A SENSOR

Abstract

The present invention relates to a sensor for detecting one or more target analytes, the sensor comprising: at least one polymeric sensing element capable of selectively and reversibly binding to a target analyte; at least one working electrode having the polymeric sensing element disposed thereon; at least one reference electrode that is electrically communicated with said working electrode; and means for measuring a change in an electrical property across said working electrode and said reference electrode. In particular, the target analyte is Na+, urea or creatinine. Also disclosed is a multi-layered sensor, comprising at least one working electrode layer and at least one reference electrode layer, said working electrode layer and said reference electrode layer being separated by at least one electrically insulating layer.

Claims

1.-17. (canceled)

18. A sensor for detecting one or more target analytes, the sensor comprising: (a) at least one polymeric, sensing element capable of selectively and reversibly binding to a target analyte; (b) at least one working electrode having the polymeric sensing element disposed thereon; (c) at least one reference electrode that is electrically communicated with said working electrode; and (d) means for measuring a change in an electrical property across said working electrode and said reference electrode.

19. The sensor of claim 18, wherein the sensor comprises at least two or more polymeric sensing elements disposed on said working electrode.

20. The sensor of claim 19, wherein each polymeric sensing element is independently configured to detect the same or different target analyte.

21. The sensor of claim 18, wherein each polymeric sensing element is disposed on a surface of the working electrode, said polymeric sensing elements being exposed to an external environment when said sensor is in use.

22. The sensor of claim 18, wherein the electrodes are composed of copper.

23. The sensor of claim 18, wherein each polymeric sensing element is independently selected from an ion-selective polymer membrane or a molecularly imprinted polymer (MIP) film.

24. The sensor of claim 23, wherein the ion-selective polymer membrane comprises an ionophore dispersed within a polymer matrix, said ionophore capable of reversibly forming a complex with said target analyte.

25. The sensor of claim 24, wherein the polymer matrix further comprises at least one additive selected to repel non-target molecules or ions, which are not of the same charge as the target analyte, from the ion-selective polymer membrane.

26. The sensor of claim 23, wherein the ion-selective polymer membrane is prepared from a polymer coating composition comprising at least one polymer, a plasticizer, an ionophore and at least one lipophilic ion additive.

27. The sensor of claim 23, wherein the MIP film is prepared by: casting a polymer film from a composition comprising a polymer and a target analyte intended for detection by the MIP film; drying the film; and removing the target analyte from the dried film to generate cavities thereon, wherein the cavities are specifically adapted to receive the target analyte.

28. The sensor of claim 18, wherein the target analyte is selected from one or more of the group consisting of: Na+, urea, and creatinine.

29. The sensor of claim 18, wherein the working electrode and reference electrode are separated by an electrically insulating layer.

30. The sensor of claim 18, wherein the means for measuring the potential difference comprises at least one transmitter capable of relaying the measured electrical property as electrical signals to an external computer.

31. The sensor of claim 18, wherein said electrical property being measured is selected from voltage, potential difference, impedance or resistance.

32. A multi-layered sensor comprising: at least one working electrode layer and at least one reference electrode layer, said working electrode layer and said reference electrode layer being separated by at least one electrically insulating layer; at least two or more polymeric sensing elements disposed on a surface of the working electrode layer; each polymeric sensing element being configured to detect a different analyte; and means for detecting and measuring changes in an electrical property of the polymeric sensing elements.

33. An in vitro diagnostic kit or a point-of-care kit comprising a sensor for detecting one or more target analytes, the sensor comprising: (a) at least one polymeric, sensing element capable of selectively and reversibly binding to a target analyte; (b) at least one working electrode having the polymeric sensing element disposed thereon; (c) at least one reference electrode that is electrically communicated with said working electrode; and (d) means for measuring a change in an electrical property across said working electrode and said reference electrode.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0043] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0044] FIG. 1a

[0045] FIG. 1a is a schematic illustration showing one possible configuration of the sensor as disclosed herein in a cross-sectional view.

[0046] FIG. 1b

[0047] FIG. 1b is a schematic illustration showing one possible configuration of the sensor as disclosed herein in a top view.

[0048] FIG. 2a

[0049] FIG. 1(a) is a graph showing the potentiometric response of a Na.sup.+ sensor in the detection of Na.sup.+ in the presence of interference by other ionic species including K.sup.+, PO.sub.4.sup.3, Mg.sup.2+, Ca.sup.2+, urea and creatinine. Concentrations of analytes were increased every 100 s.

[0050] FIG. 2b

[0051] FIG. 2(b) is a graph showing the increase in voltage experienced by the Na+ sensor when tested with urine samples that were spiked with increasing concentrations of Na.sup.+.

[0052] FIG. 2a

[0053] FIG. 3(a) is a graph showing the electrochemical impedance spectroscopy (EIS) measurements in the presence of (a) urea.

[0054] FIG. 3b

[0055] FIG. 3(b) is a graph showing the electrochemical impedance spectroscopy (EIS) measurements in the presence of (b) creatinine.

[0056] FIG. 3c

[0057] FIG. 3(c) is a graph showing the electrochemical impedance spectroscopy (EIS) measurements in the presence of (c) uric acid.

[0058] FIG. 3d

[0059] FIG. 3(d) is a graph showing the electrochemical impedance spectroscopy (EIS) measurements in the presence of (d) Na.sup.+.

[0060] FIG. 4a

[0061] FIG. 4(a) is a graph showing the decrease in (a) impedance obtained by the urea sensor as the concentration of urea spiked into urine increases.

[0062] FIG. 4b

[0063] FIG. 4(b) is a graph showing the decrease in (b) resistance obtained by the urea sensor as the concentration of urea spiked into urine increases.

[0064] FIG. 4a

[0065] FIG. 5a is a graph showing EIS measurements in the presence of increasing concentrations of (a) creatinine from 1 to 100 mM.

[0066] FIG. 5b

[0067] FIG. 5b is a graph showing EIS measurements in the presence increasing concentrations of (b) urea from 400 to 1500 mM.

[0068] FIG. 6c

[0069] FIG. 5c is a graph showing EIS measurements in the presence of increasing concentrations of (c) Na.sup.+ from 50 to 400 mM.

[0070] FIG. 7d

[0071] FIG. 5d is a graph showing EIS measurements in the presence of increasing concentrations of (d) K.sup.+ from 50 to 400 mM.

[0072] FIG. 6a

[0073] FIG. 8a. is a graph showing the decrease in (a) impedance obtained by the creatinine sensor as the concentration of creatinine spiked into urine increases.

[0074] FIG. 6b

[0075] FIG. 9b. is a graph showing the decrease in (b) resistance obtained by the creatinine sensor as the concentration of creatinine spiked into urine increases.

[0076] FIG. 10

[0077] FIG. 7 is a schematic drawing illustrating the mechanism of voltammetric-based detection of urea by utilizing a dual-layered sensing element comprising a NH4.sup.+-selective membrane and a urease coating.

[0078] FIG. 11.

[0079] FIG. 8 is a graph showing an increase in voltage obtained by the urea sensor in the presence of an increasing concentration of ammonium acetate (NH.sub.4CH.sub.3CO.sub.2).

[0080] FIG. 9a

[0081] FIG. 9a is a graph showing the real-time potentiometric response of the urea sensor in the presence of an increasing concentration of urea, wherein the concentration of urea was increased step-wise every 100 seconds.

[0082] FIG. 9b

[0083] FIG. 9b is a graph illustrating the increase in voltage obtained with an increasing concentration of urea for a urea sensor.

DETAILED DESCRIPTION OF DRAWINGS

[0084] FIG. 1 shows one configuration of a sensor 10 according to the present disclosure. The modified copper working electrode 12 and reference electrode 18 are separated by an isolation material 22 (e.g. a plastic film), which is electrically insulating, in a three-layered structure. The working electrode 12 may be modified by at least one layer of a polymeric sensing element 14. The reference electrode 18 may also be optionally modified wherein at least one layer of a reference coating 16 is disposed thereon. In a particular embodiment, the reference coating 16 is a Ag/AgCl+ coating.

[0085] A portion of the reference electrode 18 wraps around one end of the isolation material 22, so that the connecting points of both the working and reference electrodes are on the same side of the sensor, which can be connected to the connecting pins 24 of the transmitter box 26. The transmitter box 26 measures an electrical signal generated by the sensor strips, and can transmit the data wirelessly to a monitoring computer and software for data analysis (not shown).

[0086] The different sensors can be attached onto a single strip of isolation material, and connected to dedicated channels on the transmitter box for multiplexed detection. An embodiment of this is schematically illustrated in FIG. 1b wherein the at least three different sensing elements 32, 34, and 36 are disposed on the copper working electrode 12. Each sensing element is configured to detect a different analyte. Sensing element 32 may be a ion-selective polymer membrane configured to detect sodium ions. Sensing element 34 may be a MIP film configured to detect the presence of urea molecules. Sensing element 36 may be a MIP film configured to detect the presence of creatinine molecules. Each respective sensing element may be connected separately to the transmitted box 26 to provide independent and separate electrical input to the transmitter such that each target analyte can be detected independently.

[0087] Another embodiment of the disclosed sensor is illustrated in FIG. 7 wherein a multi-layered polymer sensing element is provided on the electrode. In particular, the schematic illustrates the mechanism of voltammetric-based detection of urea. Similar to the Na.sup.+ sensor, an ion-selective membrane is required. In this case, ammonium (NH4.sup.+)-selective membrane is utilized, in addition to a urease coating that is deposited on top of the NH4.sup.+-selective membrane. In the presence of target urea molecules, urea may be hydrolyzed to NH4.sup.++HCO.sub.3.sup.. The NH4.sup.+ generated can then diffuse into the NH4.sup.+-selective membrane, resulting in a voltage change.

EXAMPLES

[0088] Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Materials and Methods

Example 1

Preparation of Sodium-Selective Polymeric Sensing Element

[0089] The following describes the preparation of 1 mL of a Na.sup.+-selective membrane, which can be scaled up according to the volume required. 241.5 L of tetrahydrofuran (THF) was mixed with 100 L of sodium ionophore X (15 g/L in THF), 50 L of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (Na-TFPB, 16 g/L in THF), 500 L of PVC (100 g/L in THF), and 108.5 L of bis(2-ethylhexyl) sebacate (DOS, neat). The solution was mixed thoroughly, drop-casted onto the surface of the copper tape, and left to dry for at least 1 h at ambient conditions. The modified copper tape can then be used as the working electrode for the detection of Na.sup.+ in sample solution.

[0090] In order to prepare the reference electrode for Na.sup.+ measurement, another piece of copper tape was coated with Ag/AgCl ink. The coated copper tape was then dried at 120 C. for 1 h.

Example 2

Preparation of MIP Films for Urea Sensor

[0091] To fabricate the working electrode, a solution of 10 wt % poly(vinyl alcohol-co-ethylene) (10% EVAL) was first prepared in dimethyl sulfoxide (DMSO). Next, template urea molecules were dissolved in the prepared 10% EVAL solution such that urea has a final concentration of 2 wt %. The mixture was then drop-cased on the copper tape, and left to dry overnight at ambient conditions.

[0092] For the reference electrode, 1 wt % of template urea molecules were dissolved in 10% EVAL, drop-casted on the copper tape, and left to dry overnight at ambient conditions.

[0093] Subsequently, the MIP-coated copper tapes were washed in 50% ethanol solution with mild shaking for 2 h to remove the template urea molecules.

Example 3

Preparation of MIP Films for Creatinine Sensor

[0094] To prepare the working and reference electrodes, 0.1 and 0.05 wt. % of template creatinine molecules were dissolved in 10% EVAL solution, respectively. The mixtures were then coated on separate copper tapes and left to dry overnight at ambient conditions. The copper tapes were then washed in 50% ethanol solution with mild shaking for 2 h to remove the template creatinine molecules.

Performance Characterization

Example 4

Sodium Sensor

[0095] The Na.sup.+ sensor was prepared by coating the sodium-selective membrane on the copper electrode. FIG. 2a illustrates the open circuit potential (OCP) response of the sodium-selective membrane for the detection of Na.sup.+ and in the presence of interfering ions and compounds, such as K+, PO.sub.4.sup.3, Mg.sup.2+, Ca.sup.2+, urea and creatinine.

[0096] A distinct increase in voltage was observed with each increase in Na+ concentration. K+ is a common interfering ion due to its similarity in size as compared to Na.sup.+, but only a slight increase in voltage was observed at 400 mM of K+, which was much higher than the normal daily maximum value of 50-125 mM.

[0097] Hence, we do not expect any significant interference from the presence of K+ when trying to detect high concentrations of Na+ during dehydration. In addition, there were relatively insignificant voltage/p.d. changes when electrodes were subjected to other interfering ions and compounds. Therefore, the results show that the disclosed sensor and sensing element is capable of the selective detection of Na+.

[0098] To validate the functionality of the Na+-selective sensor, various concentrations of Na+ were spiked into urine collected from a volunteer using NaCl (5 M), and the OCP response in the spiked urine samples was measured.

[0099] FIG. 2b illustrates the increase in voltage obtained when the Na+ sensor was tested with urine spiked with an increasing concentration of Na+, demonstrating the feasibility of our sensor in measuring Na+ concentration in physiological urine.

Example 5

Urea Sensor

[0100] FIG. 3a shows a decrease in impedance as urea concentration increased. In contrast, there were relatively insignificant impedance changes in the presence of interferences, such as creatinine, uric acid and Na+(FIGS. 3b-d, respectively). Therefore, the results show that the disclosed sensor is capable of the specific detection of urea.

[0101] Subsequently, we spiked various concentrations of urea into urine collected from a volunteer. FIG. 4a shows a decrease in the impedance obtained when the concentration of spiked urea increased. The resistance was also observed to decrease in the presence of higher urea concentration (FIG. 4b).

Example 6

Creatinine Sensor

[0102] Likewise, FIG. 5a shows a decrease in impedance as creatinine concentration increased. In contrast, there were relatively insignificant impedance changes in the presence of interferences, such as urea, Na+ and K+(FIGS. 5b-d, respectively). Therefore, specific creatinine detection was achieved.

[0103] Various concentrations of creatinine were subsequently spiked into urine collected from a volunteer. FIG. 6a shows a decrease in impedance when the concentration of spiked creatinine in urine was increased. The resistance also decreased in the presence of higher spiked creatinine concentration (FIG. 6b). As the normal physiological creatinine concentration in the urine ranges from 5-20 mM, the disclosed sensor can be used to detect an excess/abnormal amount of creatinine in urine, which can be indicative of renal problems.

Example 7

[0104] Urea Sensor with Multi-Layer Sensing Element

Preparation of Ammonium-Selective Membrane

[0105] The following describes the preparation of 1 mL of the NH4+-selective membrane, which can be scaled up according to the volume required. 397.5 L of THF was mixed with 120 L of ammonium ionophore I (15 g/L in THF), 50 L of Na-TFPB (16 g/L in THF), 366 L of PVC (100 g/L in THF), and 66.5 L of DOS (neat). The solution was mixed thoroughly, drop-casted onto the surface of the copper tape, and left to dry for at least 1 h at ambient conditions.

Preparation of Urease Coating

[0106] The volumes described below can be scaled up according to volume required.

Solution A (30 mg/ml Urease in 4% BSA)

TABLE-US-00001 Volume Component (L) 60 mg/ml urease in 50 mM maleic acid-NaOH buffer 30 pH 6.5 10% bovine serum albumin (BSA) 24 50 mM maleic acid-NaOH buffer pH 6.5 6

Solution B (0.625% Glutaraldehyde)

[0107]

TABLE-US-00002 Component Volume (L) 50% glutaraldehyde (stock solution) 1 50 mM maleic acid-NaOH buffer pH 79 6.5

[0108] Solutions A and B were mixed in a ratio of 12:3 v/v, and drop casted onto the surface of the NH4+-selective membrane. The mixture is left to dry overnight at ambient conditions.

[0109] The urease and NH4.sup.+-selective coatings formed the working electrode of the urea sensor. The reference electrode was prepared as described in Section 2.1.

Performance Characterization

[0110] FIG. 8 below shows the increase in voltage obtained when the urea sensor was subjected to an increasing concentration of ammonium acetate standards.

[0111] FIG. 9a shows the real-time potentiometric response of the urea sensor in the presence of an increasing concentration of urea. We observed a distinct increase in voltage with each increase in urea concentration. FIG. 9b illustrates the increase in voltage obtained with an increasing concentration of urea.

INDUSTRIAL APPLICABILITY

[0112] In this work, copper tapes, modified with target-specific polymeric membranes are used as inexpensive material to develop multiplexed sensors that can be integrated with diapers for health screening and monitoring, as well as for the early diagnosis of diseases, such as renal failure. The sensors have been validated for the detection of Na+, urea and creatinine spiked in human urine samples. While the detection of sodium ions, urea and creatinine are expressly exemplified, the sensor may be configured for detecting other types of analytes by making corresponding modifications of the polymeric sensing element (i.e., the MIP film or the ion-selective polymer membrane).

[0113] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.