DISPOSABLE ELECTROCHEMICAL SENSING STRIPS AND ASSOCIATED METHODS

Abstract

Electrochemical sensing device (S) for measuring the content of ions in biological fluid samples (D) comprising two membrane half cells, a salt bridge (3) connecting them, means for bringing a biological fluid sample (D) in contact with the measuring cell, wherein the first and second membranes (11, 21) of the half-cells are selective to the same ions, the first volume (13) and second volume (23) adjacent to the membranes are filled with known concentrations (C1, C2) of the ions to which the membranes (11, 21) are selective, these known concentrations being different such that a voltage can be measured between the first electrode (12) and the second electrode (22) that allows calibrating the sensing device (S) and then measuring the ion-content of the sample. The invention also refers to a method using such sensing devices. The sensing devices are useful especially in the area of so-called home monitoring.

Claims

1. An electrochemical sensing device for measuring the content of ions in a biological fluid sample comprising: a first half cell provided with a first ion-selective electrode made of a first ion-selective membrane and a first conductive support, and a first volume in contact with the first ion-selective membrane; a second half cell provided with a second ion-selective electrode made of a second ion-selective membrane and a second conductive support, and a second volume in contact with the second ion-selective membrane; a salt bridge connecting the first volume and the second volume; and means for bringing a biological fluid sample in contact with the second volume; wherein: the salt bridge comprises a diffusion limiter, which allows opening the salt bridge when it is removed; wherein the first and second membranes are selective to the same ions; and the first volume and second volume are filled with aqueous solutions of known concentrations of the ions to which the membranes are selective, these known concentrations being different; such that, after opening the salt bridge by removal of the diffusion limiter, a voltage can be measured between the first conductive support and the second conductive support, said measured voltage thus allowing calibrating the electrochemical sensing device and then measuring the ion-content of the biological fluid sample.

2. The electrochemical sensing device according to claim 1, wherein the difference of ion concentration between the aqueous solutions of known concentrations comprises the range of concentrations to be measured.

3. The electrochemical sensing device according to claim 1, wherein the diffusion limiter is mechanical, thermal or chemical.

4. The electrochemical sensing device according to claim 1, wherein the means for bringing a biological fluid sample in contact with the second volume comprise a sample inlet, which connects the outside with the second volume.

5. The electrochemical sensing device according to claim 4, which comprises a gas diffusion layer in the sample inlet, such that the sample must cross it to reach the second volume.

6. The electrochemical sensing device according to claim 1, wherein the ion selective membranes are made of a polymer with a plasticizer in which the compounds that selectively interact with the ions to be measured are dissolved or immobilized.

7. The electrochemical sensing device according to claim 1, wherein the first volume and second volumes and the salt bridge are filled with aqueous solutions of known concentrations embedded in a hydrogel.

8. The electrochemical sensing device according to claim 1, wherein the conductive supports are made of a conductive metal, composite conductive polymer filled with metallic nanoparticles, graphite, carbon nanotubes, graphene, conductive polymer or a conductive ink.

9. The electrochemical sensing device according to claim 1, which is formed by the following layers: a bottom enclosing layer; the conductive supports and measuring terminals; a first intermediate enclosing layer provided with through holes for housing the membranes, and cuts for accessing the measuring terminals; a second intermediate enclosing layer comprising a through hole that defines two housings for the first volume and the second volume and a channel, which connects the housings and that houses the salt bridge and cuts for accessing the measuring terminals; and a top enclosing layer comprising a through hole for depositing the biological fluid sample and cuts for accessing the measuring terminals.

10. The electrochemical sensing device for measuring the content of ions in a biological fluid sample comprising: a first half cell provided with a first ion-selective electrode made of a first ion-selective membrane and a first conductive support; a second half cell provided with a second ion-selective electrode made of a second ion-selective membrane a second conductive support; a salt bridge connecting first ion-selective membrane and the second ion-selective membrane; means for bringing a biological fluid sample in contact with the salt bridge in the vicinity of the second ion-selective membrane; wherein the first and second membranes are selective to the same ions, and which comprises a first calibration volume with a known concentration of the ions to which the membranes are selective, the calibration volume being placed in contact with the salt bridge in the vicinity of the first ion-selective membrane, the salt bridge being filled with a known concentration of the ions to which the membranes are selective, and which comprises a diffusion limiter between the calibration volume and the salt bridge, such that a voltage can be measured between the first electrode and the second electrode that allows calibrating the electrochemical sensing device when the diffusion limiter is removed, and then measuring the ion-content of the biological fluid sample.

11. The electrochemical sensing device according to claim 10, which comprises a second calibration volume with a known concentration of the ions to which the membranes are selective, the second calibration volume being placed in contact with the salt bridge in the vicinity of the second ion-selective membrane.

12. The electrochemical sensing device according to claim 10 configured as a strip.

13. A method for measuring the content of ions in a biological fluid sample by using the electrochemical sensing device according to claim 10, which after removing the diffusion limiter(s) comprises the steps of: a) measuring the voltage (V.sub.CAL) between the first half cell and the second half cell for calibrating the device in order to determine the calibration equation; b) placing a biological fluid sample in contact with the second volume; c) measuring the voltage (V.sub.SAMP) between first half cell and the second half cell after a sufficient time has lapsed for the ions of the fluid sample to diffuse into the second ion-selective membrane such that a stable measure can be taken; and d) determining the ion concentration in the biological fluid sample.

14. The method according to claim 13, wherein the step of removing the diffusion limiter(s) is carried out while coupling the electrochemical sensing device to a reading terminal.

15. An electrochemical sensing device for measuring the content of ions in biological fluid samples comprising: a first half cell provided with a first ion-selective electrode made of a first ion-selective membrane and a first conductive support, and a first volume in contact with the first ion-selective membrane; a second half cell provided with a second ion-selective electrode made of a second ion-selective membrane and a second conductive support, and a second volume in contact with the second ion-selective membrane; a salt bridge connecting the first volume and the second volume; and means for bringing a biological fluid sample in contact with the second volume, wherein the salt bridge comprises a diffusion limiter, which allows opening the salt bridge when it is removed, and wherein the first volume, the second volume and the salt bridge are a hydrogel.

16. The electrochemical sensing device according to claim 1 configured as a strip.

17. A method for measuring the content of ions in a biological fluid sample by using the electrochemical sensing device according to claim 1, which after removing the diffusion limiter(s) comprises the steps of: a) measuring the voltage (V.sub.CAL) between the first half cell and the second half cell for calibrating the device in order to determine the calibration equation; b) placing a biological fluid sample in contact with the second volume; c) measuring the voltage (V.sub.SAMP) between first half cell and the second half cell after a sufficient time has lapsed for the ions of the fluid sample to diffuse into the second ion-selective membrane such that a stable measure can be taken; and d) determining the ion concentration in the biological fluid sample.

18. The method according to claim 17, wherein the step of removing the diffusion limiter(s) is carried out while coupling the electrochemical sensing device to a reading terminal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0117] To complete the description and in order to provide for a better understanding of the invention, a set of drawings is provided. Said drawings form an integral part of the description and illustrate an embodiment of the invention, which should not be interpreted as restricting the scope of the invention, but just as an example of how the invention can be carried out. The drawings comprise the following figures:

[0118] FIG. 1 is a schematic cross-section of an already existing sensing device.

[0119] FIG. 2 is a schematic cross-section of a sensing device according to an embodiment of the invention, wherein the salt bridge is already composed of volumes with different ion concentrations.

[0120] FIG. 3 is a schematic cross-section of a sensing device according to an embodiment of the invention, which comprises a gas membrane, specially adapted to measure the NH.sub.4.sup.+ (Ammonium ion) content.

[0121] FIG. 4 is a schematic cross-section of a sensing device according to another embodiment of the invention, wherein calibration volumes can be brought in contact with the ends of the salt bridge.

[0122] FIG. 5 is an exploded perspective view of an embodiment of the invention based in a layered design.

[0123] FIG. 6 is analogous to FIG. 5, but it shows an embodiment provided with a gas membrane.

[0124] FIG. 7 shows a sequence of diffusion of ions through a salt bridge.

[0125] FIG. 8 shows a sequence of diffusion of ions through a tortuous salt bridge, which slows down the diffusion.

[0126] FIG. 9 shows an experimental device used in the calibration experiment.

[0127] FIG. 10 shows the experimental arrangement used for demonstrating the viability of the inventive sensor.

[0128] FIG. 11 is a graph of the experimental data.

[0129] FIG. 12 Time evolution graph of the response of a sensor using hydrogel as a barrier to delay signal interfering substances.

DESCRIPTION OF WAYS OF CARRYING OUT THE INVENTION

[0130] FIG. 2 depicts a preferred embodiment of the electrochemical sensing device S for measuring the content of ions in biological fluid samples D, which comprises two half-cells 1, 2, one of which will play the role of the reference half-cell, and the other of which will play the role of the measuring or indicator half-cell.

[0131] The first half cell comprises a first ion-selective electrode 1 in turn made of a first ion-selective membrane 11 and a first conductive support 12 (both components enclosed in a dashed line rectangle), and a first volume 13 in contact with the first ion-selective membrane 11.

[0132] Accordingly, the second half cell comprises a second ion-selective electrode 2 made of a second ion-selective membrane 21 and a second conductive support 22, and a second volume 23 in contact with the second ion-selective membrane 21.

[0133] In all the embodiments, a salt bridge 3 connects the first volume 13 and the second volume 23, thus closing the sensing circuit.

[0134] The device is completed with means for bringing a biological fluid sample D in contact with the second volume 23, which in this case is a receptacle connected with the second volume through a sample inlet M.

[0135] According to the invention, the first and second membranes 11, 21 are selective to the same ions and the first volume 13 and second volume 23 are filled with known concentrations C1, C2 of the ions to which the membranes 11, 21 are selective, these known concentrations being different.

[0136] In this way, it is possible to measure a voltage between the first electrode 12 and the second electrode 22 that allows calibrating the electrochemical sensing device S prior to measuring the ion-content of the biological fluid sample D.

[0137] The device works as shown in FIGS. 7 and 8. The details of the materials forming the device are specified below in relation to FIGS. 5 and 6.

[0138] At a time t=0 s the drop D is deposited on the inlet M. First, the analyte diffuse through the volume 23 and reaches the membrane 21. Then the measurements start. In the meanwhile, the analyte diffuses through the salt bridge, and while the analyte has not reached the volume 13, the measurements are taken. This allows the concentrations to stabilize and the reading terminal, where the device is inserted, can identify the plateaus and then, determine the concentrations. The analysis time usually lasts no longer than 5 min, so it is ensured that the ions will never reach volume 13.

[0139] The plateaus are the zones, where the voltage measured stabilizes in a voltage vs time graph.

[0140] In the right picture of FIG. 7 it is shown that after 15 min there is still time remaining for measuring, since the analyte front has only travelled 40% of the whole bridge.

[0141] As seen in FIG. 8, where the times are indicated, a diffusion limiter based on a labyrinth allows slowing down the diffusion, and then better controlling the time windows for the measurements.

[0142] FIGS. 9 to 11 illustrate an experiment carried out to demonstrate the viability of the inventive device.

[0143] The experiment, as shown in FIG. 10, makes use of a recipient with a liquid sample, the concentration of which can be accurately controlled via the dispenser DI. The device S is connected to a potentiometer through the terminals E1 and E2.

[0144] FIG. 9 shows the device S used for the experiment, which is shown partially submerged in FIG. 10, and where the reference half-cell (Ref) has been covered, such that only the indicator or measurement electrode (Ind) will be accessible for the sample D, through the inlet M. In the experiment, the sample is part of the liquid contained in the recipient, and it is supposed to have the same concentration (i.e., the liquid is uniform).

[0145] Then, before the analyte has reached the reference membrane, by travelling through the salt bridge 3, the concentration of the analyte D is varied by adjusting it with the dispenser DI. A stepped voltage graphic (a graphic with plateaus corresponding to stable concentrations in the volume 23) is obtained where the voltages are collected: The latter represented in the graphic shown in FIG. 11.

[0146] The graphic of FIG. 11 shows the strong linear correlation between the logarithm of the concentration and the voltage, and thus the viability of the inventive sensor.

[0147] As an example, the calibration and measurement process is as follows:

[0148] First, a device comprising a diffusion limiter 4 (or 5) if conceived with calibration volumes apart) is manufactured in factory.

[0149] The Nernst equation applies to such device, namely:

E=a+b.Math.log[NH.sub.4.sup.+]

[0150] Where, theoretically for Ammonium ion b=59.2 mV/decade.

[0151] Then the device is packaged, stored and distributed. Then, days, weeks or months later, the user unpacks it and couples it to a reading terminal. When coupling, preferably, the diffusion limiter 4 (or 5) will be removed, and then the measuring circuit will be electrically closed. Then the measurements will start.

[0152] So, in factory the different concentrations of the ion to be measured are those as shown in FIG. 9, 10.sup.4 in the first volume 13 and 10.sup.5 M in the second volume 23.

[0153] Then, for example, the potentiometer indicates 55.8 mV, instead of the theoretical value mentioned above. This difference is only due to the different concentrations already prepared in factory. The user has still not dropped its sample.

[0154] Starting from this value, the pre-calibration model yields:

55.8=a+b.Math.log[10.sup.5](b.Math.log[10.sup.4]+a)

55.8=b.Math.(log[10.sup.5]log[10.sup.4])

b=55.8 mV

55.8=55.8.Math.log[10.sup.5]+a

a=223.2

And thus: E=55.8.Math.log[NH.sub.4.sup.+]+223.2

[0155] Then the user places a blood drop, and after 1 or 2 minutes, that is, when an equilibrium has been reached, the potentiometer could for instance indicate 38 mV.

[0156] Then, assuming that the hydrogel volume equals the volume of the sample (i.e. thanks to the volume of the dosing reservoir DR interposed between the inlet M and the volume 23):

[NH.sub.4.sup.+](C.sub.hydrogel+C.sub.sample)/2

[0157] The concentration of Ammonium ion measured is:

38=55.8.Math.log[NH.sub.4.sup.+]+223.2

[NH.sub.4.sup.+]=2.1.Math.10.sup.5 M

[0158] And then, the concentration of Ammonium ion in blood is:

[NH.sub.4.sup.+].sub.s=(10.sup.5+C.sub.sample)/2;

C.sub.sample=3.2.Math.10.sup.5M

[0159] According to the preferred embodiment of the invention, the salt bridge 3 comprises a diffusion limiter 4, which allows opening the salt bridge 3 when it is removed.

[0160] Therefore, the diffusion limiter 4 is a component that can delay the connection between the two ends of the bridge.

[0161] According to a practical implementation, the electrochemical sensing device is formed by the following layers, as shown in FIGS. 5 and 6: [0162] a bottom enclosing layer L1; In a practical embodiment, this layer (1 mm thick) could be COC (Cyclic Olefin Copolymer, a thermoplastic polymer). It has a 300 m bas-relief on the upper surface, where the conductive supports 12, 22 and the measuring terminals E1, E2 (which made up the conductive layer) are deposited, for example by screen-printing; [0163] a first intermediate enclosing layer L2 provided with through holes L21, L22 for housing the selective membranes 11, 21, and cuts L23, L24 for accessing the measuring terminals E1, E2. This layer is a 300 m COC layer; [0164] a second intermediate enclosing layer L3 comprising a through hole, which defines two housings L31, L32 for the first volume 13 and the second volume 23 and a microchannel L33, which connects the housings L31, L32 and that houses the salt bridge 3 and cuts L33, L34 for accessing the measuring terminals E1, E2. This layer (100 m thick) is also COC. For example, to determine ammonium ion content, 0.01 M TRIS buffer pH 7.4 with 10.sup.5 M ammonium ion is used for the salt bridge 3 (microchannel between housings) and in the L32 chamber (indicator electrode) whereas 0.01 M TRIS buffer pH 7.4 with 10.sup.4 M ammonium ion is used in the left chamber (reference electrode). [0165] a top enclosing layer L4 comprising a through hole L41 for depositing the biological fluid sample D and cuts L43, L44 for accessing the measuring terminals E1, E2. This layer is also formed by Cyclic Olefyn Copolymer. As seen, the sample inlet is divided in two holes, one for the introduction, by capillarity, of the sample and another to evacuate the air. There is another intermediate layer, which defines the dosing reservoir DR, so that when the sample is deposited on one of the holes, it enters the sample dosing reservoir DR and once there, that is, with the volume of sample to be analyzed defined, the analyte is diffused through the volume 23.

[0166] Obviously, any other plastic that meets the manufacturing needs of the device could be used.

[0167] As shown in FIGS. 3 and 6, the electrochemical sensing device can comprise a gas diffusion layer (membrane) MG between volumes 24 and 23, such that when the drop of sample enters through the inlet M and reaches the volume 24, the analyte to be determined reacts with the reagents present in volume 24 in order to form a gas compound, which is the only one able to diffuse through the MG and reach the volume 23, where it reacts with another reagents present in volume 23 to recover the original form, which can be measured by the electrode 2.

[0168] This procedure allows a highly selective measurement but can be applied only with analytes showing acid-base properties and in which one of these forms is a gas.

[0169] For example, considering the specific case of NH.sub.4.sup.+ (Ammonium ion) determination in biological samples with an ammonium selective electrode, when the sample is introduced through the inlet M, it reaches the volume 24, which contains a hydrogel with a basic pH (NaOH). Ammonia gas is formed from ammonium ion, which diffuses through the MG reaching the volume 23. Volume 23 is a hydrogel with a trishydroxymethyl aminomethane (TRIS) buffered solution set to pH 7.4, so that the ammonia gas is converted again to ammonium ion, which can be determined by the ammonium selective electrode 2.

[0170] Obviously, the only way to the inside of the device must be this access, that is the sample inlet, for the sample (blood) drop, and all the remaining volumes should be correctly encapsulated to guarantee stability and avoid biohazards.

[0171] The sensing device shown in FIG. 3 was used to measure ammonia amounts in blood. Ammonia is a relevant metabolite/biomarker in many diseases such as urea cycle disorders. The composition of the hydrogel used was 1% of agarose and 99% of a buffered dissolution of Tris 0.01M at pH 7.4 with 10 M NH.sub.4+, filling the volumes 23 and 13. The volume 24 was filled using a dissolution of NaOH 0.1 M. The response time was 4 min. Volumes of 1 L of standard dissolutions of Li+ of increasing concentration were added. The linear range obtained was 75-1564 mol/L NH.sub.4+(threshold to discriminate between a normal and a pathological ammonium concentration is 60 mol/L in adults and 75-100 mol/L in newborns, over 200 mol/L can cause severe consequences such as mental illness or dead) thus proving that the device is useful to determine toxic amounts of ammonia in blood samples.

[0172] This device was also used to measure urea amounts in blood. Urea is also a relevant metabolite/biomarker in many diseases such as urea cycle disorders. Urea measurement with the present sensing device can be performed by indirectly measuring Ammonium ion as result of the enzymatic conversion of urea into CO.sub.2 and Ammonia through urease enzyme. The composition of the hydrogel used was 1% of agarose and 99% of a buffered dissolution of Tris 0.01M at pH 7.4 with 100 M NH.sub.4+, filling the volumes 23 and 13. The volume 24 was filled using a dissolution of urease 0.66 mg/ml. The response time was 4 min. Volumes of 1 L of standard dissolutions of Li+ of increasing concentration were added. The linear range obtained was 325-2260 mol/L NH4+ being enough to determine its concentration in real blood samples (around 2000 mol/L).

[0173] According to another embodiment, shown in FIG. 4, the electrochemical sensing device S for measuring the content of ions in biological fluid samples D comprises: [0174] a first half cell provided with a first ion-selective electrode 1 made of a first ion-selective membrane 11 and a first conductive support 12; [0175] a second half cell provided with a second ion-selective electrode 2 made of a second ion-selective membrane 21 and a second conductive support 22; [0176] a salt bridge 3 connecting first ion-selective membrane 11 and the ion-selective membrane 21; [0177] means for bringing a biological fluid sample D in contact with the salt bridge 3 in the vicinity of the ion-selective membrane 21; where the first and second membranes 11, 21 are selective to the same ions, and which comprises a first calibration volume 13, which is filled with an aqueous solution with a known concentration C1 of the ions to which the membranes 11, 21 are selective, the calibration volume 13 being placed in contact with the salt bridge 3 in the vicinity of the first ion-selective membrane 11, the salt bridge 3 being filled with a known concentration C2 of the ions to which the membranes 11, 21 are selective, and which comprises a diffusion limiter 5 between the calibration volume 13 and the salt bridge 3, such that a voltage can be measured between the first electrode 12 and the second electrode 22 that allows calibrating the electrochemical sensing device S when the diffusion limiter 5 is removed, and then measuring the ion-content of the biological fluid sample D.

[0178] Optionally, the sensing device, based in calibration volumes different from the salt bridge, comprises a second calibration volume 23 with a known concentration C2 of the ions to which the membranes 11, 21 are selective, the second calibration volume 23 being placed in contact with the salt bridge 3 in the vicinity of the second ion-selective membrane 21.

[0179] Both variants of the inventive sensing device allow carrying out a method which comprises the steps of:

[0180] previously removing the diffusion limiter 4 of the salt bridge 3 in the case of the first embodiment, or breaking the seals 5 in the case of the second embodiment;

[0181] a) measuring the voltage V.sub.CAL between the first half cell 1 and the second half cell 2 for calibrating the device S in order to determine the calibration equation;

[0182] b) placing a biological fluid sample D in contact with the second volume 23;

[0183] c) measuring the voltage V.sub.SAMP between first half cell 1 and the second half cell 2 after a sufficient time has lapsed for the ions of the fluid sample D to diffuse into the second ion-selective membrane 21 such that a stable measure can be taken; and

[0184] d) determining the ion concentration in the biological fluid sample D.

[0185] The steps of removing the diffusion limiter 4 or breaking the seals 5, and step a) are carried out after coupling the electrochemical sensing device S to a reading terminal or reading platform, and preferably the removal of the diffusion limiter 4 or the seals 5 will be done automatically during this coupling step, such that the user will not have to worry about it. This can be done, for example, by displacing a lancet that will open the communication between the two sides of the salt bridge 3. The reading terminal can have a protrusion in its coupling slot that induces a force on the coupling end of the sensing device, where the lancet is placed. Another possibility is to place a thermal source in the slot of the reading terminal such that it heats a thermal diffusion limiter 4, for example a wax, and melts it, thus initiating the calibration process.

[0186] The invention also relates to an electrochemical sensing device S for measuring the content of ions in biological fluid samples D comprising: [0187] a first half cell provided with a first ion-selective electrode 1 made of a first ion-selective membrane 11 and a first conductive support 12, and a first volume 13 in contact with the first ion-selective membrane 11; [0188] a second half cell provided with a second ion-selective electrode 2 made of a second ion-selective membrane 21 and a second conductive support 22, and a second volume 23 in contact with the second ion-selective membrane 21; [0189] a salt bridge 3 connecting the first volume 13 and the second volume 23; and [0190] means for bringing a biological fluid sample D in contact with the second volume 23, and wherein the salt bridge 3 comprises a diffusion limiter 4, which allows opening the salt bridge 3 when it is removed, and wherein the first volume 13, the second volume 23 and the salt bridge are a hydrogel.

[0191] This device has been used with a hydrogel having a composition of 1% of agarose and 99% of distilled water and the results depicted in FIG. 12 were obtained.

[0192] Herein, three data series are shown. The first one (black dots) corresponds to the addition of a pure dissolution of 1 mM Li+(Sigma-Aldrich). It takes up to 100 seconds to reach the maximum potential. The second data series (dark grey dots) corresponds to an addition of a 40 g/L BSA (Roche) dissolution simulating the plasma protein medium. The protein takes longer to reach the sensor, making the E grow more slowly. Finally, the light grey series corresponds to an addition of a solution of Li+ and BSA, simulating a synthetic sample of plasma. As it can be seen, at 100 s the maximum potential corresponding to Li+ is reached, so the E value corresponding to that time should be taken. If we wait longer, proteins will reach the sensor and that will cause an increase of the potential, causing an overestimation of Li+ concentration. This means that if the potential is measured at any time before 100 s, the measurement will be free of the interference of the proteins, thanks to the filtering effect of the hydrogel. In any case, the values of E to reach the desired limit of detection have to be taken into account. This result is positive for the utility of the sensing device, because the measurement by the final user will be within this time.

[0193] In this text, the term comprise and its derivations (such as comprising, etc.) should not be understood in an excluding sense, that is, these terms should not be interpreted as excluding the possibility that, what is described and defined, may include further elements, steps, etc. On the other hand, the invention is obviously not limited to the specific embodiment(s) described herein, but also encompasses any variations that may be considered by any person skilled in the art (for example, as regards the choice of materials, dimensions, components, configuration, etc.), within the general scope of the invention as defined in the claims.