WO2016156941A1 - Solid state electrolyte biosensor - Google Patents

Solid state electrolyte biosensor Download PDF

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Publication number
WO2016156941A1
WO2016156941A1 PCT/IB2015/056920 IB2015056920W WO2016156941A1 WO 2016156941 A1 WO2016156941 A1 WO 2016156941A1 IB 2015056920 W IB2015056920 W IB 2015056920W WO 2016156941 A1 WO2016156941 A1 WO 2016156941A1
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WIPO (PCT)
Prior art keywords
ion
membrane
selective
ionophore
target
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PCT/IB2015/056920
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French (fr)
Inventor
Vijaywanth MATHUR
Vivek Pandey
Ramesh MAMDRAPURKAR
Dhanada DESHPANDE
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Diasys Diagnostics India Private Limited
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Publication of WO2016156941A1 publication Critical patent/WO2016156941A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/333—Ion-selective electrodes or membranes

Definitions

  • the present disclosure pertains to technical field of biosensors.
  • the present disclosure pertains to disposable solid state biosensor for measuring clinically important ionic species such as potassium ion (K + ), sodium ion (Na + ), chloride ion (CI ⁇ ), calcium ion (Ca 2+ ), and pH in blood samples.
  • electrolytes such as potassium ion (K + ), sodium ion (Na + ), chloride ion (CI ), calcium ion (Ca 2+ ).
  • K + potassium ion
  • Na + sodium ion
  • CI chloride ion
  • Ca 2+ calcium ion
  • electrolytes regulate human body's nervous system, metabolic processes, renal function, vision, cardiac operation, pH balance, and olfactory senses.
  • Human body requires a precise balance of electrolytes in intracellular and extracellular fluids to function properly, and through absorption by the intestines or excretion by the kidneys, the body adjusts electrolyte levels accordingly. Neither the intestines nor the kidneys can function, however, if either the electrolytes are not present to be absorbed or are overabundant and cannot be excreted.
  • the major disadvantage is that the known potentiomtric based sensors are not precise and often produce inaccurate results due to false signals caused by interfering active species present in test samples. Lifetime of these sensors are short and cost per analysis is also relatively high. Fabrication of these potentiomtric based biosensors often requires complicated manufacturing technologies and not suitable for application to disposable electrochemical biosensors. A further drawback of the known sensors is that the various layers of the sensors become detached from one another or form cracks and thereby affecting the service life of the sensors. Further, diagnostic devices that utilize the known potentiomtric based sensors require calibration both before and after measurement. In clinical setting it is desirable to maximize the amount of data obtainable from a sample having a volume as small as possible, typically a sample on the order of micro-liters. However, the known sensors require large volume of test sample.
  • aspects of the present disclosure relate to membrane formulation from which an ion- selective electrode membrane can be formed and used in electrochemical biosensors for potentiometric measurement of clinically important electrolytes in biological fluids.
  • the present disclosure provides a disposable electrochemical biosensor incorporating an ion-selective membrane for potentiometric measurement of clinically important electrolytes such as potassium ion, sodium ion, chloride ion, calcium ion and pH in biological fluids such as whole blood, blood serum and blood plasma.
  • clinically important electrolytes such as potassium ion, sodium ion, chloride ion, calcium ion and pH in biological fluids such as whole blood, blood serum and blood plasma.
  • the ion-selective membrane can be formed from a membrane formulation which can include an ionophore selective for a target ion, at least one polymeric material, at least one plasticizer, and a pore former.
  • the biological fluid to be tested can contain one or more target ions
  • the membrane formulation used to form ion-selective membrane can include a variety of ionophores selective for each of the target ions.
  • suitable ionophores can be selected from those selective for sodium, potassium or chloride ions.
  • the polymeric material that can be used to formulate the membrane formulation can be selected from polyvinyl chloride, polyvinyl acetate, silicone rubber, cellulose acetate or copolymers thereof.
  • the plasticizer that can be used to formulate the membrane formulation can be selected from the group consisting of dialkyl aryl phosphonates, trialkyl phosphates, trialkyl phosphites, dialkyl sebacates, dialkyl adipates, dialkyl phthalates, nitrophenyl alkyl ethers and 2-nitrophenyl aryl ethers.
  • the present disclosure provides an ion selective membrane for use in an electrochemical biosensor for measuring sodium, potassium, and chloride ions in blood samples, wherein the ion selective membrane can be formed from a membrane formulation which can include calixarene, valinomycin, tetradimethylammonium chloride, polyvinyl chloride, dioctyl sebacate and potassium tetrakis(4-chlorophenyl)borate.
  • the membrane components can be dissolved in suitable solvent, for example in tetrahydrofuran, and the resulting solution can be deposited on sensor electrodes by spin casting to form an ion selective membrane layer.
  • the present disclosure provides a disposable electrochemical biosensor for measuring target ion in a biological fluid
  • the biosensor can include (a) a polymeric base plate, (b) at least one ion selective electrode and a reference electrode disposed on the polymeric base plate, and (c) an ion selective membrane covering at least a portion of the ion selective electrode and a reference electrode, wherein the ion selective membrane is formed from a membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former.
  • the present disclosure provides a method for making a disposable electrochemical biosensor having an ion-selective membrane for measuring target ion in a biological fluid, wherein the method can include the steps of (a) providing a polymeric base plate, (b) disposing at least one ion selective electrode and a reference electrode on the polymeric base plate, (c) optionally depositing a plurality of nanostructures over the at least one ion selective electrode and the reference electrode to form a layer, (d) applying onto the ion selective electrode and the reference electrode a membrane formulation in solution with a volatile organic solvent, wherein the membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former, and (e) evaporating the volatile organic solvent from the membrane formulation to form a single film on the electrodes.
  • FIG. l illustrates an exploded view of a diagnostic device including an electrochemical biosensor in accordance with embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.
  • inventive subject matter provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
  • the present disclosure provides a membrane formulation from which an ion-selective electrode membrane can be formed and used in electrochemical biosensors for potentiometric measurement of clinically important electrolytes such as potassium ion (K + ), sodium ion (Na + ), chloride ion (CI ⁇ ), calcium ion (Ca 2+ ) and pH in biological fluids.
  • biological fluid can include, but not limited to, whole blood, blood serum, blood plasma, other body fluids such as ISF (interstitial fluid), urine, saliva and sweat.
  • the ion-selective membrane can be formed from a membrane formulation which can include an ionophore selective for a target ion, at least one polymeric material, at least one plasticizer, and a pore former.
  • the membrane formulation from which an ion selective membrane is formed can include an ionophore having high selectivity for the target ion.
  • the membrane formulation can include a variety of ionophores selective for each of the different ionic species present in the test sample.
  • suitable ionophores can be selected from those selective for sodium, potassium or chloride ions.
  • the ionophore selective for sodium ions may be present in an amount of from 1 to 5 percent by weight, preferably from 2 to 4 percent by weight, more preferably 3 percent by weight based on the total weight of the membrane formulation.
  • the ionophore selective for potassium ions may be present in an amount of from 1 to 5 percent by weight, preferably from 2 to 4 percent by weight, more preferably 3 percent by weight based on the total weight of the membrane formulation.
  • the ionophore selective for chloride ions may be present in an amount of from 10 to 70 percent by weight, preferably from 40 to 60 percent by weight, more preferably 50 percent by weight based on the total weight of the membrane formulation.
  • the polymeric material that can be used in the membrane formulation can be selected from polyvinyl chloride, polyvinyl acetate, silicone rubber, cellulose acetate or copolymers thereof.
  • the polymeric material can serve the purpose of providing support and structure to the ion selective membrane, and can act as a matrix into which the ion-selective compounds i.e. the ionophores are incorporated.
  • the polymeric material may be present in an amount of from 20 to 50 percent by weight, preferably from 30 to 40 percent by weight, more preferably from 33-35 percent by weight based on the total weight of the membrane formulation.
  • the polymeric material used to formulate the membrane formulation can be polyvinyl chloride.
  • the membrane formulation of the present disclosure can include one or more plasticizers.
  • the plasticizer may influence the relative rates of partitioning of different ionic species of test sample into the membrane.
  • the plasticizer may also contribute to the dissolution of the ionophore and it may also facilitate the compounding or production of the membrane formulation and improving the membrane's flexibility.
  • Suitable plasticizers can include dialkyl aryl phosphonates, trialkyl phosphates, trialkyl phosphites, dialkyl sebacates, dialkyl adipates, dialkyl phthalates, nitrophenyl alkyl ethers, 2-nitrophenyl aryl ethers and mixtures thereof.
  • the plasticizer may be present in an amount of from 20 to 70 percent by weight, preferably from 60 to 70 percent by weight, more preferably from 63-67 percent by weight based on the total weight of the membrane formulation.
  • the plasticizer used to formulate the membrane formulation can be dioctyl sebacate.
  • the membrane formulation can include a pore former in an amount preferably ranges from 20 to 30 mol percent of ionophore concentration in the formulation.
  • the pore former can be a salt of a tetraphenylborate or substituted versions thereof.
  • the pore former can be potassium tetrakis (4-chlorophenyl) borate.
  • the membrane formulation from which an ion selective membrane can be formed for potentiometric measurement of sodium ion in blood samples wherein the membrane formulation can include calixarene as sodium ionophore, polyvinyl chloride as polymer matrix, dioctyl sebacate as plasticizer and potassium tetrakis(4- chlorophenyl)borate as pore former.
  • the membrane formulation from which an ion selective membrane can be formed for potentiometric measurement of potassium ion in blood samples wherein the membrane formulation can include valinomycin as potassium ionophore, polyvinyl chloride as polymer matrix, dioctyl sebacate as plasticizer and potassium tetrakis(4- chlorophenyl)borate as pore former.
  • the membrane formulation from which an ion selective membrane can be formed for potentiometric measurement of chloride ion in blood samples wherein the membrane formulation can include tetradimethylammonium chloride as chloride ionophore, polyvinyl chloride as polymer matrix, dioctyl sebacate as plasticizer and potassium tetrakis(4-chlorophenyl)borate as pore former.
  • the membrane formulation can include more than one ionophores to detect different ionic species in a test sample.
  • the ionophores can be selected such that each ionophore can exhibit good selectivity for the respective target ions.
  • the selectivity coefficient for an ion selective membrane containing more than one ionophores can be varied by varying the relative amounts of the ionophores in the membrane.
  • an ion selective membrane can be formed from a membrane formulation for potentiometric measurement of sodium and potassium ions in blood samples, wherein the membrane formulation can include calixarene, valinomycin, polyvinyl chloride, dioctyl sebacate and potassium tetrakis(4-chlorophenyl)borate.
  • an ion selective membrane can be formed from a membrane formulation for potentiometric measurement of sodium, potassium and chloride ions in blood samples, wherein the membrane formulation can include calixarene, valinomycin, tetradimethylammonium chloride, polyvinyl chloride, dioctyl sebacate and potassium tetrakis(4- chlorophenyl)borate.
  • the membrane formulation from which an ion selective membrane can be formed for potentiometric measurement of sodium and potassium ions in blood samples can include 3% by weight of calixarene, 3% by weight of valinomycin, 30-35% by weight of polyvinyl chloride, 63-67% by weight of dioctyl sebacate, and 20 to 30 mol% of potassium tetrakis(4-chlorophenyl)borate.
  • the membrane formulation of the present disclosure can be prepared as a homogeneous solution in a suitable solvent such as, but not limited to, tetrahydrofuran or dimethylformamide, which is suitable for casting into a thin film.
  • a suitable solvent such as, but not limited to, tetrahydrofuran or dimethylformamide
  • the homogeneous solution containing the membrane components may then be deposited on sensor electrodes preferably using a spin cast instrument.
  • the solvent can be allowed to evaporate to form an ion selective membrane on electrode surface.
  • the ion selective membrane can preferably be in the range of from 0.5 ⁇ to 50 ⁇ thick.
  • the present disclosure provides a disposable electrochemical biosensor incorporating an ion-selective membrane for potentiometric measurement of target ion in a biological fluid
  • the biosensor can include (a) a polymeric base plate, (b) at least one ion selective electrode and a reference electrode disposed on the polymeric base plate, and (c) an ion selective membrane covering at least a portion of the ion selective electrode and a reference electrode, wherein the ion selective membrane is formed from a membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former.
  • the biosensor of the present disclosure can include a polymeric base plate upon which one or more ion selective electrodes and a reference electrode can be deposited.
  • the polymeric base plate can be of any desirable shape, thickness and size and can be made of any suitable polymeric material.
  • the polymeric base plate can act as a bottom support for electrodes of the sensor.
  • the polymeric base plate can be made of polycarbonate polymer.
  • the ion selective electrodes of the present disclosure can be potentiometric in nature and can be selective in their responses to ionic species of interest due to the presence of ion selective membrane on their surface.
  • the ion selective electrodes and the reference electrode may be deposited on the polymeric base plate by screen printing, vapor deposition, electro-deposition, chemical vapor deposition, sputtering, or any other suitable deposition method known in the art.
  • the electrochemical biosensor of the present disclosure can include three ion selective electrodes and a reference electrode for multiplexed detection of sodium, potassium and chloride ions in blood samples simultaneously.
  • the membrane formulation can be prepared as a homogeneous solution in a suitable solvent such as tetrahydrofuran or dimethylformamide, and the resulting solution can be deposited on the surfaces of the electrodes preferably using a spin cast instrument.
  • the deposition of membrane formulation on the electrodes may produce a thin layer of ion selective membrane at micrometer or sub-micrometer thickness.
  • the ion selective membrane layer may preferably be dried and the electrodes deposited with ion selective membrane may be stored in a desiccated container before final lamination to form the electrochemical biosensor.
  • the ion-selective membrane formed from the membrane formulation can be selectively permeable to an ion whose concentration is to be determined. Interaction of ions (electrolytes) in the test sample with corresponding ionophores in the membrane can alter the electrical potential across the membrane which can be measured as a change in potential between the ion selective electrodes and the reference electrode.
  • the surfaces of ion selective electrodes and the reference electrode can be deposited with a plurality of nanostructures prior to coating the electrodes with the membrane formulation.
  • the nanostructure coating can increase the surface area of the electrodes.
  • the nanostructures may be deposited only on the surface of ion selective electrodes or it may be deposited on both ion selective electrodes and reference electrode. The increase in surface area of ion selective electrodes can enhance the sensitivity and accuracy of the biosensor even with test samples containing very low concentration of target ionic species.
  • nanostructures refer to solid particles or hollow-core particles, which can have particle size less than 500 nm, preferably less than 100 nm, more preferably less than 50 nm.
  • the non-limiting exemplary nanostructures according to the present disclosure can be selected from carbon nanotubes (CNTs) or gold nanoparticles.
  • the nanostructures can be gold nanoparticles deposited over the ion selective electrodes using the electro deposition technique.
  • the nanostructures can be carboxylated carbon nanotubes and the percentage of carboxylation of carbon nanotubes can range from 3% to 5%.
  • the nanostructures can be deposited over the surfaces of ion selective electrodes and the reference electrode by drop casting a solution of nanostructure in a suitable buffer.
  • multi-walled carboxylated carbon nanotubes can be mixed with diethanolamine buffer and the resulting solution can be drop casted on surfaces of ion selective and reference electrodes to form a nanostructure layer on the electrode surfaces.
  • the present disclosure provides a disposable electrochemical biosensor incorporating an ion-selective membrane for potentiometric measurement of target ion in a biological fluid
  • the biosensor can include (a) a polymeric base plate, (b) at least one ion selective electrode and a reference electrode disposed on the polymeric base plate, (c) a plurality of nanostructures deposited over the at least one ion selective electrode and the reference electrode to form a layer, and (d) an ion selective membrane covering at least a portion of the nanostructure layer, wherein the ion selective membrane is formed from a membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former.
  • the present disclosure provides a disposable electrochemical biosensor incorporating an ion-selective membrane for potentiometric measurement of sodium ion, potassium ion and chloride ions in a biological fluid
  • the biosensor can include (a) a polymeric base plate, (b) three ion selective electrodes and a reference electrode disposed on the polymeric base plate, (c) an ion selective membrane covering at least a portion of the ion selective electrodes and the reference electrode, wherein the ion selective membrane is formed from a membrane formulation comprises calixarene, valinomycin, tetradimethylammonium chloride, polyvinyl chloride, dioctyl sebacate and potassium tetrakis(4-chlorophenyl)borate.
  • the present disclosure provides a method for making a disposable electrochemical biosensor having an ion-selective membrane for measuring target ions in a biological fluid, wherein the method can include the steps of (a) providing a polymeric base plate, (b) disposing at least one ion selective electrode and a reference electrode on the polymeric base plate, (c) optionally depositing a plurality of nanostructures over the at least one ion selective electrode and the reference electrode to form a layer, (d) applying onto the electrodes a membrane formulation in solution with a volatile organic solvent, wherein the membrane formulation comprises an ionophore selective for the target ions, at least one polymeric material, at least one plasticizer, and a pore former, and (e) evaporating the volatile organic solvent from the membrane formulation to form a single film on the electrode surface.
  • the disposable biosensor of the present disclosure can be directly or indirectly connected to a display device and can allow the users to view the test results immediately in a more direct, economic and efficient manner.
  • FIG. 1 illustrates a preferred configuration of a diagnostic device assembly.
  • the diagnostic device 100 can be constructed of a top cover 102, sample adhesive layer 104, channel adhesive layer 106, an electrochemical biosensor 108, vent hole layer 110 and bottom cover 112.
  • the top cover 102 of the diagnostic device 100 can be made of a rigid material, preferably plastic, capable of repetitive deformation without cracking and can include a sample entry port through which a test sample can be introduced.
  • the top cover 102 can be joined to the bottom cover 112 in a sealing operation to complete the assembly.
  • the channel adhesive layer 106 can comprises a channel that is laser or die cut therein.
  • the channel adhesive layer 106 can serve as a pneumatic seal around the channel to limit the volume of test sample exposed to the electrochemical biosensor 108.
  • the design of the diagnostic device 100 and the components thereof are purely exemplary and the diagnostic device 100 and its components can take any desired size, shape and thickness to suite configuration of matching parts.
  • the electrochemical biosensor of the present disclosure can allow for multiplexing and enable simultaneous measurement of electrolytes such as potassium ion (K + ), sodium ion (Na + ),
  • the electrochemical biosensor is highly selective and can reduce the change in electric potential caused by interfering electroactive substances present in biological samples. Further, the electrochemical biosensor of the present disclosure can be highly resistant to change in temperature, pH, or other stimuli, that can have adverse effect on the selectivity, responsiveness, and accuracy of sensor.
  • Example 1 Sodium ion sensing membrane formulation
  • Example 3 Chloride ion sensing membrane formulation:
  • Multi-walled carboxylated carbon nanotubes (COOH-CNTs) solution was prepared by adding COOH-CNTs in 0.1M diethanolamine buffer to get a final concentration of lOmg/ml. 6 ⁇ 1 of COOH-CNTs solution was drop casted on electrode surface. The COOH-CNTs was dried at 60°C for 10 minutes.
  • Example 6 Membrane formulation for sensing sodium and potassium ions
  • a single membrane formulation for sensing multiple ionic species such as sodium and potassium ions in a test sample was prepared in the same manner as described in above examples.
  • the formulation was prepared by using same percentage by weight of polyvinyl chloride, dioctyl sebacate, potassium tetrakis(4-chlorophenyl)borate and sodium and potassium ionophores.
  • the present disclosure provides a membrane formulation for use in electrochemical biosensors having excellent selectivity for target ions such as potassium ion (K + ), sodium ion (Na + ), chloride ion (CI ⁇ ), calcium ion (Ca 2+ ) and pH present in biological fluids.
  • target ions such as potassium ion (K + ), sodium ion (Na + ), chloride ion (CI ⁇ ), calcium ion (Ca 2+ ) and pH present in biological fluids.
  • the present disclosure provides an electrochemical biosensor having an ion-selective electrode membrane that enables high-precision measurement of potassium ion (K + ), sodium ion (Na ), chloride ion (CI ), calcium ion (Ca ) and pH in biological fluids.
  • the present disclosure provides an electrochemical biosensor that has high selectivity and accurately senses the target ionic species even in low concentration range of blood samples.
  • the present disclosure provides an electrochemical biosensor that is disposable but very cost-efficient to manufacture.
  • the present disclosure provides an electrochemical sensor that has good reproducibility and multiplexing capabilities.
  • the present disclosure provides an electrochemical sensor that requires relatively lower sample volumes than known sensors.
  • the present disclosure provides an electrochemical biosensor that is highly stable and has excellent storage stability.
  • the present disclosure provides an electrochemical sensor that overcomes the drawbacks of the prior art.

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Abstract

The present disclosure provides an ion-selective membrane for use in electrochemical biosensors for potentiometric measurement of clinically important electrolytes such as potassium ion, sodium ion, chloride ion, calcium ion and p H in biological fluids, wherein the ion-selective membrane can be formed from a membrane formulation which can include an ionophore selective for target ion, at least one polymeric material, at least one plasticizer, and a pore former. The present disclosure further provides an electrochemical biosensor for measuring target ion in a biological fluid, wherein the biosensor can include (a) a polymeric base plate, (b) at least one ion selective electrode and a reference electrode disposed on the polymeric base plate, (c) an ion selective membrane covering at least a portion of the ion selective electrode and the reference electrode, wherein the ion selective membrane is formed from a membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former.

Description

SOLID STATE ELECTROLYTE BIOSENSOR
FIELD OF THE I VENTION
[0001] The present disclosure pertains to technical field of biosensors. In particular, the present disclosure pertains to disposable solid state biosensor for measuring clinically important ionic species such as potassium ion (K+), sodium ion (Na+), chloride ion (CI~), calcium ion (Ca2+), and pH in blood samples.
BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] In clinical setting it is important to monitor certain blood analytes that are highly relevant to normal physiological functions and homeostasis, particularly electrolytes such as potassium ion (K+), sodium ion (Na+), chloride ion (CI ), calcium ion (Ca2+). These electrolytes regulate human body's nervous system, metabolic processes, renal function, vision, cardiac operation, pH balance, and olfactory senses. Human body requires a precise balance of electrolytes in intracellular and extracellular fluids to function properly, and through absorption by the intestines or excretion by the kidneys, the body adjusts electrolyte levels accordingly. Neither the intestines nor the kidneys can function, however, if either the electrolytes are not present to be absorbed or are overabundant and cannot be excreted.
[0004] There have been substantial efforts in the prior art to provide systems and methods for measuring these electrolytes in bodily fluids such as, whole blood, plasma, serum, urine or saliva. One method employs electrochemical sensor that measures electrolyte concentrations in blood samples by potentiometry with membrane-based ion selective electrodes.
[0005] The major disadvantage is that the known potentiomtric based sensors are not precise and often produce inaccurate results due to false signals caused by interfering active species present in test samples. Lifetime of these sensors are short and cost per analysis is also relatively high. Fabrication of these potentiomtric based biosensors often requires complicated manufacturing technologies and not suitable for application to disposable electrochemical biosensors. A further drawback of the known sensors is that the various layers of the sensors become detached from one another or form cracks and thereby affecting the service life of the sensors. Further, diagnostic devices that utilize the known potentiomtric based sensors require calibration both before and after measurement. In clinical setting it is desirable to maximize the amount of data obtainable from a sample having a volume as small as possible, typically a sample on the order of micro-liters. However, the known sensors require large volume of test sample.
[0006] There is thus a need in the art for a very precise, fast response, disposable, highly stable and cost effective sensor for measurement of clinically important electrolytes such as potassium ion (K+), sodium ion (Na+), chloride ion (CI~), calcium ion (Ca2+), and pH in blood samples. Also there is a need in the art for method of manufacturing such sensors.
[0007] The present disclosure satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.
OBJECTS OF THE INVENTION
[0008] It is an object of the present disclosure to provide an ion-selective electrode membrane for use in electrochemical biosensors for measuring target ions in biological fluids.
[0009] It is a further object of the present disclosure to provide an electrochemical biosensor having an ion-selective electrode membrane for measurement of potassium ion (K+), sodium ion (Na ), chloride ion (CI ), calcium ion (Ca ) and pH in biological fluids.
[0010] It is another object of the present disclosure to provide an electrochemical biosensor that can be used to measure the concentrations of multiple electrolytes in a single analysis.
[0011] It is another object of the present disclosure to provide an electrochemical biosensor that can facilitate quick and high accuracy measurement of potassium ion (K+), sodium ion (Na+), chloride ion (CI~), calcium ion (Ca2+) and pH in biological fluids.
[0012] It is another object of the present disclosure to provide a disposable solid-state electrochemical biosensor for measurement of potassium ion (K+), sodium ion (Na+), chloride ion (CI~), calcium ion (Ca2+) and pH in biological fluids.
[0013] It is another object of the present disclosure to provide a diagnostic device including an electrochemical biosensor for convenient and repeated clinical measurements of potassium ion (K+), sodium ion (Na+), chloride ion (CI~), calcium ion (Ca2+) and pH in biological fluids. [0014] It is another object of the present disclosure to provide a method of producing stable and reproducible electrochemical biosensor.
SUMMARY OF THE INVENTION
[0015] Aspects of the present disclosure relate to membrane formulation from which an ion- selective electrode membrane can be formed and used in electrochemical biosensors for potentiometric measurement of clinically important electrolytes in biological fluids.
[0016] In an aspect, the present disclosure provides a disposable electrochemical biosensor incorporating an ion-selective membrane for potentiometric measurement of clinically important electrolytes such as potassium ion, sodium ion, chloride ion, calcium ion and pH in biological fluids such as whole blood, blood serum and blood plasma.
[0017] According to embodiments of the present disclosure, the ion-selective membrane can be formed from a membrane formulation which can include an ionophore selective for a target ion, at least one polymeric material, at least one plasticizer, and a pore former.
[0018] According to embodiments, the biological fluid to be tested can contain one or more target ions, and the membrane formulation used to form ion-selective membrane can include a variety of ionophores selective for each of the target ions.
[0019] According to embodiments, suitable ionophores can be selected from those selective for sodium, potassium or chloride ions.
[0020] According to embodiments, the polymeric material that can be used to formulate the membrane formulation can be selected from polyvinyl chloride, polyvinyl acetate, silicone rubber, cellulose acetate or copolymers thereof.
[0021] According to embodiments, the plasticizer that can be used to formulate the membrane formulation can be selected from the group consisting of dialkyl aryl phosphonates, trialkyl phosphates, trialkyl phosphites, dialkyl sebacates, dialkyl adipates, dialkyl phthalates, nitrophenyl alkyl ethers and 2-nitrophenyl aryl ethers.
[0022] In an exemplary embodiment, the present disclosure provides an ion selective membrane for use in an electrochemical biosensor for measuring sodium, potassium, and chloride ions in blood samples, wherein the ion selective membrane can be formed from a membrane formulation which can include calixarene, valinomycin, tetradimethylammonium chloride, polyvinyl chloride, dioctyl sebacate and potassium tetrakis(4-chlorophenyl)borate. [0023] According to embodiments, the membrane components can be dissolved in suitable solvent, for example in tetrahydrofuran, and the resulting solution can be deposited on sensor electrodes by spin casting to form an ion selective membrane layer.
[0024] In another aspect, the present disclosure provides a disposable electrochemical biosensor for measuring target ion in a biological fluid, wherein the biosensor can include (a) a polymeric base plate, (b) at least one ion selective electrode and a reference electrode disposed on the polymeric base plate, and (c) an ion selective membrane covering at least a portion of the ion selective electrode and a reference electrode, wherein the ion selective membrane is formed from a membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former.
[0025] In another aspect, the present disclosure provides a method for making a disposable electrochemical biosensor having an ion-selective membrane for measuring target ion in a biological fluid, wherein the method can include the steps of (a) providing a polymeric base plate, (b) disposing at least one ion selective electrode and a reference electrode on the polymeric base plate, (c) optionally depositing a plurality of nanostructures over the at least one ion selective electrode and the reference electrode to form a layer, (d) applying onto the ion selective electrode and the reference electrode a membrane formulation in solution with a volatile organic solvent, wherein the membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former, and (e) evaporating the volatile organic solvent from the membrane formulation to form a single film on the electrodes.
[0026] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0028] FIG. l illustrates an exploded view of a diagnostic device including an electrochemical biosensor in accordance with embodiments of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION
[0029] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0030] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.
[0031] Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."
[0032] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0033] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.
[0034] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.
[0035] Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0036] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0037] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0038] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0039] The present disclosure provides a membrane formulation from which an ion-selective electrode membrane can be formed and used in electrochemical biosensors for potentiometric measurement of clinically important electrolytes such as potassium ion (K+), sodium ion (Na+), chloride ion (CI~), calcium ion (Ca2+) and pH in biological fluids. [0040] As used herein, the term "biological fluid" can include, but not limited to, whole blood, blood serum, blood plasma, other body fluids such as ISF (interstitial fluid), urine, saliva and sweat.
[0041] According to embodiments of the present disclosure, the ion-selective membrane can be formed from a membrane formulation which can include an ionophore selective for a target ion, at least one polymeric material, at least one plasticizer, and a pore former.
[0042] The membrane formulation from which an ion selective membrane is formed can include an ionophore having high selectivity for the target ion. In case two or more different ions to be detected in a test sample, the membrane formulation can include a variety of ionophores selective for each of the different ionic species present in the test sample.
[0043] According to embodiments, suitable ionophores can be selected from those selective for sodium, potassium or chloride ions.
[0044] The ionophore selective for sodium ions may be present in an amount of from 1 to 5 percent by weight, preferably from 2 to 4 percent by weight, more preferably 3 percent by weight based on the total weight of the membrane formulation.
[0045] The ionophore selective for potassium ions may be present in an amount of from 1 to 5 percent by weight, preferably from 2 to 4 percent by weight, more preferably 3 percent by weight based on the total weight of the membrane formulation.
[0046] The ionophore selective for chloride ions may be present in an amount of from 10 to 70 percent by weight, preferably from 40 to 60 percent by weight, more preferably 50 percent by weight based on the total weight of the membrane formulation.
[0047] The polymeric material that can be used in the membrane formulation can be selected from polyvinyl chloride, polyvinyl acetate, silicone rubber, cellulose acetate or copolymers thereof. The polymeric material can serve the purpose of providing support and structure to the ion selective membrane, and can act as a matrix into which the ion-selective compounds i.e. the ionophores are incorporated.
[0048] The polymeric material may be present in an amount of from 20 to 50 percent by weight, preferably from 30 to 40 percent by weight, more preferably from 33-35 percent by weight based on the total weight of the membrane formulation.
[0049] In an exemplary embodiment, the polymeric material used to formulate the membrane formulation can be polyvinyl chloride. [0050] The membrane formulation of the present disclosure can include one or more plasticizers. The plasticizer may influence the relative rates of partitioning of different ionic species of test sample into the membrane. The plasticizer may also contribute to the dissolution of the ionophore and it may also facilitate the compounding or production of the membrane formulation and improving the membrane's flexibility. Suitable plasticizers can include dialkyl aryl phosphonates, trialkyl phosphates, trialkyl phosphites, dialkyl sebacates, dialkyl adipates, dialkyl phthalates, nitrophenyl alkyl ethers, 2-nitrophenyl aryl ethers and mixtures thereof.
[0051] The plasticizer may be present in an amount of from 20 to 70 percent by weight, preferably from 60 to 70 percent by weight, more preferably from 63-67 percent by weight based on the total weight of the membrane formulation.
[0052] In an exemplary embodiment, the plasticizer used to formulate the membrane formulation can be dioctyl sebacate.
[0053] The membrane formulation can include a pore former in an amount preferably ranges from 20 to 30 mol percent of ionophore concentration in the formulation.
[0054] In an exemplary embodiment, the pore former can be a salt of a tetraphenylborate or substituted versions thereof. In a preferred exemplary embodiment, the pore former can be potassium tetrakis (4-chlorophenyl) borate.
[0055] In a preferred exemplary embodiment, the membrane formulation from which an ion selective membrane can be formed for potentiometric measurement of sodium ion in blood samples, wherein the membrane formulation can include calixarene as sodium ionophore, polyvinyl chloride as polymer matrix, dioctyl sebacate as plasticizer and potassium tetrakis(4- chlorophenyl)borate as pore former.
[0056] In a preferred exemplary embodiment, the membrane formulation from which an ion selective membrane can be formed for potentiometric measurement of potassium ion in blood samples, wherein the membrane formulation can include valinomycin as potassium ionophore, polyvinyl chloride as polymer matrix, dioctyl sebacate as plasticizer and potassium tetrakis(4- chlorophenyl)borate as pore former.
[0057] In a preferred exemplary embodiment, the membrane formulation from which an ion selective membrane can be formed for potentiometric measurement of chloride ion in blood samples, wherein the membrane formulation can include tetradimethylammonium chloride as chloride ionophore, polyvinyl chloride as polymer matrix, dioctyl sebacate as plasticizer and potassium tetrakis(4-chlorophenyl)borate as pore former.
[0058] The membrane formulation can include more than one ionophores to detect different ionic species in a test sample. The ionophores can be selected such that each ionophore can exhibit good selectivity for the respective target ions. The selectivity coefficient for an ion selective membrane containing more than one ionophores can be varied by varying the relative amounts of the ionophores in the membrane.
[0059] In a preferred exemplary embodiment, an ion selective membrane can be formed from a membrane formulation for potentiometric measurement of sodium and potassium ions in blood samples, wherein the membrane formulation can include calixarene, valinomycin, polyvinyl chloride, dioctyl sebacate and potassium tetrakis(4-chlorophenyl)borate.
[0060] In another exemplary embodiment, an ion selective membrane can be formed from a membrane formulation for potentiometric measurement of sodium, potassium and chloride ions in blood samples, wherein the membrane formulation can include calixarene, valinomycin, tetradimethylammonium chloride, polyvinyl chloride, dioctyl sebacate and potassium tetrakis(4- chlorophenyl)borate.
[0061] In a more preferred exemplary embodiment, the membrane formulation from which an ion selective membrane can be formed for potentiometric measurement of sodium and potassium ions in blood samples, the membrane formulation can include 3% by weight of calixarene, 3% by weight of valinomycin, 30-35% by weight of polyvinyl chloride, 63-67% by weight of dioctyl sebacate, and 20 to 30 mol% of potassium tetrakis(4-chlorophenyl)borate.
[0062] According to embodiments, the membrane formulation of the present disclosure can be prepared as a homogeneous solution in a suitable solvent such as, but not limited to, tetrahydrofuran or dimethylformamide, which is suitable for casting into a thin film. The homogeneous solution containing the membrane components may then be deposited on sensor electrodes preferably using a spin cast instrument. After depositing the membrane formulation solution, the solvent can be allowed to evaporate to form an ion selective membrane on electrode surface. The ion selective membrane can preferably be in the range of from 0.5 μηι to 50 μηι thick.
[0063] In another aspect, the present disclosure provides a disposable electrochemical biosensor incorporating an ion-selective membrane for potentiometric measurement of target ion in a biological fluid, wherein the biosensor can include (a) a polymeric base plate, (b) at least one ion selective electrode and a reference electrode disposed on the polymeric base plate, and (c) an ion selective membrane covering at least a portion of the ion selective electrode and a reference electrode, wherein the ion selective membrane is formed from a membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former.
[0064] The biosensor of the present disclosure can include a polymeric base plate upon which one or more ion selective electrodes and a reference electrode can be deposited. In accordance with embodiments of the present disclosure, the polymeric base plate can be of any desirable shape, thickness and size and can be made of any suitable polymeric material. The polymeric base plate can act as a bottom support for electrodes of the sensor.
[0065] In an exemplary embodiment, the polymeric base plate can be made of polycarbonate polymer.
[0066] The ion selective electrodes of the present disclosure can be potentiometric in nature and can be selective in their responses to ionic species of interest due to the presence of ion selective membrane on their surface. The ion selective electrodes and the reference electrode may be deposited on the polymeric base plate by screen printing, vapor deposition, electro-deposition, chemical vapor deposition, sputtering, or any other suitable deposition method known in the art.
[0067] In an exemplary embodiment, the electrochemical biosensor of the present disclosure can include three ion selective electrodes and a reference electrode for multiplexed detection of sodium, potassium and chloride ions in blood samples simultaneously.
[0068] The membrane formulation can be prepared as a homogeneous solution in a suitable solvent such as tetrahydrofuran or dimethylformamide, and the resulting solution can be deposited on the surfaces of the electrodes preferably using a spin cast instrument. The deposition of membrane formulation on the electrodes may produce a thin layer of ion selective membrane at micrometer or sub-micrometer thickness. After deposition, the ion selective membrane layer may preferably be dried and the electrodes deposited with ion selective membrane may be stored in a desiccated container before final lamination to form the electrochemical biosensor.
[0069] The ion-selective membrane formed from the membrane formulation can be selectively permeable to an ion whose concentration is to be determined. Interaction of ions (electrolytes) in the test sample with corresponding ionophores in the membrane can alter the electrical potential across the membrane which can be measured as a change in potential between the ion selective electrodes and the reference electrode.
[0070] In accordance with embodiments of the present disclosure, the surfaces of ion selective electrodes and the reference electrode can be deposited with a plurality of nanostructures prior to coating the electrodes with the membrane formulation. The nanostructure coating can increase the surface area of the electrodes. The nanostructures may be deposited only on the surface of ion selective electrodes or it may be deposited on both ion selective electrodes and reference electrode. The increase in surface area of ion selective electrodes can enhance the sensitivity and accuracy of the biosensor even with test samples containing very low concentration of target ionic species.
[0071] As used herein, the term "nanostructures" refer to solid particles or hollow-core particles, which can have particle size less than 500 nm, preferably less than 100 nm, more preferably less than 50 nm.
[0072] The non-limiting exemplary nanostructures according to the present disclosure can be selected from carbon nanotubes (CNTs) or gold nanoparticles.
[0073] In an exemplary embodiment, the nanostructures can be gold nanoparticles deposited over the ion selective electrodes using the electro deposition technique.
[0074] In another exemplary embodiment, the nanostructures can be carboxylated carbon nanotubes and the percentage of carboxylation of carbon nanotubes can range from 3% to 5%.
[0075] The nanostructures can be deposited over the surfaces of ion selective electrodes and the reference electrode by drop casting a solution of nanostructure in a suitable buffer.
[0076] In an exemplary embodiment, multi-walled carboxylated carbon nanotubes can be mixed with diethanolamine buffer and the resulting solution can be drop casted on surfaces of ion selective and reference electrodes to form a nanostructure layer on the electrode surfaces.
[0077] In another aspect, the present disclosure provides a disposable electrochemical biosensor incorporating an ion-selective membrane for potentiometric measurement of target ion in a biological fluid, wherein the biosensor can include (a) a polymeric base plate, (b) at least one ion selective electrode and a reference electrode disposed on the polymeric base plate, (c) a plurality of nanostructures deposited over the at least one ion selective electrode and the reference electrode to form a layer, and (d) an ion selective membrane covering at least a portion of the nanostructure layer, wherein the ion selective membrane is formed from a membrane formulation comprises an ionophore selective for the target ion, at least one polymeric material, at least one plasticizer, and a pore former.
[0078] In another aspect, the present disclosure provides a disposable electrochemical biosensor incorporating an ion-selective membrane for potentiometric measurement of sodium ion, potassium ion and chloride ions in a biological fluid, wherein the biosensor can include (a) a polymeric base plate, (b) three ion selective electrodes and a reference electrode disposed on the polymeric base plate, (c) an ion selective membrane covering at least a portion of the ion selective electrodes and the reference electrode, wherein the ion selective membrane is formed from a membrane formulation comprises calixarene, valinomycin, tetradimethylammonium chloride, polyvinyl chloride, dioctyl sebacate and potassium tetrakis(4-chlorophenyl)borate.
[0079] In another aspect, the present disclosure provides a method for making a disposable electrochemical biosensor having an ion-selective membrane for measuring target ions in a biological fluid, wherein the method can include the steps of (a) providing a polymeric base plate, (b) disposing at least one ion selective electrode and a reference electrode on the polymeric base plate, (c) optionally depositing a plurality of nanostructures over the at least one ion selective electrode and the reference electrode to form a layer, (d) applying onto the electrodes a membrane formulation in solution with a volatile organic solvent, wherein the membrane formulation comprises an ionophore selective for the target ions, at least one polymeric material, at least one plasticizer, and a pore former, and (e) evaporating the volatile organic solvent from the membrane formulation to form a single film on the electrode surface.
[0080] According to embodiments, the disposable biosensor of the present disclosure can be directly or indirectly connected to a display device and can allow the users to view the test results immediately in a more direct, economic and efficient manner.
[0081] In yet another aspect, the present disclosure provides a diagnostic device including the electrochemical biosensor of the present disclosure for convenient and repeated measurement of clinically important electrolytes in biological fluids. FIG. 1 illustrates a preferred configuration of a diagnostic device assembly. As shown in FIG. 1, the diagnostic device 100 can be constructed of a top cover 102, sample adhesive layer 104, channel adhesive layer 106, an electrochemical biosensor 108, vent hole layer 110 and bottom cover 112. [0082] The top cover 102 of the diagnostic device 100 can be made of a rigid material, preferably plastic, capable of repetitive deformation without cracking and can include a sample entry port through which a test sample can be introduced. The top cover 102 can be joined to the bottom cover 112 in a sealing operation to complete the assembly. The channel adhesive layer 106 can comprises a channel that is laser or die cut therein. The channel adhesive layer 106 can serve as a pneumatic seal around the channel to limit the volume of test sample exposed to the electrochemical biosensor 108.
[0083] It is to be appreciated that the design of the diagnostic device 100 and the components thereof are purely exemplary and the diagnostic device 100 and its components can take any desired size, shape and thickness to suite configuration of matching parts.
[0084] The electrochemical biosensor of the present disclosure can allow for multiplexing and enable simultaneous measurement of electrolytes such as potassium ion (K+), sodium ion (Na+),
2+
chloride ion (CI ), calcium ion (Ca ) and pH in biological fluids with very low sample volume. The electrochemical biosensor is highly selective and can reduce the change in electric potential caused by interfering electroactive substances present in biological samples. Further, the electrochemical biosensor of the present disclosure can be highly resistant to change in temperature, pH, or other stimuli, that can have adverse effect on the selectivity, responsiveness, and accuracy of sensor.
EXAMPLES
[0085] The present invention is further explained in the form of following examples. However it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.
Example 1: Sodium ion sensing membrane formulation
[0086] Polyvinyl chloride (31 wt%), dioctyl sebacate (63 wt%) and potassium tetrakis(4- chlorophenyl)borate (20 mol % of ionophore) were weighed into a glass vial and dissolved in 3 ml of tetrahydrofuran. To this mixture was added calixarene (3 wt%) and the resulting mixture was stirred further to get a homogeneous solution. Example 2: Potassium ion sensing membrane formulation:
[0087] Polyvinyl chloride (33 wt%), dioctyl sebacate (63 wt%) and potassium tetrakis(4- chlorophenyl)borate (20 mol % of ionophore) were weighed into a glass vial and dissolved in 3 ml of tetrahydrofuran. To this mixture was added Valinomycin (3.6 wt%) and the resulting mixture was stirred further to get a homogeneous solution.
Example 3: Chloride ion sensing membrane formulation:
[0088] Polyvinyl chloride (50 wt%) weighed into a glass vial and dissolved in 3 ml of tetrahydrofuran. To this mixture was added tetradimethylammoniumchloride (TDMAC) (50wt%) and the resulting mixture was stirred further to get a homogeneous solution.
Example 4: Membrane deposition
[0089] For each sensor, 3 microlitres of the membrane formulation was deposited on electrode surfaces previously coated with nanostructures, using a spin cast instrument. The solvent was allowed to evaporate overnight before use.
Example 5: Nanostructure deposition on electrode surface
[0090] Multi-walled carboxylated carbon nanotubes (COOH-CNTs) solution was prepared by adding COOH-CNTs in 0.1M diethanolamine buffer to get a final concentration of lOmg/ml. 6μ1 of COOH-CNTs solution was drop casted on electrode surface. The COOH-CNTs was dried at 60°C for 10 minutes.
Example 6: Membrane formulation for sensing sodium and potassium ions
[0091] A single membrane formulation for sensing multiple ionic species such as sodium and potassium ions in a test sample was prepared in the same manner as described in above examples. The formulation was prepared by using same percentage by weight of polyvinyl chloride, dioctyl sebacate, potassium tetrakis(4-chlorophenyl)borate and sodium and potassium ionophores. ADVANTAGES OF THE PRESENT INVENTION
[0092] The present disclosure provides a membrane formulation for use in electrochemical biosensors having excellent selectivity for target ions such as potassium ion (K+), sodium ion (Na+), chloride ion (CI~), calcium ion (Ca2+) and pH present in biological fluids.
[0093] The present disclosure provides an electrochemical biosensor having an ion-selective electrode membrane that enables high-precision measurement of potassium ion (K+), sodium ion (Na ), chloride ion (CI ), calcium ion (Ca ) and pH in biological fluids.
[0094] The present disclosure provides an electrochemical biosensor that has high selectivity and accurately senses the target ionic species even in low concentration range of blood samples.
[0095] The present disclosure provides an electrochemical biosensor that is disposable but very cost-efficient to manufacture.
[0096] The present disclosure provides an electrochemical sensor that has good reproducibility and multiplexing capabilities.
[0097] The present disclosure provides an electrochemical sensor that requires relatively lower sample volumes than known sensors.
[0098] The present disclosure provides an electrochemical biosensor that is highly stable and has excellent storage stability.
[0099] The present disclosure provides an electrochemical sensor that overcomes the drawbacks of the prior art.

Claims

We Claim:
1. An ion-selective membrane for use in electrochemical biosensor for measuring a target ion in a biological fluid, wherein said membrane is formed from a membrane formulation comprising: an ionophore selective for said target ion;
at least one polymeric material;
at least one plasticizer; and
a pore former.
2. The membrane of claim 1, wherein said biological fluid contains one or more target ions, and said membrane formulation comprises an ionophore selective for each of said target ions.
3. The membrane of claim 1, wherein said ionophore is selective for an ion selected from the group consisting of sodium ion, potassium ion and chloride ion.
4. The membrane of claim 2, wherein said ionophore selective for sodium ion is calixarene.
5. The membrane of claim 2, wherein said ionophore selective for potassium ion is valinomycin.
6. The membrane of claim 2, wherein said ionophore selective for chloride ion is tetradimethylammonium chloride.
7. The membrane of claim 1, wherein said at least one polymeric material is selected from polyvinyl chloride, polyvinyl acetate, silicone rubber, cellulose acetate or copolymers thereof.
8. The membrane of claim 7, wherein said at least one polymeric material is polyvinyl chloride.
9. The membrane of claim 1, wherein said at least one plasticizer is selected from the group consisting of dialkyl aryl phosphonates, trialkyl phosphates, trialkyl phosphites, dialkyl sebacates, dialkyl adipates, dialkyl phthalates, nitrophenyl alkyl ethers and 2-nitrophenyl aryl ethers.
10. The membrane of claim 9, wherein said at least one plasticizer is dioctyl sebacate.
11. The membrane of claim 1, wherein said pore former is potassium tetrakis(4- chlorophenyl)borate.
12. The membrane of claim 1, wherein said biological fluid is selected from whole blood, blood serum or blood plasma.
13. An ion-selective membrane for use in electrochemical biosensor for measuring sodium ion and potassium ion in a biological fluid, wherein said membrane is formed from a membrane formulation comprising: calixarene and valinomycin;
polyvinyl chloride;
dioctyl sebacate; and
potassium tetrakis(4-chlorophenyl)borate.
14. The membrane of claim 13, wherein said membrane formulation comprises:
3% by weight of calixarene and 3% by weight of valinomycin;
30 to 35 % by weight of polyvinyl chloride;
60 to 67 percent by weight of dioctyl sebacate; and
20 to 30 mol % of potassium tetrakis(4-chlorophenyl)borate.
15. An electrochemical biosensor for measuring target ion in a biological fluid, said biosensor comprising: a polymeric base plate;
at least one ion selective electrode and a reference electrode disposed on said polymeric base plate; and
an ion selective membrane covering at least a portion of said at least one ion selective electrode and said reference electrode, wherein said ion selective membrane is formed from a membrane formulation comprising: an ionophore selective for said target ion, at least one polymeric material, at least one plasticizer, and a pore former.
16. The sensor of claim 15, wherein said biological fluid contains one or more target ions, and said membrane formulation comprises an ionophore selective for each of said target ions.
17. The sensor of claim 15, wherein said ionophore is selective for an ion selected from the group consisting of sodium ion, potassium ion and chloride ion.
18. The sensor of claim 17, wherein said ionophore selective for sodium ion is calixarene.
19. The sensor of claim 17, wherein said ionophore selective for potassium ion is valinomycin.
20. The sensor of claim 17, wherein said ionophore selective for chloride ion is tetradimethylammonium chloride.
21. The sensor of claim 15, wherein said polymeric material is polyvinyl chloride.
22. The sensor of claim 15, wherein said plasticizer is dioctyl sebacate.
23. The sensor of claim 15, wherein said pore former is potassium tetrakis(4- chlorophenyl)borate.
24. An electrochemical biosensor for measuring target ion in a biological fluid, said biosensor comprising: a polymeric base plate;
at least one ion selective electrode and a reference electrode disposed on said polymeric base plate;
a plurality of nanostructures deposited over said at least one ion selective electrode and said reference electrode to form a layer; and
an ion selective membrane covering at least a portion of said nanostructure layer, wherein said ion selective membrane is formed from a membrane formulation comprising: an ionophore selective for said target ion, at least one polymeric material, at least one plasticizer, and a pore former.
25. A method for making an electrochemical biosensor having an ion-selective membrane for measuring target ion in a biological fluid, said method comprising the steps of: providing a polymeric base plate;
disposing at least one ion selective electrode and a reference electrode on said polymeric base plate;
optionally depositing a plurality of nanostructures over said at least one ion selective electrode and said reference electrode to form a layer;
applying onto said at least one ion selective electrode and said reference electrode a membrane formulation in solution with a volatile organic solvent, wherein said membrane formulation comprises an ionophore selective for said target ion, at least one polymeric material, at least one plasticizer, and a pore former; and
evaporating said volatile organic solvent from said membrane formulation to form a single film on said ion selective electrode and said reference electrode.
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