub.rel: relative re-emission of the PMo dye (%) and EI: Laser and UCP emission intensity (arbitrary units, AU). X-Axis: λ: wavelength (nm).

(6) FIG. 4 shows kinetic curves of test strips in the presence of various amounts of glucose portrayed as relative signals for UCP-based measurement (upper panel) and re-emission of a standard PMo dye (lower panel) Glucose concentrations are indicated. Y-Axes: UCP.sub.rel: relative UCP-Signal (%) and RE.sub.rel: relative re-emission (%); X-Axes: t: Timepoints (s).

(7) FIG. 5 shows kinetic curves of test strips lacking UCPs in the presence of various amounts of glucose portrayed as relative signals for UCP-based measurement. Y-Axis: UCP.sub.rel: relative UCP-Signal (arbitrary units) and RE.sub.rel: relative RE-Emission (%); X-Axes: t: Timepoints (s).

(8) FIG. 6 shows standard curves for UCP-based and re-emission based measurements and their corresponding linear regression lines. R.sup.2 values are 0.96 for UCP-based measurement and 0.997 for re-emission based measurement. Y-Axis: I.sub.rel: mean relative signal (%), endpoint was at <2% slope; and CV %: coefficient of variation. X-Axis: c: glucose concentration (mg/dl).

(9) FIG. 7 shows spectra of the IR laser, of UV-UCP excitation and emission, and cNADH absorption and emission. 168: UCP excitation; 162: UCP emission; 170: cNADH absorption; 172: cNADH emission. X-Axis: λ: wavelength (nm). Y-Axis: A: Absorption, E: Excitation (both: arbitrary units).

(10) FIG. 8 relative signal intensities at 365 and 800 nm of UV-UCPs in assay mixtures comprising various amounts of glucose. X-Axis: c: glucose concentration (mM); Y-Axis: I.sub.rel: relative intensity (=signal after addition of glucose/signal before addition of glucose).

(11) FIG. 9 correlation between the signals at 365 nm and at 800 nm on a glucose test strip comprising UV-UCP before addition of glucose.

(12) FIG. 10 relative normalized signal intensities detectable from a UV-UCP test strip in the presence of various amounts of glucose. X-Axis: n: amount of glucose per test strip (μmol); Y-Axis: I.sub.rel,n: relative normalized signal intensity (=(signal at 365 nm after addition of glucose/signal at 800 nm after addition of glucose)/(signal at 365 nm before addition of glucose/signal at 800 nm before addition of glucose).

(13) FIG. 11 relative signal intensities detectable from a UV-UCP test strip (graph UCP) in the presence of various amounts of glucose dissolved in D.sub.2O, compared to conventional cNAD test strips (graph “reflectance”). X-Axis: c: glucose concentration in the sample applied to the test strip; Y-Axis: I.sub.rel: relative signal intensity.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(14) In FIG. 1, a cross-sectional view of an exemplary embodiment of a test element 120 is depicted. In this exemplary embodiment, the test element 120 is designed as a test strip. However, additionally or alternatively, other types of test elements 120 may be used, such as test tapes and/or test discs.

(15) The test element 120, as outlined above, comprises at least one test field 128 and at least one capillary element 126. The capillary element 126 is adapted to guide the sample 122 of the body fluid across the test field 128 in a flow direction 146. Thus, the capillary element 126 may suck the sample 122 over the test field 128 by capillary forces. For improving the capillary forces, the test element 120 may further comprise one or more venting openings 148.

(16) The test field 128 comprises at least one detection layer 150 comprising the at least one detector matrix 130. The test field 128 may further comprise one or more additional layers, such as at least one separation layer 152 covering the detection layer 150 on the side facing the capillary element 126. The separation layer 152 may comprise one or more pigments, preferably inorganic pigments, such as an inorganic oxide, which may provide a white optical background for optical measurement. Further, the separation layer 152 may be adapted for separating off at least one particular component contained in the body fluid.

(17) The test element 120 comprises at least one detection window in a substrate 156, through which a change of optical properties in the test field 128 may be detected by using the detector 132. It shall be noted that, in the embodiment depicted in FIG. 2, an optical test element 120 is depicted, in which the detector matrix 130 is adapted to change at least one optical property in the presence of the analyte to be detected.

(18) In FIG. 2, a cross-sectional view of an embodiment of a test device 112 and a test system 114 according to the present invention is depicted. The test device 112, preferably, is embodied as a hand-held device. The test device 112 preferably comprises a casing 116, which may have a volume of less than 1000 cm.sup.3, preferably of less than 500 cm.sup.3, in order to be carried by a person. The test device 112 comprises a receptacle 118 for receiving a test element 120, which, besides the test device 112, forms a component of the test system 114. The receptacle is adapted to locate the test element 120 in at least one application position in which a sample 122 of the body fluid is applicable to the test element 120, such as to an application opening 124 of a capillary element 126, which will be explained in further detail below. The test element 120 comprises at least one test field 128 having at least one detector matrix 130 adapted to change at least one optical property in the presence of an analyte to be detected by the test system 114, such as glucose.

(19) The test device 112 further comprises a detector 132 which, in this specific embodiment, comprises at least one light source 134 for illuminating the test field 128 and at least one optically sensitive element 136 adapted to measure detection light emitted and/or transmitted and/or reflected from the test field 128.

(20) The test device 112 further comprises at least one evaluation unit 138 which is adapted to determine the concentration of the analyte by using an evaluation algorithm. The evaluation unit 138 preferably may be or may comprise at least one data processing device, such as at least one computer and/or at least one application-specific integrated circuit. As an example, the evaluation unit 138 may comprise a microcomputer. Further, the evaluation unit 138 may comprise one or more further elements, such as at least one data storage device and/or other components. The evaluation unit 138 is unidirectionally or bidirectionally connected to the detector 132, such as for receiving measurement values from the detector 132. Further, the evaluation unit 138 may be adapted to control the overall functionality of the test device 112, such as for controlling the measurement process performed by the detector 132.

(21) The test device 112 may further comprise one or more human-machine-interfaces, such as at least one display 140 and/or at least one control element 142, such as at least one push button. The elements 140, 142 may also be connected to the evaluation unit 138. The test device 112 may further comprise one or more additional sensors for detecting one or more ambient parameters, such as one or more temperature sensors 145 adapted for determining an ambient temperature. As outlined above, these one or more ambient parameters may be used by the evaluation unit 138 for choosing an appropriate algorithm. The test device 112 may further comprise at least one electronic interface 144, for unidirectional and/or bidirectional exchange of data and/or commands with one or more external devices, such as a wireless and/or a wire-based interface.

(22) In the following, several measurements will be shown for demonstrating that detector matrices comprising UCP can be used to determine the concentration of an analyte, in particular glucose, in a sample.

EXAMPLES

I. UCPs

Example 1: Materials and Methods

(23) Commercially available UCP particles (Honeywell Lumilux Green UC 2, Honeywell Specialty Chemicals Seelze GmbH) were mixed with polyacrylic acid (poly(acrylic acid, sodium salt), Sigma-Aldrich) and then embedded in to the bottom chemical layer by mixing them with the standard coating mixture as described below. These particles were composed of Y.sub.2O.sub.2S:Yb,Er and were excited with an infrared radiation (980 nm). The test strips were then used to investigate the correlation between the detected UCP emission as a function of glucose concentration under infrared laser excitation.

Example 2: Coating of Test Strips

(24) The coating mixture of the chemical layers was as follows (components are in aqueous solution):

(25) Bottom layer: 0.99 g/m.sup.2 L-glycerol-3-phosphate-di-sodium; 1.89 g/m.sup.2 HCl; 0.38 g/m.sup.2 Keltrol F; 0.070 g/m.sup.2 TEACl; 0.19 g/m.sup.2 Mega 8; 0,025 g/m.sup.2 Geropon T77; 0.98 g/m.sup.2 PVP 2500; 5.8 g/m.sup.2 Transpafill; 11.1 g/m.sup.2 Propiofan; 0.11 g/m.sup.2 N,N-bis-hydroxyethyl-p-nitrosoanilin; 0.33 g/m.sup.2 PMo; 0.0037 g/m.sup.2 PQQ disodium salt; 0.27 g/m.sup.2 mut Q-GDH2; 0.80 g/m.sup.2 RbOH; 0.020 g/m.sup.2 CaCl.sub.2*2 H.sub.2O; 0.020 g/m.sup.2 K.sub.3[Fe(CN).sub.6]; 0,083 g/m.sup.2 NaOH; 1.0 g/m.sup.2 2-methyl-2-butanol; 8.15 g/m.sup.2 UCP; 0.40 g/m.sup.2 PAA. pH 6.85.

(26) Top layer: 1.4 g/m.sup.2 Gantrez S97; 0.29 g/m.sup.2 NaOH; 0.55 g/m.sup.2 TEACl; 0.33 g/m.sup.2 Mega 8; 0,090 g/m.sup.2 Geropon T77; 1.8 g/m.sup.2 PVP2500; 1.9 g/m.sup.2 silicic acid FK320DS; 18 g/m.sup.2 TiO.sub.2; 11 g/m.sup.2 Propiofan; 0.080 g/m.sup.2 BM 31.1008; 2.3 g/m.sup.2 PMo; 0.050 g/m.sup.2 CaCl.sub.2*2 H.sub.2O; 0.082 g/m.sup.2 LiOH; 1.0 g/m.sup.2 2-methyl-2-butanol. pH 6.75.

(27) Coating was performed on Pocalon N343 EM (125 μm thick, Lonza, Basel, Germany). Bottom layer coating thickness was 113 μm and top layer 54 μm.

Example 3: Measurements

(28) The emission of the UCP was monitored with a J&M TIDAS S DAD—spectrometer by recording the detected spectra with 200 ms integration time and 250 ms datapoint interval. The IR excitation source was a 100 mW continuous wave diode laser system (LRD-0980-PFR, Laserglow Technologies, Toronto, Canada) that was attenuated down to 1.6 mW. After the excitation and recording of the spectra were started, a 5 μl sample of glucose solution was introduced to the test strip.

(29) From the recorded spectra the emission intensity from 650-690 nm in different timepoints was integrated and each of the values was compared to the initial one to obtain the relative intensity in different timepoints. These values were then used to determine the point on the kinetic curve, where the slope is <2%/s, that was used as a reaction endpoint for the glucose concentration in question. The average values of these endpoints (5 replicates) were then plotted as a function of glucose concentration. The same test strips were also used for a reference measurement that was performed with the current technique by observing the relative amount of re-emitted light at 660 nm.

Example 4: Spectra

(30) The spectra of the IR laser, UCP emission and the absorption of the PMo dye are presented in FIG. 3. The 980 nm laser band is used to excite the UCP particles in the test strips that emit around 550 and 660 nm. The PMo dye has a broad absorption spectrum overlapping the UCP emission but also with the laser line.

Example 5: Results

(31) Kinetic curves obtained are displayed in FIG. 4 for the UCP (FIG. 4A) and re-emission based measurement (FIG. 4B). Key findings are an increased dynamic signal range and an increase in the relative signal with low glucose concentrations in the UCP signals in comparison to re-emission measurement.

(32) Test strips that did not contain any UCP did not produce any detectable signal under IR excitation. The kinetic data from these test strips show that there is also no change in the relative signal over time as glucose sample is applied (FIG. 5).

(33) The code curves obtained from UCP-based and re-emission measurements are presented in FIG. 6. As can be derived from FIG. 6, the UCP-based measurement has much wider relative signal range as compared to the traditional re-emission based measurement. Specifically, the relative signal difference between values measured at 7.5 and at 500 mg/dl glucose was 98.8% for the UCP-based measurement, but only 66.3% for the traditional re-emission based measurement.

II. UV-UCPs

Example 6: UV-Emitting UCPs in Glucose Measurement

(34) FIG. 7 shows the emission spectrum of UCP particles consisting of Na/KYF.sub.4:Yb, Tm (20 mol % K.sup.+) and shows a strong emission around 360 nm after laser excitation at 980 nm. This UV-emission is conveniently located in the same region where the indicators and redox cofactors NADH and cNADH have their absorption maxima. There are also emission lines in the 800-900 nm region that are not affected by the indicator absorption or emission, and can therefore be used as an internal control to compensate fluctuations caused by the excitation source.

(35) A glucose assay was conducted using the above-mentioned UV-UCP-particles together with NAD or cNAD and glucose dehydrogenase enzyme in a buffered aqueous solution on microtitre wells. Each well contained 50 ng of UCP, 1.88 μmol of indicator (NAD or cNAD) and 1 μg enzyme in 200 μl volume. The UV emission from the UCPs was measured in the beginning at two wavelengths (365 and 800 nm UCP emission) to acquire the zero-level signal. After this, a 10 μl sample of glucose was added, incubated for 15 min and measured again. The results were then calculated by comparing the emission intensities in each sample concentration to the respective zero-signal intensity. FIG. 8 shows the relative signals calculated for each of the glucose concentrations.

(36) As FIG. 8 indicates, the emission at 365 nm of the UCP decreases as the indicator is reduced due to the addition of glucose, whereas the emission at 800 nm remains unaffected. cNAD was used in the same concentration as NAD; since cNAD has a different equilibrium in the reaction, the decrease in UCP emission is less pronounced.

Example 7: UV-UCP in Glucose Test Strips

(37) For these experiments, the same UV-UCP as in Example 6 were used in a coating mixture having the composition indicated below. These particles are quenched by water, such that in the presence of water, a part of the emission below 800 nm is lost. In order to show that the basic principle is working, water was removed from the strips by drying and then the final signal was measured.

(38) The coating mixture of the chemical layers was as follows (components are in aqueous solution):

(39) Bottom layer: 2.5 g/m.sup.2 Sipernat FK 320DS; 0.20 g/m.sup.2 poly(acrylic acid, sodium salt); 0.38 g/m.sup.2 Gantrez S97; 1.0 g/m.sup.2 Propiofan 70D; 0.040 g/m.sup.2 Geropon 177; 0.26 g/m.sup.2 Na/KPO.sub.4; 1.60 g/m.sup.2 cNAD-Na and 0.80 g/m.sup.2 GlucDH2. The pH of the coating mixture adjusted to 7.5.

(40) Top layer: 16.5 g/m.sup.2 ZrO2 TZ-3YS; 0.35 g/m.sup.2 poly(acrylic acid, sodium salt); 1.30 g/m.sup.2 Gantrez S97; 2.17 g/m.sup.2 Propiofan 70D; 6 g/m.sup.2 UCP and 0.04 g/m.sup.2 Geropon. pH=7.5.

(41) Coating was performed on Bayfol CR210 (125 μm thick, Bayer MaterialScience AG, Germany). Bottom layer coating thickness was 50 μm and top layer 100 μm.

(42) The UV-UCPs were coated in the upper layer of cNAD test strips. The emission of UCPs without glucose was first measured under an infrared laser excitation with a power of 100 mW, providing proof that the emissions at 365 nm and at 800 nm are linearly correlated (FIG. 9). After this, 5 μl of a glucose sample were introduced onto the strips, with glucose concentrations varying from 0 to 800 mg/ml (0-19.4 μmol of glucose per strip) with three replicates for each concentration. The strips were then dried in 50° C. for 20 minutes in order to remove water from the strips. After drying, the strips were measured as described above.

(43) The UV-emission was first normalized calculating the ratio with the emission measured at 800 nm. The normalized signals from before and after the addition of glucose and drying were then compared to calculate the relative signal. This was finally plotted as a function of the amount of glucose introduced to the test strip (FIG. 10), showing that the detected UCP emission is inversely correlated with the amount of glucose introduced to the test strip.

Example 8: UV-UCP in Glucose Test Strips with D.SUB.2.O

(44) For these experiments, the same test strips as in Example 7 were used. As another way of avoiding quenching by water, D.sub.2O was used as a solvent for dissolving glucose. Measurements were performed as described in Example 7, however, without drying the test strips after application of glucose solutions. As shown in FIG. 11, correlation between the relative signal measured and the amount of glucose applied in UV-UCP test strips (“UCP”) is as good as in conventional measurement of blood glucose with cNAD test strips (“reflectance”).

(45) The work leading to this invention has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 264772 (CHEBANA).

LIST OF REFERENCE NUMBERS

(46) 112 test device 114 test system 116 casing 118 receptacle 120 test element 122 sample 124 application opening 126 capillary element 128 test field 130 detector matrix 132 detector 134 light source 136 optically sensitive element 138 evaluation unit 140 display 142 control element 144 interface 145 temperature sensor 146 flow direction 148 venting opening 150 detection layer 152 separation layer 154 detection window 156 carrier element (support) 160 light reflected from a conventional teststrip in the presence of glucose 162 UCP emission 164 light reflected from a conventional teststrip in the absence of glucose 166 IR-laser spectrum 168 UCP excitation 170 cNADH absorption 172 cNADH emission