WO2016038529A1

WO2016038529A1 - Device and method for non-enzymatic and electrochemical detection of glucose bioanalyte

Info

Publication number: WO2016038529A1
Application number: PCT/IB2015/056854
Authority: WO
Inventors: Vinay Kumar; Navakant BHAT
Original assignee: Indian Institute Of Science
Priority date: 2014-09-08
Filing date: 2015-09-08
Publication date: 2016-03-17

Abstract

An electrochemically active device is provided for collecting and retaining a biological sample with electrodes connected to conductive tracks arranged on a substrate. The device is provided with a non- enzymatic and an electrochemically active receptor and the receptor is in chemical contact with the electrodes and a biological sample with a bioanalyte. The present invention also provides a holder and a point-of-care biosensor for the device of the present invention and a method for measuring glucose bioanalyte in a reduce volume of the biological sample. The device, point-of-care biosensor and the method of the present invention facilitate accurate measurements concentrations of glucose bioanalytes by determining redox current values in the biological samples.

Description

DEVICE AND METHOD FOR NON-ENZYMATIC AND ELECTROCHEMICAL DETECTION OF GLUCOSE BIOANALYTE

Field of Invention

[001] This invention generally relates to biosensors and methods for quantitative measurement of bioanalytes in biological samples. More particularly, the present invention relates to a non-enzymatic and electrochemically active biosensor, for an accurate detection and quantitative measurement of glucose in biological samples of reduced volume.

Background of the invention

[002] Self-blood glucose monitoring is very important part of diabetes management. Glucometers are a very important tool for diabetic patients as well as for doctors because patients can adjust their medicine doses based on the blood glucose trends, particularly in the case of insulin dependent type-1 diabetic patients. According to American Diabetes Association (ADA) recommendations for insulin dependent patients, blood glucose measurements are required to be performed at least three or more times a day. Patients treated with less frequent insulin injections, oral glucose- lowering drugs or diet alone are also encouraged to use self-monitoring blood glucose (SMBG) as an aid in achieving target glycemia.

[003] Diabetes is the main driving force behind the point-of-care glucometer industry. Most of these glucometers are based on the enzymatic detection of blood glucose. The enzymatic detection of blood glucose is based on the glucose oxidase (GOx) electrochemistry. The blood glucose is oxidized into gluconolactane while oxygen is reduced into H₂O₂. Despite the fact that is widely used, glucose oxidase still has problems like critical operating conditions such as optimum temperature below 44°C, pH range 2-8, chemical instability and high cost. According to the international diabetes federation, 80% of people with diabetes live in low and middle-income countries and per test cost of enzymatic glucometer strips is still not economical for this population.

[004] With the advancement in technology, lots of point-of-care glucometer options are available. These are based on electrochemical as well as photometric techniques. Most of these glucometers use the glucose oxidase enzyme for electrochemical detection of blood glucose. [005] US6475750B 1 discloses the hydrogel based glucose biosensor based on glucose binding molecule such as concanavalin A (Con A) and D-mannopyranoside. The immobilized hexose saccharide competitively binds with free glucose to the glucose- binding molecules, thus changing the number of crosslinks in the hydrogel, which changes hydrogel swelling tendency and the pressure of the hydrogel in its confined space in proportion to the concentration of free glucose. By measuring the change in hydrogel pressure with a pressure transducer, the biosensor is able to accurately measure the concentration of the tree glucose molecule.

[006] US6033866A discloses the amperometric glucose sensor based on two redox mediator, glucose oxidase and potassium ferrocyanide.

[007] US20070131549A1 discloses a system for more accurately measuring glucose in a sample includes a first glucose-sensing electrode incorporating a quantity of glucose oxidase, a second glucose-sensing electrode incorporating a quantity of PQQ- glucose dehydrogenase, a reference electrode, and means for selecting between a first glucose measurement made with the first glucose-sensing electrode and a second glucose measurement made with the second glucose-sensing electrode.

[008] US20140061044A1 discloses a non-enzymatic glucose sensor and method based on CuO nano particles in alkaline medium. The glucose sensor contains at least one non-enzymatic electrode configured to catalyze the electro-oxidation of glucose in preference to other bio-molecules.

[009] US20150144486A1 discloses non-enzymatic glucose sensors based on a polymeric film composition that includes a functional polymeric film. The polymeric film composition includes polyurethane and perfluorosulfonic acid polymer.

[010] US5217691A discloses a semi quantitative determination of glucose based on non-enzymatic technique. Glucose concentration in an aqueous test sample can be determined by preparing a test solution by contacting an aqueous test sample and a di hydroxide component, at an initial pH above 6.5, capable of forming a complex with glucose, which releases a proton, and determining the final pH of the test solution.

[011] All the above methods involve use of enzymes and implementation of complex functionalization steps on electrode surface.

[012] Therefore, there is a need to provide a non-enzymatic and electrochemically active biosensor, for an accurate detection and quantitative measurement of glucose in biological samples of reduced volume.

Objects of the present invention

[013] The primary object of the present invention is to provide an electrochemically active device with a non-enzymatic receptor, for collection and retention of biological samples of reduced volume, for a subsequent quantitative detection of glucose bioanalyte.

[014] An object of the present invention is to provide a device holder, adapted to receive the electrochemically-active device with a non-enzymatic receptor of the present invention.

[015] Another object of the present invention is to provide a point-of-care biosensor, adapted to receive the electrochemically-active device with a non-enzymatic receptor of the present invention, for the detection and quantitative measurement of glucose bioanalyte, in biological samples of reduced volume, through a measurement of redox current flowing through the electrochemically active device, on the application of an redox potential.

[016] It is also an object of the present invention to provide a method for the detection and quantitative measurement of glucose bioanalyte concentrations, through an accurate measurement of redox current flowing through the electrochemically-active and non-enzymatic device.

Brief description of the drawings

[017] FIG.l is a schematic exploded view of an electrochemically-active device with a non-enzymatic receptor, depicting a two-electrode arrangement, in accordance with an aspect of the present invention.

[018] FIG.2 is a schematic exploded view of a three-electrode arrangement, of an electrochemically-active device with a non-enzymatic receptor, in accordance with another aspect of the present invention.

[019] FIG.3 is a schematic exploded view of two pairs of three-electrode arrangement with trays, of an electrochemically-active device with a non-enzymatic receptor, in accordance with yet another aspect of the present invention.

[020] FIG.4 (a) is schematic top view of an electrochemically-active device with a non-enzymatic receptor with a three -electrode arrangement for quantitative measurement of urine glucose and blood glucose.

[021] FIG.4(b) is a cross-sectional view of an electrochemically active device with a non-enzymatic receptor, where the non-enzymatic receptor is arranged on the surface of a receptor-membrane.

[022] FIG.4(c) is a cross-sectional view of an electrochemically active device, with a non-enzymatic receptor, where the non-enzymatic receptor is arranged on the surface of electrodes.

[023] FIG.4(d) is a cross-sectional view of an electrochemically active device, where non-enzymatic receptor, is an integral part of the electrode.

[024] FIG.5 is a perspective view of device holder holding the device of the present invention, for quantitative measurement and display of urine glucose and blood glucose.

[025] FIG.6(a) is a perspective view of point-of-care biosensor holding the device of the present invention.

[026] FIG.6(b) is a schematic depiction of broad internal electronic architecture of the point-of-care biosensor.

[027] FIG.7 shows the UV-VIS spectra of methylene blue (MB) and leucomethylene blue (LMB) with NaOH in deionised water (DI) water.

[028] FIG.8 shows a decrease in absorption spectrum of methylene blue (MB) with different glucose concentrations in DI water.

[029] FIG.9 shows the decrease in absorption spectrum of MB with different glucose concentrations in human blood plasma.

[030] FIG.10 shows the cyclic voltamogram of methylene blue in phosphate buffer saline.

[031] FIG.ll is a high-level flow chart depicting process steps to measure quantitatively the concentration of glucose bioanalyte by using the device and point-of- care biosensor of the present invention.

[032] FIG.12(a) is a cyclic voltamogram of MB-NaOH with different concentrations of glucose in human blood plasma.

[033] FIG.12(b) depicts reduction currents plot versus glucose concentration in human blood plasma.

Summary of the present invention

[034] The present invention provides an electrochemically active device for collecting and retaining a biological sample with a glucose bioanalyte, the device comprising at least a two-electrode member and an electrochemically active and non-enzymatic receptor in chemical contact with the two-electrode members. The present invention also provides a point-of-care biosensor with device of the present invention and method of measuring glucose bioanalyte in a biological sample. The device, point-of-care biosensor and the method of the present invention facilitate accurate measurements concentrations of glucose bioanalytes by determining redox current values in the urine and blood samples.

Detailed description of the invention

[035] Accordingly, the present invention provides an electrochemically active biosensor with a non-enzymatic receptor, for an accurate detection and quantitative measurement of urine and blood glucose, in reduced volumes of biological samples.

[036] In an aspect of the present invention an electrochemically active device for collecting and retaining a biological sample is provided with at least a pair of conductive tracks that are arranged on a substrate. At least a two-electrode member is connected to the conductive tracks. A non-enzymatic and an electrochemically active receptor is arranged to be in chemical contact with the electrode member and a biological sample with a bioanalyte. The receptor, which is in chemical contact with the electrode member, is arranged to receive and retain a desired biological sample, of reduced volume.

[037] In another aspect of the present invention the preferred material for the substrate is a polymer or a paper.

[038] In yet another aspect of the present invention a a three -electrode member is arranged on the substrate.

[039] In further aspect of the present invention a plurality of two-electrode members are arranged on the substrate.

[040] It is also an aspect of the present invention where a plurality of three-electrode members are arranged on the substrate. [041] In yet another aspect of the present invention the electrode members are patterned electrodes.

[042] In further aspect of the present invention the receptor is arranged on a receptor- membrane, where the receptor-membrane is a polymer, cellulose, nitrocellulose, nylon, cotton fabric or a paper.

[043] In yet another aspect of the present invention, the receptor is arranged directly on at least two-electrode member, without having a receptor-membrane.

[044] In further aspect of the present invention, a combination of an electrochemically active organic substance and an inorganic substance is used for the formation of the receptor, where the preferred organic substance is organic substance is methylene blue and the inorganic substance is NaOH and KOH.

[045] It is also an aspect of the present invention where the device is arranged in a housing such as a cartridge or a cassette.

[046] In yet another aspect of the present invention, a holder or a device holder for holding an electrochemically active and non-enzymatic device with a biological sample, the holder is provided with a device detection and signal conditioning arrangement and the holder is disposed in a housing. A USB connector arranged at one end of the housing and an electrically conductive port arranged at the other end of the housing. An electrochemically active device for collecting and retaining a biological sample is connected to the holder through the electrically conducting port. The device is provided with at least a pair of conductive tracks, which are arranged on the substrate to which at least a two-electrode member is connected. A non-enzymatic and an electrochemically active receptor is in chemical contact with the electrode member and the biological sample with glucose bioanalyte. The receptor is arranged to receive the biological sample and detect concentrations of glucose bioanalyte.

[047] In further aspect of the present invention a point-of-care biosensor for measuring a concentration of a glucose bioanalyte in a biological sample is provided. The point-of-care biosensor comprising a housing with a display member and an electrically conducting port. The electrochemically active device for collecting and retaining the biological sample is connected to the holder through the electrically conducting port. The device with at least a pair of conductive tracks arranged on a substrate along with the electrode member. A non-enzymatic and an electrochemically active receptor is chemical contact with the member and the biological sample with a bioanalyte. The receptor is arranged to receive the biological sample for detection of glucose bioanalyte. The point-of-care biosensor is provided with a digital controller and is configured to apply a redox potential to the electrode and measure redox current from said device. The corresponding concentration levels of glucose bioanalyte are retrieved and displayed by linearly matching the concentrations of the glucose bioanalyte.

[048] In further aspect of the present invention a database member with stored standard values of concentrations glucose bioanalyte in biological samples along with reciprocal redox currents, connected to said digital controller, of the point-of-care biosensor.

[049] In yet another aspect of the present invention, the biological sample that is used in conjunction with the point-of-care biosensor is urine or blood.

[050] In further aspect of the present invention a method for measuring concentrations of glucose bioanalyte in a biological sample is provided. In this method, a redox potential is applied to the electrochemically active device of the present invention with an non-enzymatic receptor, which is exemplarily connected to the device holder through the electrically conducting port. The receptor is in chemical contact with the electrode member and the biological sample of reduced volume with glucose bioanalyte. The concentration of the glucose bioanalyte is determined in the biological sample, by measuring a corresponding redox current and linearly matching it to concentrations of glucose bioanalyte.

[051] In the method of present invention the reduced volume is in the range of 1-300 microlitres (μί).

[052] Now, the preferred embodiments of the invention are now described by referring to the accompanied drawings. FIG.l depicts an electrochemically active device with a non-enzymatic receptor, adapted to collect and retain a biological sample, for subsequent measurement of glucose analyte present in the biological sample.

[053] The device 100 as shown in FIG.l is provided with a substrate 101, which acts as base on which other constituents of the device are constructed. The substrate 101, in this embodiment is exemplarily shown as an elongated rectangular structure. However, it is understood here that the substrate 101 can take other shapes such as square, circular depending on the shape and configuration of a biosensor that holds the device 100. The substrate 101 can be made of any suitable rigid or flexible material that is suitable for the incorporation of patterned electrodes. For instance, materials such as polyvinylchloride (PVC), polyethylene terephthalate (PET), polymethylmethacrylate (PMMA), epoxy fiber composites, polyamides composites, and paper can be used as preferred materials for the substrate 101. Whereas, the preferred rigid materials for the substrate 101 can be ceramic, glass or any other like materials. In any case, the selection of suitable material for the substrate 101 is made to ensure that the substrate 101 can not only provide a desirable strength and flexibility but also can act as an electrical insulator. Advantageously the substrate 101 is hydrophilic in nature to prevent percolation of the biological sample, when it comes in physical contact with the substrate 101. The surface of the substrate 101 is generally provided with a smooth texture. However, the substrate 101 can also be provided with a rough surface and/or with cavities or wells. The edges of the substrate 101 are also provided with suitable profiles, such as tapered or curved, to facilitate an easy ingress into and egress out of the biosensor.

[054] A pair of conductive tracks 102a and 102b are arranged on the substrate 101. The conductive tracks 102a and 102b are formed by using any patterning method such as screen printing, lithography, thermal evaporation, sputtering, laser patterning, preferably screen-printing. In an exemplary aspect, in FIG.l, pair of conductive tracks 102a and 102b is formed for implementation. However, the required number of conductive tracks can be suitably increased or varied. The routing of the conductive tracks 102a and 102b are exemplarily shown as straight tracks in FIG.l. Other suitable configurations for the conducting tracks such as polygons can be used. The material for the conductive tracks 102a and 102b can be an electrically conductive materials such as copper, aluminum, gold, silver, platinum, carbon, or any other suitable electrically conducting material or alloys of these materials. The material for the conducting tracks 102a and 102b can also be electrochemically active such as gold, platinum, mercury, carbon, glassy carbon and graphite. The conducting tracks 102a and 102b are used to establish an electrical connection with the biosensor of the present invention as hereinafter described.

[055] Pair of electrodes 103a and 103b is electrically connected to the conducting tracks 102a and 102b respectively, as shown in FIG.l. The electrodes 103a and 103b are overlaid on the conducting tracks 102a and 102b and arranged at the terminal ends of the conducting tracks 102a and 102b, so as to form a layer above the conducting tracks 102a and 102b, as shown in FIG.l.The material for the electrodes 103a and 103b are selected from metals, organics or alloys, which are electrochemically active, such as gold, platinum, mercury, carbon, glassy carbon and graphite. In the arrangement of electrodes as shown in FIG.l, the electrode 103a acts as a working electrode and whereas the electrode 103b is a counter electrode.

[056] A receptor-membrane 104 is arranged on the pair of electrodes 103a and 103b as shown in FIG.l, which acts as base member for the integration of a receptor as hereinafter described. The receptor-membrane 104 as used is with desired porosity, which is larger than the size of the glucose bioanalyte, which is preferably in the range of 7nm to 14 microns, to facilitate the desired level of permeability of glucose bioanalyte. The receptor-membrane 104 can also be made an integral part of the receptor 105. The material for the membrane 104 can be polymer, cellulose, nitrocellulose, nylon, cotton fabric, filter paper or any other commercially available membranes such as BIODYNE membrane from PALL life-sciences and GE Healthcare membranes.

[057] The device 100 of present invention is used for the detection and quantitative measurement of glucose bioanalyte, in human biological samples, such as urine and blood. Accordingly, in the present invention, an electrochemically active device with a non-enzymatic receptor 105, is in chemical contact with the membrane 104. The receptor 105, in this preferred embodiment, is shown as a layer of electrochemically active non-enzymatic substance. The electrochemically active and non-enzymatic substance that is used as the non-enzymatic receptor 105 to detect glucose bioanalyte in urine and blood samples, is a combination of an organic and inorganic substances. The preferred organic substance is methylene blue and the inorganic substance can be one of KOH or NaOH.

[058] The initiation of chemical contact of the receptor 105 with the electrodes 103a and 103b is performed in the following manner. A solution of receptor 105 is prepared and dispensed on the electrodes 103a and 103b and/or membrane and dried to form a solid chemical layer on the electrodes 103a and 103b and membrane 104.

[059] Alternately, the receptor solution is pre-mixed with a desired biological sample and a reduced volume of the pre-mixed solution is dispensed on the electrodes 103b and 103b and/or membrane 104, for the measurement of glucose bioanalyte.

[060] In another aspect of the present invention, the receptor solution is prepared separately and dispensed on the electrode/membrane. Thereafter, the desired biological sample having glucose bioanalyte is applied on the electrode, for its measurement.

[061] A passivation layer 106 is arranged to cover the conductive tracks as shown in FIG.l. The passivation layer 106 is used to provide protection for the conductive elements of the device and to precisely define the electrode region.

[062] The device 100 is housed in a housing (not shown in FIG.l), which can be a cassette or cartridge.

[063] Therefore, the present invention provides a device with a non-enzymatic receptor for receiving and holding a biological sample, of reduced volume for a subsequent measurement of glucose bioanalyte.

[064] In yet another aspect of the present invention, as shown in FIG.2, an arrangement of set of three electrodes 103a, 103b and 103c is implemented in conjunction with a receptor (as shown in FIG.l), where the electrodes 103a, 103b and 103c are connected to the conducting tracks 102a, 102b and 102c respectively, to collect and retain a biological sample. The increased number of electrodes facilitates the detection of a single bio-analyte in the biological sample with an increased accuracy. In this implementation the electrode 103c acts as a reference electrode. The preferred material for the reference electrode 103c is silver (Ag), a silver chloride (AgCl), silver/silver chloride (Ag/AgCl) or saturated calomel, where the potential of the electrodes does not change with time.

[065] In yet another aspect of the present invention as shown in FIG.3 two pairs of three -electrodes 103a, 103b, 103c, 103d, 103e and 103f are arranged on the conducting tracks 102a, 102b, 102c, 102d, 102e and 102f and are adapted for use to measure the concentration of bioanalytes in multiple biological samples. In this aspect, in case, the desired biological samples are blood and urine, shielded wells or trays are arranged on the electrodes to demarcate two different sensing areas, to facilitate an independent sensing of the biological samples. Accordingly, two receptors are provided in conjunction with each of the pair of the electrodes to receive these samples and separate measurement of concentrations of glucose in these two different biological samples is performed. In addition, if deemed necessary, physical partitions may be provided to separate the electrodes.

[066] As shown in FIG.4(a), depicts a top view of the electrochemically active device with a non-enzymatic receptor for the detection of glucose in blood and urine samples.

[067] FIG.4(b), which is a corresponding cross-sectional view depicting a substrate 101 on the surface of which conducting tracks 102a, 102b, 102c are arranged. A 3- electrode arrangement with a working electrode 103a, counter electrode 103b and reference electrode 103c is connected to the conducting tracks 102a, 102b, 102c. The membrane 104 is arranged on surface of the electrodes 103a, 103b and 103c. The receptor layer 105 is arranged on the surface of the membrane 104.

[068] FIG.4(c), which is a corresponding cross-sectional view depicting a substrate 101 on the surface of which conducting tracks 102a, 102b, 102c are arranged. A 3- electrode arrangement with a working electrode 103a, counter electrode 103b and reference electrode 103c connected to the conducting tracks 102a, 102b, 102c. The receptor layer 105 is arranged on surface of the electrodes 103a, 103b and 103c.

[069] FIG.4(d), which is a corresponding cross-sectional view depicting a substrate 101 on the surface of which conducting tracks 102a, 102b, 102c are arranged. A 3- electrode arrangement with a working electrode 103a, counter electrode 103b and reference electrode 103c connected to the conducting tracks 102a, 102b, 102c, where the electrodes are treated with the receptor 105. In this aspect, the electrochemically active device, the non-enzymatic receptor 105, is integrated with the electrode.

[070] Accordingly, the constructive embodiments as shown in FIG.4(a) (b), (c) and (d) are used to measure glucose bioanalyte in urine and blood samples.

[071] The foregoing embodiments illustrate the electrochemically active and non- enzymatic device of the present invention. In yet another aspect of the present invention, as shown in FIG.5, a device holder 200 for sensing a bioanalyte in a bio- sample, comprises a housing 201 with a device detection and internal circuitry and the housing 201 is adapted to connect to a processor and a display member. A device insertion port 203 is provided in the housing 201. The device 100, which is permitted to pass through the device insertion port 203, includes a substrate with at least a two- electrode member along with a non-enzymatic receptor, connected to the housing 301, and the receptor configured to receive a bio sample 204. A USB plug 202 is connected to the housing 201 as shown in FIG.5. The device holder 200 is used to collect and retain the biological sample for subsequent testing. The device holder 200 is also provided with device detection, signal conditioning and data acquisition features to identify the type of bioanalyte that is stored on the device 100. The device holder 200 enables the user to insert the holder 200 into a biosensor and collect the biological sample for measurement.

[072] The device holder 200 of the present invention, which is adapted to interface with a processor and a display unit, is connected to the processor and powered. The device 100 is then loaded into the device holder 200. The device detection circuitry inside the housing is adapted to indicate the detection of the designated device. When the device holder detects the device, the device is loaded with the biological sample and a desired redox potential is applied by the internal circuitry in the digital-to-analog converter (DAC) to the working electrode of the device with respect to the reference electrode. The redox current that is passing through the counter and working electrodes is measured by internal circuitry by using I to V converter. In this aspect, the device holder 200 is exemplarily shown as a dongle. However, it is understood that other suitable electronic devices, including wireless devices, can be suitably adapted for use as a device holder.

[073] The point-of-care biosensor 300 for sensing a glucose bioanalyte, in a biological sample, as shown in FIG.6 (a), comprises a housing 301. A micro USB 302 and micro SD card 303 are arranged in conjunction with the housing 300. The micro USB 302 is used to charge the biosensor 300 and micro SD card is used as a storage device. The housing 301 is also provided with a display member 304, which can be an LCD, LED, OLED, OMLED, TFT or any other such display devices, including touch-sensitive devices. A device insertion port 305 is provided in the housing 301. The device insertion port 305 is provided with metallic contacts to engage the device electrically. In other words, the insertion port 305 is provided to receive the device 100, through the electrode members of the device 100. The point-of-care biosensor 300 is provided to facilitate a user to use the device 100, in a simple way. The device 100 is initially inserted into the point-of-care biosensor 300 and loaded with a selected biological sample, in reduced volume, in the range of 1-300 uL, which entails a minimum invasive means in collecting the biological sample. The user is also at liberty to use the biosensor 300 at a room temperature and without concerning about other environmental factors such as humidity, temperature variation and storage conditions. The user by using the biosensor 300 is able to measure the concentration levels of the desired bioanalytes, in a substantially shorter period of time. The user is provided with an instantaneous and accurate display of the concentration of the glucose bioanalyte on the display member 304, since the inherent binding nature of bioanalyte is used in the biosensor 300 to measure the concentration levels. By using the biosensor 300 of the present invention, the user is enabled to use the biosensor without a need for active preparation of the biological sample before it is tested.

[074] Now, referring to FIG.6(b), an internal electronic hardware architecture of the biosensor 300 is described. A database member 306 is provided in the housing 301 to store standard values of redox current and bioanalyte concentration of glucose present in the biological samples. The database 306 also incorporates the data pertaining to historical and current data of concentrations of the bioanalytes. The executables that are required to perform the various functions of the biosensor 300 are stored on a medium of the biosensor 300. A digital controller 307 is provided in the housing 301 and connected to the database member 306 and configured to apply a redox potential to at least a two-electrode member having an electrochemically active and non-enzymatic receptor with a biological sample having glucose bioanalyte and to measure the corresponding redox current. The digital controller 307 is arranged to measure a redox current of the glucose bioanalyte by linearly matching with the value of concentration and display the value of measured concentration of the glucose bioanalyte.

[075] A power supply to the biosensor 300 is regulated by a power supply unit 308, which is connected to the biosensor 300. The power supply unit 308 includes both online and offline rechargeable battery with charging circuitry. A signal conditioning and device detection unit 309 is connected to the microcontroller 307 to detect the presence of the device 100 in the biosensor 300 and to apply the redox potential to the electrodes and measuring the redox current from the selected biological sample. Humidity and temperature sensors 310 and 311 are arranged in the housing 301. Once the measurement of the concentration levels of the bioanalyte is completed by the microcontroller 307, the concentration levels are displayed on the display member 304, along with historical data of the concentration levels of the bioanalyte.

[076] The present invention also provides a method for an accurate detection and quantitative measurement of glucose bioanalyte in a bio-sample. The desired biological samples such as blood or urine are collected in very small volumes i.e., in the range of micro litres (μί), from human subjects, with a minimally invasive means, by following standard protocols. In the method of present invention the preferred volume of the biological sample that can be used for the measurement of bioanalyte is preferably in the range of 1-300 micro litres (μί). The required volume of the biological sample is subject to the size of the surface area of the receptor of the device. The reduced collection of sample substantially reduces trauma in the subjects, since it is obtained through a minimally invasive sample extraction technique. The reduced volume of biological samples avoids the need for a user to phlebotomy collection products.

[077] In the method of the present invention, the determination and accurate measurement of a bioanalyte, is performed by implementing the principle of electrochemistry. Accordingly, the bioanalyte that is advantageously selected for its measurement is glucose through a measurement of redox current flowing through electrochemically-active devices, on the application of an electric potential.

[078] In the present invention the receptor substance is selected from a combination of organic and inorganic substances.

[079] In the method of present invention the receptor substance is prepared, advantageously as a solution of preferred chemical substances as hereinafter described. For instance, in case MB and NaOH is selected as a preferred receptor, MB is dissolved preferably in an alkaline aqueous solution (NaOH/KOH) or any other solvents which can dissolve these substances.

[080] The receptor solution thus prepared is applied to the electrode members or membranes of the device of the present invention to form a dry chemical layer of receptor, prior to the application of biological samples.

[081] The receptor solution thus prepared is applied to the electrode members or membranes of the device of the present invention, prior to the application of biological samples.

[082] Alternately, the receptor solution can also be premixed with the biological samples and the mixed solution is applied to the electrode members or membranes of the device.

[083] In an exemplary aspect the method for detection and measurement of human blood plasma glucose is now described. It is understood here that the same method can be equally made applicable to test the glucose bioanalyte in urine sample.

[084] In order to test the presence of glucose in a plasma sample, the reduced volume of the biological sample (plasma) is brought in chemical contact with the receptor of the device of the present invention. The receptor is a combination of organic and inorganic substance, which is MB and NaOH.

[085] The organic substance methylene blue (MB) is a water-soluble cationic dye that has been widely used in many applications ranging from medicine for malaria, methemoglobinemia, cancer, cyanides poisoning and for staining of biological samples. Methylene blue shows the adsorption peak at 660 nm under UV-VIS spectroscopy as shown in FIG.7. The absorption spectra of methylene blue and NaOH in DI water solution decreases with glucose concentration, as shown in FIG.8.

[086] Methylene blue and NaOH demonstrate UV-VIS absorption spectra at around 665 nm in human blood plasma. Whereas, the absorption of MB and NaOH in human blood plasma solution decreases with an increase in glucose concentration, as shown in FIG.9

[087] Methylene blue (MB) also demonstrates reversible peaks under cyclic voltammetry (CV), as shown in FIG.10. In the electrochemical and non-enzymatic detection of glucose of the present invention, in the alkaline medium, methylene blue reduces into leucomethylene blue in the presence of glucose. A combination of electrochemical reduction of methylene blue into leucomethylene blue, on the electrode surface and the chemical reduction of methylene blue into leucomethylene blue in the presence of glucose, is used to determine the level of glucose in the blood. The cyclic voltammetry reversible peak reduction reaction of methylene blue in alkaline medium decrease with glucose concentration. The reason for this decrease in peak current is due to reduction of a portion of methylene blue by chemical blue bottle reaction.

[088] Reduced quantity of whole blood or blood plasma is applied on to the electrodes and process steps as shown in FIG.ll. Redox potential is applied to both the sets of the electrodes and the corresponding redox current is measured from these electrodes.

[089] The measured redox current is matched with the stored redox current values and the matching glucose concentration is secured and displayed by the biosensor. Alternately, the linear-fit equation can also be used to compute the concentration of bioanalyte by using the redox current value. The biosensor after having extracted the value of concentration of glucose in the blood sample displays the value.

[090] It is also within purview of this invention, a photometric technique can also be used, as an alternate to or in addition to the electrochemical technique of measuring Redox current, to track the absorption spectra around 665nm, which follows the glucose concentration as illustrated in FIG.8 and FIG.9. Then the absorption spectra value can be linearly matched to the glucose concentration.

[091] The subject matter of the invention is now illustrated in the form of the following examples. These examples are provided for purpose of illustration and shall not be construed as limiting the scope of the invention.

Example 1: Determination of glucose concentration and corresponding reduction current using MB-NaOH as a receptor

[092] A master solution of NaOH is prepared by dissolving the 3.2 g NaOH per 10ml of saline water. A master solution of MB is prepared by dissolving the 50mg MB in 5 ml of DI water. 10 solution of MB and 10 solution of NaOH is mixed and used as a receptor for the detection of glucose bioanalyte in the human blood plasma sample. Equilibration time given before running the experiment: 1 minute 22 seconds.

[093] A 300 volume of the human plasma sample is taken and dispensed on the electrode of the biosensor device and the corresponding cyclic voltammogram is obtained by values using the CHI Electrochemical workstation using the potential window varies from -0.15 V to -0.6 V, as shown in FIG.12(a) .

[094] The plasma solution contains glucose, sodium hydroxide and methylene blue. In the first step an enolate of glucose is formed. The next step is a redox reaction of the enolate with methylene blue. The glucose is oxidized to gluconic acid, which in alkaline solution is in the form of sodium gluconate. Methylene blue is then reduced to colorless leucomethylene blue, thereby demonstrating a linear decrease in peak reduction current with glucose concentration as shown in FIG.12(a) and FIG.12(b). If the concentration of glucose in blood plasma sample is increased then the more methylene blue reduces into leucomethylene resulting in the decrease in peak reduction current of MB.

[095] The values of concentrations of the blood plasma glucose (mg/dL) along with corresponding reduction current values (μΑ) are recorded and tabulated as shown in Table 1.

[096] Table 1 is prepared from linear fit equation as given below, which is derived from the repeatability data plots:

j/ = -0.0994x + 60.19

In the above equation "y" represents the redox current value and "x" represents the concentration of analyte.

Table 1

Example 2: Measurement of glucose with methylene blue - NaOH receptor

[097] A sample volume of synthetic urine of 300 is placed on the electrode having the MB+NaOH then the peak reduction current value is noted from cyclic voltamogram specifying a potential window from -0.15V to -0.60V in CHI-Electrochemical workstation. The value of peak reduction current is 47.77 μΑ . This current value is looked in the Table 1 and the corresponding concentration of plasma glucose is obtained as 112 mg/dL.

[098] In the present invention non-enzymatic, non-antibody based and electrochemically active receptors are used in conjunction with electrodes, for quantitative measurement glucose bioanalyte in a biological sample.

[099] In the quantitative measurement of glucose bioanalyte of the present invention a minimal invasive technique where a reduced volume of sample volume is used.

[0100] It is also understood that the following claims are intended to cover all the generic and specific features of the invention herein described and all statements of the scope of the invention, which as a matter of language might be said to fall there between.

Claims

An electrochemically active device for collecting and retaining a biological sample, comprising:

(i) at least a pair of conductive tracks disposed on a substrate;

(ii) at least a two-electrode member connected to said at least pair of conductive tracks; and

(iii) a non-enzymatic and an electrochemically active receptor, said receptor in chemical contact with said at least two-electrode member and a biological sample with a bioanalyte.

The device as claimed in claim 1, wherein said substrate is a polymer or a paper.

The device as claimed in claim 1, wherein a three-electrode member disposed on said substrate.

The device as claimed in claim 1 , wherein a plurality of two-electrode members disposed on said substrate.

The device as claimed in claim 1, wherein a plurality of three-electrode members disposed on said substrate.

The device as claimed in claim 1, wherein said electrode members are patterned electrodes.

The device as claimed in claim 1, wherein said receptor disposed on a receptor- membrane, said receptor-membrane is a polymer, cellulose, nitrocellulose, nylon, cotton fabric or a paper.

The device as claimed in claim 1, wherein said receptor disposed directly on said at least two-electrode member, without said receptor-membrane.

The device as claimed in claim 1, wherein said receptor is a combination of an electrochemically active organic substance and an inorganic substance.

The device as claimed in claim 8, wherein said organic substance is methylene blue. The device as claimed in claim 8, wherein said inorganic substance is NaOH and KOH. The device as claimed in claim 1, wherein said device disposed in a housing, said housing is a cartridge or a cassette.

A holder for holding an electrochemically active device with a non-enzymatic device with a biological sample, said holder comprising: (i) a device detection and signal conditioning means disposed in a housing;

(ii) a USB connector disposed at one end of said housing and an electrically conductive port disposed at the other end of said housing; and

(iii) an electrochemically active device for collecting and retaining a biological sample is connected to said holder through said electrically conducting port, said device with at least a pair of conductive tracks disposed on a substrate; at least a two- electrode member connected to said at least pair of conductive tracks; and a non- enzymatic and an electrochemically active receptor, said receptor in chemical contact with said at least two-electrode member and a biological sample with a bioanalyte and said receptor disposed to receive said biological sample for detection of concentrations of glucose bioanalyte.

A point-of-care biosensor for measuring a concentration of a bioanalyte in a biological sample, comprising:

(i) a housing with a display member and an electrically conducting port;

(ii) an electrochemically active device for collecting and retaining a biological sample is connected to said holder through said electrically conducting port;

(iii) said device with at least a pair of conductive tracks disposed on a substrate; at least a two-electrode member connected to said at least pair of conductive tracks; and a non-enzymatic and an electrochemically active receptor, said receptor in chemical contact with said at least two-electrode member and a biological sample with a bioanalyte and said receptor disposed to receive said biological sample, detect concentrations of glucose bioanalyte; and

(iv) a digital controller disposed in said housing and configured to apply a redox potential and measure redox current from said device, retrieve and display concentration levels of glucose bioanalyte, by linearly matching the concentrations of the glucose bioanalyte.

The point-of-care biosensor, as claimed in claim 14, wherein a database member with stored standard values of concentrations glucose bioanalyte in biological samples along with reciprocal redox currents, connected to said digital controller.

The point-of-care biosensor as claimed in claim 14, wherein said biological sample is urine or blood. A method for measuring concentrations of glucose bioanalyte in a biological sample, comprising the steps of:

(i) applying a redox potential to an electrochemically active device with an non- enzymatic receptor and is connected to said holder through said electrically conducting port, said device with at least a pair of conductive tracks disposed on a substrate; at least a two-electrode member connected to said at least pair of conductive tracks; and a non-enzymatic and an electrochemically active receptor, said receptor in chemical contact with said at least two-electrode member and a biological sample with a bioanalyte and said receptor disposed to receive said biological sample; and

(ii) determining a concentration of glucose bioanalyte in said biological sample, by measuring a corresponding redox current and linearly matching it to concentrations of glucose bioanalyte.

The method as claimed in claim 17, wherein said biological sample is urine or blood. The method as claimed in claim 17, wherein said reduced volume is in the range of 1-300 microlitres (μΐ_^).

The method as claimed in claim 17, wherein said receptor disposed directly on said at least two-electrode member, without said receptor-membrane.