Means for the quantitative determination of sodium concentration and creatinine concentration

Abstract

The present invention relates to a single-use test-strip for the quantitative determination of sodium concentration and creatinine concentration and for the subsequent determination of their ratio, and to a non-invasive point-of-care (POC) device for detecting sodium depletion and/or sodium overload in a patient's body. Furthermore, the present invention relates to a method for simultaneously and quantitatively determining sodium concentration and creatinine concentration in a patient's urine sample and to a method of detecting sodium depletion and/or sodium overload in a patient's body.

Claims

1. A single-use test-strip for the quantitative determination of sodium concentration and creatinine concentration in a patient's urine sample, said test-strip comprising: a substrate which either is electrically insulating or which has an electrically insulating layer applied thereon, an electrode assembly applied on said substrate or on said electrically insulating layer, if present, said electrode assembly comprising at least one sodium-selective working electrode; one creatinine-selective working electrode; either one joint reference electrode for both said sodium-selective working electrode and said creatinine-selective working electrode, or a reference electrode for said sodium-selective working electrode and a separate reference electrode for said creatinine-selective working electrode; one or two neutral electrodes for measuring and eliminating interferences, wherein the one or two neutral electrodes comprise a membrane comprising a polymeric matrix without any sodium-selective carrier and without any creatinine-selective carrier; and an interface for electrically connecting said electrode assembly to a readout-meter device.

2. The single-use test-strip according to claim 1, wherein said working electrodes, said reference electrode(s) and said neutral electrode(s) have been applied on said substrate or on said electrically insulating layer, if present, by a suitable deposition technique, selected from printing, sputtering, evaporating, electro-less plating, affixation, gluing and lithography, thus forming an electrode assembly on said substrate or on said electrically insulating layer, and wherein said sodium-selective working electrode comprises a sodium-selective membrane, and said creatinine-selective working electrode comprises a creatinine-selective membrane, and wherein said neutral electrode(s) comprises (comprise) a membrane that is not sodium-selective and not creatinine-selective.

3. The single-use test-strip according to claim 1, wherein said substrate is made of a material selected from plastic, ceramic, alumina, paper, cardboard, rubber, textile, carbon-based polymers, fluoropolymers, silicon-based substrates, silicon based polymers, semiconducting materials, dielectric materials, inorganic dielectric materials, and wherein said electrically insulating layer, if present, is made of a dielectric material, wherein, if said electrically insulating layer is present on said substrate, said electrode assembly is located on said electrically insulating layer.

4. The single-use test-strip according to claim 2, wherein said sodium-selective working electrode comprises a sodium-selective membrane that comprises a sodium-selective carrier in a polymer matrix, and said creatinine-selective membrane comprises a creatinine-selective carrier in a polymer matrix.

5. The single-use test-strip according to claim 1, wherein each of said electrodes in said electrode assembly has an electrical lead, respectively, wherein said electrical lead connects said electrode with said interface for electrically connecting said electrode assembly to a readout-meter device.

6. The single-use test-strip according to claim 1, wherein said joint reference electrode has a surface larger than the surface of each of said working electrodes, or each of said separate reference electrodes has a surface larger than the surface of each of said working electrodes.

7. A non-invasive point-of-care (POC) device for detecting sodium depletion and/or sodium overload in a patient's body, said POC device comprising: a readout-meter device for the quantitative and selective measurement of sodium and creatinine concentrations in a urine sample and for determining a ratio of sodium-to-creatinine, said readout-meter device comprising a receiving module for receiving an interface of a single-use test-strip according to claim 1 and for establishing electrical contact between said readout-meter device and an electrode assembly of said single-use test-strip, thus allowing the detection and transmission of electrical signal(s) from said single-use test-strip to said readout-meter device, wherein said receiving module has electrical connectors for separately contacting each electrode via said interface of said test-strip a multichannel amplifier, having high input resistance, for amplifying electrical signal(s) transmitted from said single-use test-strip a controller including an analog/digital converter and a storage memory, for converting electrical signals received from said single-use test-strip into sodium concentration measurement(s) and creatinine concentration measurement(s) and for subsequently determining a ratio of sodium concentration to creatinine concentration based on said sodium concentration measurements and creatinine concentration measurements an output device for indicating concentration measurements and/or said ratio to a user, and a power supply.

8. The non-invasive point-of-care (POC) device according to claim 7, wherein said single-use test-strip is inserted into said receiving module of said readout-meter-device by way of said interface of said single-use test-strip, thus establishing electrical contact between said electrode assembly of said test-strip and said readout-meter device.

9. The non-invasive point-of-care (POC) device according to claim 7, wherein said device further comprises: a user-interface for operating said device, and/or a memory for storing a plurality of sodium and creatinine concentration measurements and calculated ratios of sodium concentration to creatinine concentration, and/or a connection interface, for transferring and/or exchanging data with an external computer or external network.

10. A method for quantitatively determining sodium concentration and creatinine concentration in a patient's urine sample, comprising the steps: a) providing a urine sample b) contacting a single-use test-strip according to claim 1 with said urine sample and allowing the electrode assembly of said test-strip to be wetted by and come into contact with said urine sample, optionally withdrawing the urine-wetted test-strip from said urine sample c) connecting said test-strip to a readout-meter device of a point-of-care (POC) device for detecting sodium depletion and/or sodium overload in a patient's body, said POC device comprising: a readout-meter device for the quantitative and selective measurement of sodium and creatinine concentrations in a urine sample and for determining a ratio of sodium-to-creatinine, said readout-meter device comprising a receiving module for receiving an interface of a single-use test-strip according to claim 1 and for establishing electrical contact between said readout-meter device and an electrode assembly of said single-use test-strip, thus allowing the detection and transmission of electrical signal(s) from said single-use test-strip to said readout-meter device, wherein said receiving module has electrical connectors for separately contacting each electrode via said interface of said test-strip a multichannel amplifier, having high input resistance, for amplifying electrical signal(s) transmitted from said single-use test-strip a controller including an analog/digital converter and a storage memory, for converting electrical signals received from said single-use test-strip into sodium concentration measurement(s) and creatinine concentration measurement(s) and for subsequently determining a ratio of sodium concentration to creatinine concentration based on said sodium concentration measurements and creatinine concentration measurements an output device for indicating concentration measurements and/or said ratio to a user, and a power supply, wherein said single-use test-strip is inserted into said receiving module of said readout-meter device, thus establishing electrical contact between said electrode assembly of said test-strip and said readout-meter device, wherein said connecting of said test strip to said readout-meter device of said POC device in step c) occurs either before or after step b), d) measuring sodium concentration and creatinine concentration in said urine sample, using said POC device assembled in step c).

11. A method of detecting sodium depletion and/or sodium overload in a patient's body, said method comprising the steps: performing the method according to claim 10 determining a ratio of sodium concentration to creatinine concentration using said point-of-care (POC) device detecting a sodium depletion, if a calculated ratio of sodium concentration to creatinine concentration in said urine sample is <8, and detecting a sodium overload, if a calculated ratio of sodium concentration to creatinine concentration is >50.

12. The method according to claim 11, wherein said sodium depletion is a sodium depletion in the plasma of a patient, or is a normonatremic sodium depletion, wherein, in such normonatremic sodium depletion, the sodium concentration in the plasma of a patient is in a normal healthy range, but the patient suffers from a depleted total body sodium pool.

13. The single-use test-strip, according to claim 2, wherein said deposition technique is screen printing or ink jet printing.

14. The single-use test-strip according to claim 3, wherein said substrate is made of a material selected from polypropylene, Teflon, glass, quartz, silicon nitride, silicon oxide, polydimethoxysiloxane, elemental silicon, polyimide, polycarbonate, polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyester, polyethylene terephthalate, polyurethane, polyvinylidene fluoride, and silicium dioxide.

Description

(1) The present invention is now further described by reference to the following figures, wherein

(2) FIG. 1 shows a schematic representation of an embodiment of a point-of-care (POC) device for detecting sodium depletion and/or sodium overload in a patient's body; EMF=electromotive force; uNa=sodium concentration in a urine sample; uCr=creatinine concentration in a urine sample; uNa/uCr=ratio sodium:creatinine concentration. 1=POC device, 2=test-strip, 3=readout-meter device, 4=electrode assembly, 5=interface for electrical connection, 6=sodium-selective electrode, 7=creatinine-selective electrode, 8=reference electrode, 9=electrical leads

(3) FIG. 2 shows top views of exemplary test strips with exemplary possible patterns of an electrode array and electrical leads applied on an insulating layer A) exemplary round shape of working electrodes+oval reference electrode 5=interface for electrical connection 6=sodium-selective electrode 7=creatinine-selective electrode 8=reference electrode 9=electrical leads B) exemplary square shape of working electrodes+rectangular reference electrode C) 9a)=exemplary contact paths at the end of electrical leads for contacting readout-meter device D) 10=neutral electrode for determining interferences

(4) FIG. 3 shows cross-sectional views of an exemplary analyte-selective electrode A) Without “inner contact layer” 11=substrate 12=insulating layer 13=conductive layer 14=analyte-selective membrane B) With “Inner contact layer” 11=substrate 12=insulating layer 13=conductive layer 14=analyte-selective membrane 15=optionally, “inner contact layer” (transducer)

(5) FIG. 4 shows an exemplary embodiment for the fabrication of an exemplary test-strip with an additional covering layer. In such exemplary fabrication method, the following steps are performed: Step 1) Provide substrate with insulating layer on top Step 2) Apply electrode assembly and electrical leads Step 3) Form analyte-selective electrodes Step 4) preferably, apply a suitable covering film, e.g. a plastic insulating material with openings for the electrodes 2a=test-strip with covering layer 4a=electrode assembly and electrical leads 5=interface for electrical connection 11a=substrate with insulating layer 15a=analyte-selective membrane solutions 16=covering film with openings for electrodes

(6) FIG. 5 shows the potentiometric response of fabricated test-strips (T1-T4) according to the present invention to different concentrations of sodium in aqueous solutions.

(7) FIG. 6 shows a comparison of sodium concentrations determined by classic conventional sodium-iron-selective electrodes (ISE), by flame photometry (i.e. atomic absorption spectroscopy, AAS), which is the reference method of the International Federation of Clinical Chemistry (IFCC), and by an exemplary sodium-selective test-strip according to the present invention.

(8) FIG. 7 shows a potentiometric response of exemplary fabricated test-strips in accordance with the present invention (C1-C5) to different creatinine concentrations in aqueous solutions.

(9) FIG. 8 shows a near-Nernst potentiometric response of exemplary fabricated test-strips in accordance with the present invention (T5-T7) to different sodium concentrations in a 0.5 M calcium chloride aqueous solutions

(10) FIG. 9 shows top view of an exemplary test strip with exemplary dimensions of the electrode array and substrate. WE1=a first working electrode; WE2=a second working electrode; RE=reference electrode.

(11) FIG. 10 shows an exemplary point-of-care (POC) device for detecting sodium depletion and/or sodium overload in a patient's body consisting of a test-strip and readout-meter displaying exemplary analysis results.

(12) Furthermore, the present invention is now further described by reference to the following examples which are given to illustrate, not to limit the present invention.

EXAMPLES

(13) Proof of Function for Sodium Specific Test-Strip

(14) Initial proof of function of Na-specific test membranes loaded on commercial Gwent® test-strips was obtained by successful potentiometric measurements of Na concentrations in fluids with predetermined Na concentrations and in native human urine samples proven linear (dynamic) range of the test strips for relevant concentrations of sodium in physiological fluids good agreement of Na-concentrations in human urine with the values measured by conventional methods including AAS and ISE proven near-Nernst potentiometric response for relevant concentrations of sodium in physiological fluids

(15) Initial proof of function of creatinine-specific test membranes loaded on commercial Gwent® test strips was obtained by successful potentiometric measurements of creatinine concentrations in fluids with predetermined creatinine concentrations and human urine

Example 1) Fabrication of Sodium-Selective Test Strips

(16) For proof-of-principle experiments, a commercially available 2-Electrode System (Gwent, UK, BE 2070921D1/007) was used. The system consists of a carbon working electrode (WE) with Ø=6 mm and a common (Ag/AgCl) counter/reference electrode (RE) that are screen printed onto a 12.0×26.5 mm plastic substrate.

(17) To realize a sodium selective electrode (Na-ISE), an ion selective membrane (Na-ISM) solution (30 μL) was casted on the area of the carbon WE. Then, the substrate was dried to remove solvent. The Na-ISM solution consisted of a mixture of 4.0 mg sodium ionophore X (4-tert-butylcalix[4]arenetetraacetic acid tetraethyl ester), 1.0 mg KTpClPB (potassium tetrakis(4-chlorophenyl)borate), 133 mg PVC (high molecular weight polyvinylchloride) and 266 mg o-NPOE (2-nitrophenyl octyl ether) in 3 mL tetrahydrofuran.

Example 2) Potentiometric Measurements with a Sodium Selective Test Strip

(18) Prior to measurement, for conditioning the Na-ISE electrode, the test strip was immersed overnight into a sodium chloride solution (1M aq. NaCl). The substrate with both electrodes was introduced into the sample solution and the potential difference (EMF) between modified WE (Na-ISE) and RE was measured with a simple digital voltmeter.

Example 3) Sensor Calibration by Measuring a Standard Sodium Solution Series

(19) Standard solutions with sodium concentrations of 1M, 10.sup.−1M, 10.sup.−2M, respectively, were prepared by dissolving sodium chloride (NaCl) in water. The EMF values were recorded and a calibration curve was set up by plotting the EMF values as a function of the minus logarithm of sodium concentrations. Four different test strips (T1-T4) were fabricated and tested. The results are summarized in Table 1.

(20) TABLE-US-00001 TABLE 1 EMF values for three different Na.sup.+ concentrations (1.0-0.01M) obtained by measurements with four different test strips (T1-T4) c(Na+) EMK EMK EMK EMK [M] [mV] T1 [mV] T2 [mV] T3 [mV] T4 1 −118 −114 −119 −119 0.1 −25 −23 −19 −21 0.01 60 64 65 63

(21) The data clearly show good reproducibility and—as shown in FIG. 5—a linear (dynamic) range between 0.01 and 1 M sodium solution. Thus, the linear range of the test strips covers medically relevant concentrations in physiological fluids, as in human urine where the normal range for Na concentrations lies between 0.02-0.25 M.

(22) Out of these data for each test strip a regression equation with the corresponding correlation coefficient R, was calculated (Table 2).

(23) TABLE-US-00002 TABLE 2 Overview of linear regression equations and correlation coefficients obtained for EMF measurements with four different sodium sensors (T1-T4) Sensor Linear regression equation R T1 y = 89.0 × −116.7 0.9987 T2 y = 89.0 × −113.3 0.9997 T3 y = 92.0 × −116.3 0.9950 T4 y = 91.0 × −116.7 0.9961

(24) Using these regression equations, the concentration of sodium in a sample can be determined from the measured EMF.

Example 4) Measurements of Native Human Urine Samples with Fabricated Sodium-Selective Test Strips and Comparison with the Results Obtained by Conventional Methods

(25) 17 different samples of native human urine (denoted with the numbers 1-17) were examined with the fabricated sodium sensor test strips. The samples were diluted 1:10 before measurement. As described above, each test strip was conditioned prior to measurement by immersion in a 1M NaCl solution. Afterwards, the test strip was dipped into the urine sample and the EMF measured by means of a potentiometer.

(26) Using the linear regression equation, the concentrations of sodium in the urine samples (Table 3) were determined as illustrated by following example: EMF of a sample measured with the T1 test strip: 27 mV Regression equation: y=89.0x−116.7 −Log [Na+]=(27+116.7)/(−89.0)=−1.6146 [Na+]=10.sup.−1.6146=0.0243

(27) Because of 1:10 dilution prior to measurement multiplying by 10.Math.0.243M=243 mM

(28) TABLE-US-00003 TABLE 3 Overview of EMFs and corresponding Na concentrations obtained from measurements using four different test strips (T1 − T4) ant the corresponding regression equations in Tab. 2. EMF [mV] c(Na+) [mM] T1 T2 T3 T4 T1 T2 T3 T4 1 27 40 30 30 243 189 264 244 2 30 42 36 37 225 180 227 205 3 47 52 47 53 147 139 172 136 4 60 61 61 66 104 110 121 98 5 51 55 49 52 131 129 164 140 6 96 99 90 91 41 41 59 52 7 70 76 69 75 80 75 99 78 8 47 51 51 52 145 143 156 140 9 127 130 134 136 18 19 19 17 10 95 102 102 105 42 38 43 37 11 36 36 36 42 193 210 226 180 12 43 43 42 45 161 175 195 167 13 60 61 58 66 104 110 131 98 14 149 152 152 158 10 10 12 10 15 158 156 159 165 8 9 10 8 16 79 78 81 77 64 71 73 74 17 78 79 75 82 65 69 85 66

(29) The same samples of human urine were analysed for their Na concentrations by conventional flame photometry (AAS) and classic Na-ISE (Table 4).

(30) TABLE-US-00004 TABLE 4 Sodium concentrations (mM) in urines from 17 children measured by fabricated Na− selectivetest-strips according to this invention, classic Na-ISE and flame photometry (AAS). c(Na+) [mM] c(Na+) [mM] c(Na+) [mM] Test strip classic Na-ISE AAS 1 221 215 188 2 197 188 158 3 140 131 99 4 101 74 71 5 132 136 112 6 44 16 50 7 77 46 55 8 138 130 166 9 17 8 12 10 38 21 41 11 193 177 171 12 166 169 162 13 105 103 92 14 10 7 10 15 9 6 11 16 68 52 70 17 70 60 97

(31) Sodium concentrations obtained by the four test-strip measurements were averaged and compared to the values determined by classic Na ISE and by flame photometry. As seen in FIG. 6 there is rather good agreement between the three methods. The differences between classic Na-ISE and flame photometry, both methods applied currently in clinical laboratories for sodium concentration determination, are in some cases even larger than between classic Na-ISE and the developed test strip.

Example 5) Fabrication of a Creatinine-Selective Test Strip

(32) For proof-of-principle experiments, a commercially available 2-Electrode System (Gwent, UK, BE 2070921D1/007) was used. The system consists of a carbon working electrode (WE) with Ø=6 mm and a common (Ag/AgCl) counter/reference electrode (RE) that are both screen printed onto a 12.0×26.5 mm plastic substrate.

(33) To realize a creatinine-selective electrode (Cr-SE), a selective membrane (Cr-SM) solution (30 μL) was casted on the area of the carbon WE. Then, the substrate was dried to remove solvent and form a selective membrane on the WE. The Cr-ISM solution consisted of a mixture of 1.8 mg Dibenzo-30-crown-10 (DB30C10), 1.8 mg potassium tetrakis (p-chlorophenyl)borate (PTp-ClPB), 65.5 mg o-nitrophenyl octyl ether (o-NPOE) and 30.9 mg PVC (high molecular weight polyvinylchloride) in 3 mL tetrahydrofurane.

Example 6) Potentiometric Measurements with a Creatinine-Selective Test Strip

(34) Prior to measurement, for conditioning the Cr-selective electrode, the test strip was immersed overnight into a 10.sup.−2 M protonated creatinine aqueous solution. The substrate with both electrodes was introduced into the sample solution and the potential difference (EMF) between modified WE (Na-ISE) and RE was measured with a simple digital voltmeter.

Example 7) Sensor Calibration by Measuring a Standard Creatinine Solution Series

(35) Standard aqueous solutions of protonated creatinine with the concentration of 1M, 10.sup.−1M, 10.sup.−2M, and 10.sup.−3M respectively, were prepared. The EMF values were recorded and a calibration curve was set up by plotting the EMF values as a function of the minus logarithm of creatinine concentrations. Five different test strips (C1-C5) were fabricated and tested. The results are summarized in Table 5.

(36) TABLE-US-00005 TABLE 5 EMF values for four different creatinine concentrations (1.0-0.001M) obtained by measurements with five different test strips (C1-C5) EMF EMF EMF EMF EMF c[Cr] [mV] [mV] [mV] [mV] [mV] M C1 C2 C3 C4 Cr5 10.sup.−3 −47 −47 −51 −49 −53 10.sup.−2 −137 −132 −138 −135 −132 10.sup.−1 −223 −212 −220 −213 −212 10.sup.0   −306 −301 −307 −305 −301

(37) The data clearly show good reproducibility and—as shown in FIG. 7—a linear (dynamic) range between 0.001 and 1 M creatinine solution. Thus, the linear range of the test strips covers medically relevant concentrations in physiological fluids, as in urine with values of 0.004-0.02 M.

(38) Out of these data for each test strip, a regression equation, listed in Table 6 with the correlation coefficient R, was calculated.

(39) TABLE-US-00006 TABLE 6 Overview of linear regression equations and correlation coefficients obtained from EMF measurements with five different creatinine sensors (C1-C5) Test strip Linear regression equation R C1 y = 86.3 × −307.7 0.9995 C2 y = 84.2 × −299.3 0.9994 C3 y = 85.0 × −306.5 0.9998 C4 y = 84.6 × −302.4 0.9986 C5 y = 82.4 × −298.1 0.9988

Example 8) Sensor Calibration with Stabilized Reference Electrode Potential Showing Near-Nernst Response in a Biologically Relevant Range

(40) The potential of the Ag/AgCl counter/reference electrode (RE) on the used commercial test-strip (Gwent, UK, BE 2070921D1/007) depends on the chloride ions concentration in the sample. To achieve a stable reference electrode potential, a saturating concentration of chloride ions are added to the standard solutions from which the sensor is calibrated.

(41) Therefore, sodium standard solutions with concentrations of 1M, 10.sup.−1M, 10.sup.−2M, 10.sup.−3M, respectively, were prepared by dissolving sodium chloride (NaCl) in a 0.5 M calcium chloride (CaCl.sub.2) aqueous solution. The sodium sensor was fabricated as described in Example 1 and the EMF values were recorded as described in Example 2. A calibration curve was set up by plotting the EMF values as a function of the minus logarithm of sodium concentrations. Three different test strips (T5-T7) were fabricated and tested. The results are summarized in Table 1.

(42) TABLE-US-00007 TABLE 7 EMF values for four different Na concentrations (1.0-0.001M) obtained by measurements with three different test strips (T5-T7) c(Na+) EMK EMK EMK [M] [mV] T5 [mV] T6 [mV] T7 1 324 320 332 0.1 272 270 275 0.01 217 216 223 0.001 157 158 161

(43) Out of these data for each test strip a regression equation with the corresponding correlation coefficient R, was calculated (Table 8).

(44) TABLE-US-00008 TABLE 8 Overview of linear regression equations and correlation coefficients obtained for EMF measurements with three different sodium sensors (T5-T7) Sensor Linear regression equation R T5 y = −55.6 × +325.9 0.9984 T6 y = −54.0 × +322.0 0.9983 T7 y = −56.5 × +332.5 0.9984

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(46) The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.