Claims:1. A biosensor strip comprising:
a plasma separating membrane; and
a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by blood sample enables quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable quantified analysis of the analyte.
2. The strip of claim 1, wherein the pre-wetting buffer is selected from one or a combination of Imidazole, Phosphate, Citrate, MOPS, HEPS, CHAPS, TRIS, Acetate and MES.
3. The strip of claim 2, wherein the pre-wetting buffer further comprises any or a combination of polymeric agents, sugars, emulsifying agents, anticoagulants, aggregating agents, and preservatives.
4. The strip of 1, wherein the plasma separating membrane is made from one or a combination of Cellulose acetate, filter paper, nylon, cotton fiber, glass fiber, polysulfone, and hydrophobicpolyester nylon mesh.
5. The strip of claim 1, wherein pre-wetting buffer is added in the range of 8-10µl drop size and blood sample is added in the range of 3-5µl drop size.
6. The strip of claim 1, wherein the analyte is selected from any of glucose, HbA1c, cholesterol, hormones, urea, triglycerides, ketone, lactate, creatinine and uric acid.
7. The strip of claim 1, wherein the reagent comprises a combination of a Dye, a Chromogenic Substrate, Enzyme(s), and an Enzyme Mix, wherein the Enzyme Mix comprises a Biomolecule Stabilizer.
8. The strip of claim 7, wherein the reagent further comprises one or a combination of Phosphate-citrate buffer, Imidazole buffer, cyclodextrin, organic solvent, Milli Q,PVP, PEG, and PMVMA.
9. The strip of claim 7, wherein the Enzyme Mix further comprises a buffer, an organic solvent, and a detergent.
10. The strip of claim 9, wherein the buffer is Sodium acetate buffer, the organic solvent is Ethylene glycol, and the detergent is Tween.
11. The strip of claim 7, wherein the Biomolecule Stabilizer comprises at least one amino-acid, at least one sugar, and at least one polymeric substance.
12. The strip of claim 10, wherein the at least one amino-acidis selected from one or a combination of DL-Aspartate, DL-Glutamate, DL-Methionine, DL-Lysin, L-Lysine, L-Arginine/L-Histidine, and wherein the at least one polymeric substance is selected from one or a combination of PMVMA, PVP, PVA, PEG, Gelatin, and BSA, and wherein the at least one sugar is selected from one or a combination of Sorbitol, Mannitol, Trehalose, Xylose, Ribose.
13. The strip of claim 1, wherein the strip comprises a strip backing material configured to hold the reaction membrane and the plasma separating membrane.
14. The strip of claim 13, wherein the strip backing material is made of thick laminated paper card.
15. A reagent composition for use in a biosensor strip to enable an efficient reaction with an analyte, said reagent composition comprising a combination of Dye(s), Enzyme(s), and an Enzyme Mix, wherein the Enzyme Mix comprises a buffer and a Biomolecule Stabilizer, and wherein the Biomolecule Stabilizer comprises at least one amino-acid, at least one sugar, and at least one polymeric substance, and wherein the reagent composition enables a minimum shelf life of 12 months at room temperature for the biosensor strip.
16. The reagent composition of claim 15, wherein the reagent composition further comprises one or a combination of Chromogenic Substrate Phosphate-citrate buffer, PVP, PEG, and PMVMA.
17. The reagent composition of claim 15, wherein the Enzyme Mix comprises Sodium acetate buffer, Ethylene glycol and Tween 20.
18. The reagent composition of claim 15, wherein the at least one amino-acid is selected from one or a combination of DL-Aspartate, DL-Glutamate, DL-Methionine, DL-Lys in, L-Lysine, L-Arginine/L-Histidine, and wherein the at least one polymeric substance is selected from one or a combination of PMVMA, PVP, PVA, PEG, Gelatin, and BSA, and wherein the at least one sugar is selected from one or a combination of Sorbitol, Mannitol, Trehalose, Xylose and Ribose.
19. The reagent composition of claim 15, wherein the Biomolecule Stabilizer further comprises one or a combination of a buffer, a chelating agent, an organic solvent, a protein, a preservative, and a salt substance.
20. The reagent composition of claim 19, wherein the buffer is any or a combination of Tris buffer, Imidazole, Sodium acetate, Phosphate, trisodium citrate, and citrate, and wherein the chelating agent is any or a combination of EDTA and Tri-Sodium citrate, and wherein the organic solvent is any or a combination of ethylene glycol, glycerol, Propylene glycol, and wherein the protein is any or a combination of Gelatin and BSA, and wherein the preservative is any or combination of thimerosol, azide, and providone, and wherein the salt substance is Ammonium sulphate.
21. The reagent composition of claim 15, wherein the analyte is selected from any or a combination of glucose, HbA1c, cholesterol, hormones, urea, triglycerides, ketone, lactate, creatinine, uric acid, HRP-IgG, HRP-Streptavidine, Protein A/G-IgG, Biotin-IgG, Recombinant Antigens and Antibodies.
22. A method for using a biosensor strip for analyzing an analyte, said method comprising the steps of:
adding a pre-wetting buffer on the biosensor strip, wherein the biosensor strip comprises a plasma separating membrane and a reaction membrane operatively coupled with the plasma separating membrane;
adding a blood sample on the biosensor strip to enable quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane;
obtaining a color on the biosensor strip based on the reaction between the reagent and the analyte after a pre-defined time interval; and
estimating the amount of the analyte based on the obtained color.
23. The method of claim 22, wherein the amount of the analyte is estimated based on one or a combination of visual chart and a computing means.
24. The method of claim 22, wherein the computing means involves any or a combination of a mobile phone, a smartphone, an optical reader device, and an image capturing device, wherein image capturing device of the computing means is configured to take an image of the colour and enable processing of the image for estimating the amount of the analyte.
25. A analyte detecting kit for semi-quantitative estimation of amount of an analyte, said kit comprising
a biosensor strip, said biosensor strip comprising:
a plasma separating membrane; and
a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by blood sample enables quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable color change of the reaction membrane; and
a chart configured to enable comparison of the colour of the reaction membrane with colours present in the chart to estimation the amount of the analyte.
26. A analyte detecting kit for quantitative estimation of amount of an analyte, said kit comprising
a biosensor strip, said biosensor strip comprising:
a plasma separating membrane; and
a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by blood sample enables quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable colour change of the reaction membrane; and
a computing device configured to take an image of the colour of the reaction membrane and analyze the colour to estimate the amount of the analyte.
, Description:TECHNICAL FIELD
[0001] The disclosure generally relates to the field of estimation of blood analyte(s). In particular, the present disclosure pertains to a biosensor strip for determining the presence, absence and/or the amount of analyte(s) in a liquid especially a biological liquid, and most especially blood.

BACKGROUND
[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] Maintaining near-normal glycaemia in patients suffering from diabetes mellitus (DM) has become a standard and a well accepted recommendation. Unfortunately, most people with DM could not achieve this clinical goal because of marked glycaemic fluctuations. Self-monitoring blood glucose (SMBG) systems have the potential to play an important role in management of diabetes and in reduction of risk of serious secondary clinical complications. Over the years, there has been a transition from simple urine sugar screening tests to more sophisticated meters and reagent strip systems to monitor blood glucose. More recently, the usage of blood glucose meters is on rise as they offer patient comfort and convenience of testing. Significant research has already been done in this area to reduce the volume of blood sample required for the test. Further, improvements in accuracy through advanced measurement algorithms have also been achieved.
[0004] Glucose meters (glucometers) as self-monitoring blood glucose (SMBG) systems have two essential parts: an enzymatic reaction and a detector. The enzyme portion of the glucose meter is generally packaged in a dehydrated state in a disposable strip. Chemical composition of these strips consists of enzymes specific for the analytes and signal transducers (in case of electrochemical methods) or chromogenic substrates (in case of reflectance based methods) along with stabilizers. Glucose present in patient’s blood sample rehydrates and reacts with enzymes to produce a product that can be detected. Some meters generate hydrogen peroxide or an intermediate product that can react with a dye, resulting in a colour change proportional to the concentration of glucose present in the sample. Other meters incorporate enzymes into a biosensor that generates an electron that can be detected by the meter. There are three principle enzymes utilized by current glucose meters: glucose oxidase, glucose dehydrogenase and hexokinase. Each enzyme has its own characteristic advantages and limitations. Electrochemical test strips have electrodes where a precise bias voltage is applied with a digital-to-analog converter (DAC), and a current proportional to the glucose in the blood is measured as a result of the electrochemical reaction on the test strip. Optical-reflectometry test strips operate on the principle of reflectance. Typically, a calibrated current passes through two light-emitting diodes (LEDs) which alternately flash onto the coloured test strip. A photodiode senses the reflected light intensity, which is dependent on the colour of the test strip, which, in turn, is dependent on the amount of glucose present in the blood sample.
[0005] Visual readout blood analyte test kits for semi-quantitative assay of glucose are also available for its utility as Point Of Care Test (POCT) at home or at clinic. During the course of analyte detection by visual read out methods, addition of blood sample to the test strip initiates reaction of specific enzymatic reagents with chromogenic substrates coated on the reaction membrane to give a colour change. Intensity of colour is directly proportional to analyte level present in the sample, which can be interpreted using a colour chart/reader within a given time frame. Exemplary visual readout semi quantitative glucose assay kits available in various brand names includes Chemcard (USA), Betacheck (Australia), Machery Nagel (Germany) etc. However, utility of such glucose meters are seriously jeopardized due to several limitations including cost of such kits and their strips, very short shelf life of strips, compatibility of such kits, user friendliness, and performance issues.
[0006] Over the years, the cost of glucometers has come down significantly. Still, initial cost of glucometer and recurring costs arising from purchase of its strips leave many patients devoid of affording such facilities. Further, additional expenses such as those arising from purchase of calibrators, batteries etc. are inevitable in case of use of any branded glucometers. In addition, most of the strips have a shelf life of only 3 months after opening the vial, so one really cannot purchase and keep large economical packs of strips.
[0007] Many glucometers operate with a coding mechanism and so, periodically such devices need to be activated or reactivated with an appropriate code for obtaining reliable results. Usage of insufficient blood sample volumes in test or improper addition of sample to the strip or failing to wipe the blood sample (30 seconds after sample addition) may give erroneous result in many glucometers or kits. Further, a variety of factors affect glucometer performance including, operator technique, environmental exposure, and patient factors such as medication, oxygen therapy, anemia, hypotension, and other disease states. Numerous reports are published till date reviewing the challenges involved in obtaining accurate glucometer results (e.g. Diabetes Science Technology vol. 3, (4), 2009).
[0008] It is a well known fact that the plasma separating membranes play a pivotal role in determining overall quality, utility and cost of glucometers. To our knowledge, following blood plasma separation techniques have been reported so far: PDMS chip format, CD format, and Membrane/paper format. Major limitations with first two microfluidic based plasma separation methods include -clogging of blood, complexities in device fabrication, slow and insufficient plasma separation, haemolysis and high cost. Comparatively, membrane/paper filter-based blood plasma separation provides a simple solution to on-chip blood plasma separation.
[0009] Initial reports in the technology domain suggest use of bibulous or moisture absorbing matrix that is impregnated with chromogenic testing reagent(s) and coated with a semi permeable barrier. The semi permeable barrier screens the cellular components of the whole blood sample and permits passage of the smaller, soluble molecules and ions to contact the chromogenic testing reagent incorporated into bibulous matrix. The analyte reacts with the chromogenic reagent to produce colour. However, colour produced in the matrix can be observed only after rinsing or wiping.
[0010] First generation blood glucose test strips use a reactive film and swelling agent in addition to detection chemistry. Exactly after one minute after addition of a drop of blood to the reactive film, blood need to be wiped off and at the end of 120 seconds, the colour developed can be read from the same side of strip. Limitation of such method includes, if RBCs are not completely wiped, they may create interference in the assay giving erroneous results. In addition, the possibility of technician coming in contact with potential infectious blood sample is high. To avoid wiping and/or rinsing, a disposable dry phase test stick with a reactive area covered by a detachable semipermeable membrane was reported. The semipermeable membrane separates cellular and particulate matter from whole blood allowing the plasma or serum to contact the reactive area. Once the plasma is separated, semipermeable membrane need to be detached from the reactive area to remove cellular components and particulate from the device and to expose the reactive area for examination of a response to a particular analyte. Another potential solution to avoid wiping and/or rinsing suggests usage of a porous membrane with porosity gradient such that the cellular components of the whole blood are retained in an area of the membrane having a low porosity. Major limitation of such a method includes interference from high molecular weight soluble plasma components such as cholesterol that may not be completely wiped from the surface of the strip.
[0011] Yang et al., (Lab Chip, 2012, 12,274)suggests use of µPAD fabricated whatman filter paper to extract plasma from whole blood and detect presence of analyte level in it. Addition of blood droplet to filter paper brings about agglutination of RBCs by specific agglutinating antibodies coated on the filter paper and thus allows the plasma to get separated by capillary diffusion. The separated plasma reaches the reaction zone, the specific analyte is acted upon by reagent coated on it, to give rise to a colour within a given time. The intensity of the colour developed is directly proportional to the anlyte level in the plasma. However, such methods to quantify blood glucose were noted to have several limitations including- this method requires at least 7 µL of blood and also requires minimum 5 minutes duration to bring about colour development. Moreover, linearity of detection (LOD) was only up to 200mg/dl and the assay sensitivity was only 50mg/dl. In addition, the device utilises very expensive agglutinating antibodies (Anti-A, Anti-B), the stability of the device at room tempt storage and its performance was not studied.
[0012] Second generation blood glucose test strips use Glass fiber membranes. However, matrices (membranes) made of coated or uncoated glass fibre filters for separating plasma or serum from whole blood incorporate agglutinating agents that exhibit several problems including, relatively slow speed of separation and only upto 50% to 60% of plasma being separated forcing the operator to apply large amount of blood sample. Moreover, RBC lysis (to some extent) was also noticed with use of such membranes in point of care assay devices. Presence of HEPES buffer impregnated in the glass fibre blood separation matrix may bring about rapid plasma separation and analyte detection. Still, such SMBG devices and processes are far from satisfactory.
[0013] Third generation blood glucose test strips use polysulfone/asymmetric membranes to achieve plasma separation. In such membranes, cellular components of blood get trapped inside the pores of the membrane to separate the plasma from whole blood sample. However, RBCs do not get trapped in these membranes. An alternative approach to blood separation involves incorporating agglutinating reagents or other separation reagents in such matrix. For example, Agglutinating agent (Lactin, concavaline A) or polyamino acids (Polylysine) or Polybrene or antibodies brings about separation of RBC in the sample. Roche Accucheck active and Accucheck trend uses nylon/polyester mesh of suitable pore size (20-200 µm) which acts as a 1st layer. This mesh is pre-treated with non-hemolytic detergent to make it hydrophilic and to enhance rate of wicking and for uniform spreading of the blood droplet
(2 µL). Furthermore, an asymmetric membrane (second layer), placed below the nylon mesh, acts as a blood separation membrane on its one side and acts as a reaction membrane on its other side. However, such membranes need to be pre-treated with hydrophilic polymers such as hydroxypropylcellulose (HPC), polyvinyl alcohol (PVA), or carboxymethylcellulose (CMC), followed by coating with one or more hemolysis inhibitors like hydrophobic aminocarboxylic acids, proteins derived from silk, Tris, TES, aminohexanoic acid, tranexamic acid, heparin etc. for performing efficient plasma separation without any haemolysis.
[0014] Several reports suggest use of polyester mesh, woven fabrics or non woven fabrics as spreading layer and/or plasma separation membrane. Patrick et al., in US patent 5296192A, have shown that use of polyester mesh (A single layer of a fabric of polyester, cotton, or a 50% polyester/50% cotton blend) pre-treated with non-hemolytic detergent and agglutinating agents like lectins or antibodies, bring about uniform spreading of blood sample and offer complete removal of RBCs without lysis. This technique helps in uniform colour development irrespective of sample application to the strip or its volume to the strip. However, adequate plasma separation in such strips can only be achieved by use of multiple membranes (spreading screen acting as a first level of filter, screen mesh acting as a second filter and reaction membrane acting as a third level of filter). Use of multiple membranes for plasma separation forces the operator to use higher amount of blood sample as these membranes absorb aqueous contents of blood increasing its viscosity. Smith John in US patent 5753452 suggests that many polymeric substance such as polyethylene glycol, poly (methylvinylether/maleic) anhydride [PMVMA], polypropylene glycol, polystyrene sulfonic acid, polyacrylic acid, polyvinyl alcohol[PVA], and polyvinyl sulfonic acid at a pH of about 4.0-8.0 can bring about clear plasma separation by sequestering RBC in the matrix and also free hemoglobins, e.g., whole blood, by sequestering red blood cells in the matrix and, preferably, also sequestering any small amounts of free hemoglobin. However, accurate measurement of blood glucose as disclosed in the document is highly dependent upon measurement of change in reflectance at about 700 nm that is substantially equivalent to that produced by the absorbance of haemoglobin in blood and a change in reflectance at about 635 nm that is indicative of the glucose concentration requiring complicated and expensive optical means for detecting intensity of light at these wavelengths.
[0015] From careful observation of the technology domain and prior art SMBG methods, one skilled in the art would immediately realize one or more challenges with currently available glucometers including, but not limited to, non-compatibility to stored venous EDTA blood (most of the point of care assays requires use of fresh venous capillary blood during testing), slow and inefficient plasma separation (most glucometers require 30 seconds to 1 minute to achieve satisfactory plasma separation), interference from cellular components of blood sample, high lipid level, varying hematocrite, haemolysis, clogging, high cost associated with glucometers and their strips, and less shelf life of consumables (test strips) for glucometers.
[0016] There is therefore a need in the art for improved glucometers, and glucose/analyte testing strips and methods that can enable semi-quantitative or quantitative visual readout of blood glucose for low cost diabetes screening and monitoring.

OBJECTS OF THE INVENTION
[0017] An object of the present disclosure is to overcome disadvantages associated with conventional glucometers and glucose testing strips.
[0018] Another object of the present disclosure is to provide a glucose testing/biosensor strip that uses commonly available low cost membranes for achieving desired plasma separation.
[0019] Another object of the present disclosure is to improve plasma separation efficiency of commonly available low cost membranes.
[0020] Another object of the present disclosure is to provide a buffer composition that can be used to pre-wet commonly available low cost membranes to improve their plasma separation efficiency rapidly in a non-hemolytic manner without hematocrite effect.
[0021] Another object of the present disclosure is to provide a glucose/analyte testing/biosensor strip with enhanced stability and longer shelf life at room temperature storage conditions in its vial.
[0022] Another object of the present disclosure is to provide a glucose/analyte testing/biosensor strip comprising only two membranes, one membrane for achieving plasma separation and other as a reaction membrane.
[0023] Another object of the present disclosure is to provide a colour chart for semi-quantitative estimation of blood glucose by Visual readout method.
[0024] Another object of the present disclosure is to provide a smartphone, its optical reader and associated software for quantitative estimation of blood glucose.
[0025] Another object of the present disclosure is to provide a glucometer with capability of detection of indirect semi-quantitative HbA1c levels using fasting blood glucose analysis.
[0026] Another object of the present disclosure is to provide a glucose/analyte testing/biosensor strip made out of low cost, eco-friendly thick paper material for sensor fabrication.

SUMMARY
[0027] The present disclosure relates to the field of estimation of blood analyte(s). In particular, the present disclosure relates to a biosensor strip for determining presence, absence and/or the amount of an analyte (such as glucose) in a liquid especially a biological liquid, and most especially blood.
[0028] In an aspect, the present disclosure relates to a biosensor strip having a plasma separating membrane, and a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by addition of a blood sample enables quick plasma separation, and wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable quantified analysis of the analyte.
[0029] In an aspect, the pre-wetting buffer can be selected from one or a combination of Imidazole, Phosphate, Citrate, MOPS, HEPS, CHAPS, TRIS, and MES. In another aspect, the pre-wetting buffer can include any or a combination of polymeric agent(s), sugar(s), emulsifying agent(s), anticoagulant(s), aggregating agent(s), and preservative(s). In another aspect, the plasma separating membrane can be made from one or a combination of Cellulose acetate, paper, nylon, cotton fiber, glass fiber, polysulfone, and hydrophobicpolyester nylon mesh.
[0030] In an aspect, the pre-wetting buffer can be added in the range of 8-10µl drop size and the blood sample can be added in the range of 3-5µl drop size. In another aspect, the analyte can be selected from any of biochemical analytes (glucose, HbA1c, cholesterol, urea, triglycerides, uric acid, ketone, lactate, hormones, antigens, antibodies, biomarkers etc.). In another aspect, the reagent can include a combination of chromogenic substrate(s), organic dye(s), enzyme(s), and an enzyme mix, wherein the enzyme mix can include an biomolecule stabilizer. In an aspect, the reagent can further include one or a combination of phosphate-citrate buffer, PVP, PEG, and PMVMA. The Enzyme Mix can further include sodium acetate buffer, Ethylene glycol, Tween, and PMVMA. In an aspect, the Biomolecule stabilizer can include at least one amino-acid, at least one sugar, and at least one polymeric substance. In another aspect, the amino-acid can be selected from one or a combination of DL-Aspartate, DL-Glutamate, DL-Methionine, DL-Lysin, L-Lysine, L-Arginine and L-Histidine, and wherein the at least one polymeric substance is selected from one or a combination of PMVMA, PVP, PVA, PEG, Gelatin, and BSA, and wherein the sugar is selected from one or a combination of sorbitol, mannitol, trehalose, xylose andribose.
[0031] The present disclosure further relates to a reagent composition for use in a biosensor strip to enable an efficient reaction with an analyte, wherein the reagent composition can include a combination of achromogenic substrate (TMB,AEC,DAB,TOOS,4-AAP, organic dyes etc),enzyme, and an enzyme mix, wherein the enzyme mix includes a buffer and an enzyme stabilizer, and wherein the Biomolecule stabilizer includes at least one amino-acid, at least one sugar, and at least one polymeric substance, and wherein the reagent composition enables a minimum shelf life of 12 months for the biosensor strip at room temperature storage inside a storage vial. The reagent can further include one or a combination of phosphate-citrate buffer, PVP, PEG, and PMVMA. The enzymemix can further include any or a combination of sodium acetate buffer, ethylene glycol, tween, and PMVMA.In an aspect, the at least one amino-acid can be selected from one or a combination of DL-Aspartate, DL-Glutamate, DL-Methionine, DL-Lysin, L-Lysine, L-Arginine and L-Histidine, and wherein the at least one polymeric substance can be selected from one or a combination of PMVMA, PVP, PVA, PEG, gelatin, and BSA, and wherein the at least one sugar can be selected from one or a combination of Sorbitol, Mannitol, Trehalose, Xylose and Ribose. In an aspect, the Biomolecule stabilizer can further include one or a combination of a Tris buffer, EDTA, Tri-Sodium citrate, thimersol, azide, providone, ammonium sulphate, propylene glycol, and ethylene glycol.
[0032] In an aspect, the present disclosure relates to a biomolecule stabilizer composition and methods to stabilize biosensor reaction membrane enzymes are not limited to glucose alone. It also provides stability to other biosensor enzymes namely eg, cholesterol, triglycerides, urea, uric acid, lactate, ketone bodies, enzyme antibody bioconjugates like streptavidine-IgG, Streptavidine-HRP, Biotin-IgG, Protein A/G-HRP etc. antigens (both recombinant/native) & its conjugates eg protein A/G –IgG conjugate used in immunoassay biosensors.
[0033] In an aspect, the present disclosure relates to a method for using a biosensor strip for analyzing an analyte, wherein method includes the step of adding a pre-wetting buffer on the biosensor strip, wherein the biosensor strip includes a plasma separating membrane and a reaction membrane operatively coupled with the plasma separating membrane. The method can further include the step of adding a blood sample on the biosensor strip to enable quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane; obtaining a colour on the biosensor strip based on the reaction between the reagent and the analyte after a pre-defined time interval; and estimating the amount of the analyte based on the obtained colour. In an aspect, the amount of the analyte can be estimated based on one or a combination of visual chart and a computing means. In another aspect, the computing means can involve any or a combination of a mobile phone, a smartphone, an optical reader device, and an image capturing device, wherein camera of the computing means is configured to take an image of the colour and enable processing of the image for estimating the amount of the analyte.
[0034] The present disclosure can further relate to an analyte detecting kit for semi-quantitative estimation of amount of an analyte, the kit including a biosensor strip, the biosensor strip including a plasma separating membrane, and a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by blood sample enables quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable colour change of the reaction membrane. The kit can further include a visual colour chart configured to enable comparison of the colour of the reaction membrane with colours present in the chart to estimate the amount of the analyte.
[0035] The present disclosure can further relate to an analyte detecting kit for quantitative estimation of amount of an analyte, wherein kit includes a biosensor strip, and wherein the biosensor strip includes a plasma separating membrane; and a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by blood sample enables quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable colour change of the reaction membrane. The kit can further include a computing device that can be configured to take an image of the colour of the reaction membrane and analyze the colour to estimate the amount of the analyte.
[0036] In an embodiment, the disclosure provides a glucose testing strip which uses commonly available low cost membranes like nylon, paper, cotton fiber etc. to achieve desired plasma separation instead of commercially available expensive membranes (made of glass fibers, polysulfone etc.).
[0037] In an embodiment, the disclosure provides a kit for semi-quantitative estimation of glucose present in a sample or a blood sample that includes: a glucose assay strip and a visual colour chart.
[0038] In an embodiment, the disclosure relates to a method of semi-quantitative estimation of glucose present in a blood sample including steps of: addition of a pre-wetting buffer composition to glucose assay strip; addition of a blood sample to the glucose assay strip; allowing the plasma separated from the blood sample to react for a pre-determined period at the reaction membrane; comparing the colour obtained at the reaction viewing window to a control or standard; and estimating the amount of glucose in the blood sample based on the comparison step.
[0039] In an embodiment, the disclosure provides a kit for quantitative estimation of glucose present in a sample or a blood sample that includes: a glucose assay strip, optionally an illuminating device (e.g. LED light source), an image capturing device and a computing device.
[0040] In an embodiment, the disclosure relates to a method of quantitative estimation of glucose present in a sample or a blood sample including steps of: addition of a drop of the pre-wetting buffer composition (8-10µl drop size) using a dropper to the blood sample receiving window of glucose assay strip; addition of a sample or a blood sample (3-5µl drop size) to the blood sample window of glucose assay strip; allowing the sample to react for a pre-determined period at the reaction membrane; capturing image(s) of at least a part of the reaction viewing window of the strip; processing images to extract Hue (H) values corresponding to the colour observed at the reaction viewing window; comparing the Hue (H) values to a control or standard; and estimating the amount of glucose in the sample based on the comparison step.
[0041] In an embodiment, the disclosure provides a kit for semi-quantitative estimation of HbA1C in a blood sample that includes: a glucose assay strip and a colour chart.
[0042] In an embodiment, the disclosure relates to a method of semi-quantitative estimation of HbA1c in a blood sample that includes steps of: addition of pre-wetting buffer composition (say 8-10µl) to the blood sample receiving window of glucose assay strip; addition of a sample or a blood sample (say 3-5µl) to the blood sample window of glucose assay strip; allowing sample or plasma separated from the blood sample to react for a pre-determined period at the reaction membrane; comparing the colour obtained at the reaction viewing window to a control or standard; and estimating the amount of HbA1c in the blood sample based on the comparison step.
[0043] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS
[0044] 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.
[0045] FIG. 1A illustrates an exemplary schematic view of a glucose testing strip in accordance with embodiments to the present disclosure.
[0046] FIG. 1B illustrates an exemplary schematic view of an unassembled glucose testing strip with bottom support strip and spreading screen in accordance with embodiments to the present disclosure.
[0047] FIG. 1C illustrates an exemplary schematic view of an unassembled glucose testing strip with bottom support strip, spreading screen and reaction membrane in accordance with embodiments to the present disclosure.
[0048] FIG. 1D illustrates an exemplary schematic view of an unassembled glucose testing strip with plasma separating membrane attached to reaction membrane in accordance with embodiments to the present disclosure.
[0049] FIG. 2A illustrates an exemplary schematic view of effect of pre-wetting on plasma separation efficiency of LF-1 in accordance with embodiments to the present disclosure.
[0050] FIG. 2B illustrates an exemplary schematic view of effect of pre-wetting on plasma separation efficiency of Nylon membrane/cotton in accordance with embodiments to the present disclosure.
[0051] FIG. 2C illustrates an exemplary schematic view of effect of pre-wetting on plasma separation efficiency of Nylon membrane/paper in accordance with embodiments to the present disclosure.
[0052] FIG. 3A illustrates a plot of effect of accelerated stability testing on activity and reaction time of Nylon reaction membrane in accordance with embodiments to the present disclosure.
[0053] FIG. 3B illustrates a plot of effect of accelerated stability testing on activity of Biodyne reaction membrane in accordance with embodiments to the present disclosure.
[0054] FIG. 3C illustrates a plot of effect of room temperature storage stability testing on activity and reaction time of Nylon reaction membrane in accordance with embodiments to the present disclosure.
[0055] FIG. 3D illustrates a plot of effect of room temperature storage stability testing on activity and reaction time of Nylon reaction membrane in accordance with embodiments to the present disclosure.
[0056] FIG. 4 illustrates an exemplary schematic view of a kit for semi-quantitative estimation of glucose present in a sample in accordance with embodiments to the present disclosure.
[0057] FIG. 5A – 5D illustrates an exemplary schematic view of method of semi-quantitative estimation of glucose present in a sample in accordance with embodiments to the present disclosure.
[0058] FIG. 6A illustrates an exemplary perspective view of a kit for quantitative estimation of glucose present in a sample in accordance with embodiments to the present disclosure.
[0059] FIG. 6B illustrates an exemplary top view of a kit for quantitative estimation of glucose present in a sample in accordance with embodiments to the present disclosure.
[0060] FIG. 6C illustrates an exemplary side view of a kit for quantitative estimation of glucose present in a sample in accordance with embodiments to the present disclosure.
[0061] FIG. 7A – 7C illustrates an exemplary schematic view of method of quantitative estimation of glucose present in a sample in accordance with embodiments to the present disclosure.
[0062] FIG. 7D illustrates an exemplary plot of comparison between blood glucose estimation output as measured by disclosed kit and as measured by accucheck in accordance with embodiments to the present disclosure.
[0063] FIG. 8 illustrates an exemplary schematic view of a kit for semi-quantitative estimation of HbA1C and estimation of associated risk profile for complications related to increased HbA1c in a blood sample in accordance with embodiments to the present disclosure.
[0064] FIG. 9 illustrates an exemplary plot of results of optimization and comparative study of positive correlation equation between fasting blood glucose sample and HbA1c values from > 800 different patient samples in accordance with embodiments to the present disclosure.
[0065] FIG. 10A illustrates a plot of outcome of pre-wetting and plasma recovery study using conventional expensive membrane (LF-1) and low cost commonly available plasma separation membranes (Nylon/paper, Nylon/cotton)in accordance with embodiments to the present disclosure.
[0066] FIG. 10B illustrates a plot of outcome of study of pre-wetting on time of plasma separation for conventional expensive membrane (LF-1) and low cost commonly available plasma separation membranes (Nylon/paper, Nylon/cotton)in accordance with embodiments to the present disclosure.
[0067] FIG. 10C illustrates an exemplary schematic view of effect of pre-wetting on plasma separation using conventional expensive membrane (LF-1) and low cost commonly available plasma separation membranes (Nylon/paper, Nylon/cotton)in accordance with embodiments to the present disclosure.
[0068] FIG. 11A – 11H illustrates exemplary schematic views of method of fabrication of glucose assay strip in accordance with embodiments to the present disclosure.
[0069] FIG. 12A – 12B illustrates outcome of point of care blood glucose assay reaction membrane composition and its batch performance study – long term room temperature storage.
[0070] FIG. 13A illustrates outcome of performance analysis of visual blood gluco dot- point of care blood glucose assay kit (Version-1) during long term storage at room temperature
[0071] FIG 13B illustrates outcome of performance analysis of Smart Gluco Scan- point of care blood glucose assay kit (Version-2) during long term storage at room temperature.
[0072] FIG. 14A illustrates outcome of performance analysis of 10mM Imidazole-Acetate with 0.01% EDTA as pre-wetting buffer composition using glass fibre (LF-1) as plasma separation matrix and nylon membrane as reaction membrane.
[0073] FIG. 14B illustrates outcome of performance analysis of 500mM Imidazole-Acetate with 0.01% EDTA, PMVMA, DHA and methyl orange, pH 6.5 as pre-wetting buffer composition using cotton fibre as plasma separation matrix and nylon membrane as reaction membrane.
[0074] FIG. 14C illustrates outcome of performance analysis of 100mM PBS with 0.01% EDTA as pre-wetting buffer composition using Whatman filter paper #1 as plasma separation matrix and nylon membrane as reaction membrane.
[0075] FIG. 14D illustrates outcome of performance analysis of 100mMES with 0.01% EDTA as pre-wetting buffer composition using Polysulfone membrane (Vivid-Pall) as plasma separation matrix and nylon membrane as reaction membrane.
[0076] FIG. 14E illustrates outcome of performance analysis of MilliQ with 0.01% EDTA as pre-wetting buffer composition using Hydrophilic nylon mesh (Satti) as plasma separation matrix and nylon membrane as reaction membrane.

DETAILED DESCRIPTION
[0077] The following is a detailed description of embodiments of the disclosure depicted in the accompanying drawings. 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.
[0078] 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.
[0079] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[0080] 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. 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.
[0081] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[0082] 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.
[0083] The present disclosure relates to the field of estimation of blood analyte(s). In particular, the present disclosure relates to a biosensor strip for determining presence, absence and/or the amount of analyte (such as glucose) in a liquid especially a biological liquid, and most especially blood.
[0084] In an aspect the present disclosure relates to a biosensor strip having a plasma separating membrane, and a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by blood sample enables quick plasma separation, and wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable quantified analysis of the analyte.
[0085] In an aspect, the pre-wetting buffer can be selected from one or a combination of Imidazole, acetate, Phosphate, Citrate, MOPS, HEPS, CHAPS, TRIS, and MES. In another aspect, the pre-wetting buffer can include any or a combination of polymeric agent(s), sugar(s), emulsifying agent(s), anticoagulant(s), aggregating agent(s), and preservative(s). In another aspect, the plasma separating membrane can be made from one or a combination of cellulose acetate, paper, nylon, cotton fiber, glass fiber, polysulfone, and hydrophobicpolyester nylon mesh.
[0086] In an aspect, the pre-wetting buffer can be added in the range of 8-10µl drop size and the blood sample can be added in the range of 3-5µl drop size. In another aspect, the analyte can be selected from any of glucose, HbA1c, cholesterol, hormones, urea, triglycerides, lactate, ketone bodies, creatinine and uric acid. In another aspect, the reagent can include a combination of a chromogenic substrate(s), organic dye(s), an enzyme, and an enzyme mix, wherein the enzyme mix can include an enzyme stabilizer. In an aspect, the reagent can further include one or a combination of Phosphate-citrate buffer, PVP, PEG, and PMVMA. The enzyme mix can further include sodium acetate buffer, ethylene glycol, Tween, and PMVMA. In an aspect, the biomolecule stabilizer can include at least one amino-acid, at least one sugar, and at least one polymeric substance. In another aspect, the amino-acid can be selected from one or a combination of DL-Aspartate, DL-Glutamate, DL-Methionine, DL-Lysin, L-Lysine, L-Arginine and L-Histidine, and wherein the at least one polymeric substance is selected from one or a combination of PMVMA, PVP, PVA, PEG, Gelatin, and BSA, and wherein the sugar is selected from one or a combination of sorbitol, mannitol, trehalose, xylose andribose.
[0087] The present disclosure further relates to a reagent composition for use in a biosensor strip to enable an efficient reaction with an analyte, wherein the reagent composition can include a combination of a chromogenic substrate(s), organic dye(s), an enzyme, and an enzyme mix, wherein the enzyme mix includes a buffer and an enzyme stabilizer, and wherein the biomolecule stabilizer includes at least one amino-acid, at least one sugar, and at least one polymeric substance, and wherein the reagent composition enables a minimum shelf life of 12 months for the biosensor strip at room temperature storage in its vial. The reagent can further include one or a combination of Phosphate-citrate buffer, PVP, PEG, and PMVMA. The Enzyme Mix can further include any or a combination of Sodium acetate buffer, Ethylene glycol, Tween, and PMVMA. In an aspect, the at least one amino-acid can be selected from one or a combination of DL-Aspartate, DL-Glutamate, DL-Methionine, DL-Lysin, L-Lysine, L-Arginine and L-Histidine, and wherein the at least one polymeric substance can be selected from one or a combination of PMVMA, PVP, PVA, PEG, Gelatin, and BSA, and wherein the at least one sugar can be selected from one or a combination of Sorbitol, Mannitol, Trehalose, Xylose and Ribose. In an aspect, the enzyme stabilizer can further include one or a combination of a Tris buffer, EDTA, Tri-Sodium citrate, Thimersol, Azide, Providone, Ammonium sulphate, Propylene glycol, and Ethylene glycol.
[0088] In an aspect, the present disclosure relates to a method for using a biosensor strip for analyzing an analyte, wherein method includes the step of adding a pre-wetting buffer on the biosensor strip, wherein the biosensor strip includes a plasma separating membrane and a reaction membrane operatively coupled with the plasma separating membrane. The method can further include the step of adding a blood sample on the biosensor strip to enable quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane; obtaining a colour on the biosensor strip based on the reaction between the reagent and the analyte after a pre-defined time interval; and estimating the amount of the analyte based on the obtained colour. In an aspect, the amount of the analyte can be estimated based on one or a combination of visual chart and a computing means. In another aspect, the computing means can involve any or a combination of a mobile phone, a smartphone, an optical reader device, and an image capturing device, wherein camera of the computing means is configured to take an image of the colour and enable processing of the image for estimating the amount of the analyte.
[0089] The present disclosure can further relate to an analyte detecting kit for semi-quantitative estimation of amount of an analyte, the kit including a biosensor strip, the biosensor strip including a plasma separating membrane, and a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by blood sample enables quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable colour change of the reaction membrane. The kit can further include a visual colourchart configured to enable comparison of the colour of the reaction membrane with colours present in the chart to estimation the amount of the analyte.
[0090] The present disclosure can further relate to an analyte detecting kit for quantitative estimation of amount of an analyte, wherein kit includes a biosensor strip, and wherein the biosensor strip includes a plasma separating membrane; and a reaction membrane operatively coupled with the plasma separating membrane, wherein addition of a pre-wetting buffer on the plasma separating membrane followed by blood sample enables quick plasma separation, wherein analyte of the separated plasma is reacted with a reagent configured in the reaction membrane to enable colour change of the reaction membrane. The kit can further include a computing device that can be configured to take an image of the color of the reaction membrane and analyze the colour to estimate the amount of the analyte.
[0091] It is to be appreciated that although most of the embodiments of the present disclosure have been explained with reference to glucose being the analyte, a person ordinarily skilled in the art would understand that the proposed techniques, methods, and biosensor strip configurations/structure/construction can used for any other analyte including but not limited to HbA1c, cholesterol, hormones, urea, triglycerides, and uric acid, and hence use of glucose is only an exemplary illustration for explaining various aspects of the present invention.
[0092] Embodiments to the present disclosure provide a glucose testing strip that uses commonly available low cost membranes such as nylon, paper, cotton fiber etc. to achieve desired plasma separation along with or instead of commercially available expensive membranes (made of glass fibers, polysulfone etc.). In an embodiment, as illustrated in FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D, the glucose testing strip102 can include at least three operative layers namely a plasma separating membrane 104, a reaction membrane 106, and a spreading screen 108 affixed to each other. The reaction membrane 106 abuts the plasma separating membrane 104 at its uppermost surface and abuts the spreading screen 108 at its lowermost surface. The stack can include the membranes 104, 106 and 108 that are sandwiched between top and bottom support strips 110 and 112. The top support strip 110 can include at least one sample receiving window114 and the bottom support strip 112 can include at least one reaction viewing window 116. The operative layers are aligned vertically with blood sample receiving window and reaction viewing window.
[0093] In an embodiment, the glucose testing strip can include at least two operative layers namely a plasma separating membrane 104 and a reaction membrane 106 affixed to each other. The plasma separating membrane 104 abuts the reaction membrane 106 at its lowermost surface. The stack can include the plasma separating membrane and the reaction membrane that are sandwiched between top and bottom support strips. The top support strip 110 can include at least one sample receiving window 114, and the bottom support strip 112 can include at least one reaction viewing window 116. The operative layers can be aligned vertically with blood sample receiving window and reaction viewing window.
[0094] In an aspect, the support strip(s) 110 and 112 can be constructed from any material(s) known to a person skilled in the art which provide rigid support to the test strip and do not interfere with the fluid sample or the test reactants. For example, pressed, non-absorbing paper or cardboard, plastics of various kinds such as mylar, polyethylene, polypropylene, or the like can be used to fabricate support strips. However, use of thick laminated paper material is preferred for construction of support strips.
[0095] The spreading screen 108 can be constructed from any material known to a person skilled in the art including nylon, silk, polyester, polyethylene, polypropylene and the use of nylon mesh (screen) is preferred as the spreading screen.
[0096] In an aspect, the plasma separating membrane104can be a low cost commonly available membrane like nylon, paper, cotton fiber etc. which when pre-wetted (just before addition of blood sample)with a buffer composition, according to the invention and as fully described below, separates a large portion of the cellular components(RBCs) from the blood sample. Thus, the material which initially reaches the uppermost surface of the reaction membrane is plasma containing minimal proportion of red blood cells. Whatever red blood cells are present at that point are separated from the plasma at the upper surface of the reaction membrane because the membrane, according to the invention and as fully described below, is coated with a reagent composition which helps in preventing passage of cellular components (RBCs) of blood to reaction surface (towards reaction viewing window). Thus the remaining portion of originally applied sample that reaches the lowermost, reading surface of the reaction membrane (towards reaction viewing window) is plasma with essentially no red blood cells detectable within the timeframe of the assay. Alternatively, any commercially available conventional plasma separating membrane made of glass fibre, polysulfone and the like, known to a person skilled in the art, can be used to separates cellular components from the blood sample.
[0097] As it would be easily realized to a person skilled in the art, the low cost commonly available membranes such as nylon, paper, cotton fiber and the like do not exhibit satisfactory plasma separation efficiency as such and hence, hampers their potential to be used for achieving desired plasma separation. In accordance with an embodiment to the present disclosure, to improve plasma separation efficiency of the low cost commonly available membranes (plasma separating membranes), the membrane 104is pre-wetted with a buffer composition before addition of sample to the sample receiving window 114. The buffer composition can include one or more of following components: water (Milli Q water or deionised water is preferred), buffer(s) like phosphate, citrate, MOPS (3-(N-morpholino) propanesulfonic acid), HEPS(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1- propanesulfonate), Imidazole, Acetate, MES, Tris or any other buffers known to a person skilled in the art, polymeric agent(s)like PVA (Poly(vinyl alcohol)), PMVMA(Poly(methyl methacrylate)) or any other polymers known to a person skilled in the art, sugar(s)like Mannitol, Sorbitol or any other sugars known to a person skilled in the art, emulsifying agent(s)like Pluronicor any other emulsifying agents known to a person skilled in the art, anticoagulant(s)like EDTA(Ethylenediaminetetraacetic acid) or any other anticoagulants known to a person skilled in the art, aggregating agent(s) like PMVMA (poly (methylvinylether/maleic) anhydride), Imidazoleor any other aggregating agent known to a person skilled in the art, inert dye(s)like Methyl Orange, Phloxin B or any other dye known to a person skilled in the art and preservative(s)like azide, thimersolor any other preservative known to a person skilled in the art either alone or in combination with other components. In an implementation, the buffer composition can include Imidazole-Acetate buffer prepared using Milli Q water (0.001- 1M and with pH 5.0-8.0), EDTA di-sodium or potassium salt (0.001% - 1%), PMVMA (0.001 % - 10 %), DHA (1: 1K – 1: 200K dilution) and Methyl Orange or other inert dye (0.001mg/ml -0.1mg/ml). In an implementation, the buffer composition can include Imidazole-Acetate buffer prepared using Milli Q water (0.01- 0.5M and with pH 6.5), EDTA di-sodium or potassium salt (0.01 % - 0.05 %), PMVMA (0.6 % - 1 %), DHA (1: 100K dilution) and Methyl Orange or other inert dye (0.008 mg/ml -0.01mg/ml).
[0098] In an aspect, the buffer composition, prepared according to an embodiment to the present disclosure that can be used for the pre-wetting of plasma separating membrane 104advantageously includes one or a combination of agglutinating polymer, anticoagulant and non-hemolytic agent, as mentioned above, in order to modulate the overall process of plasma separation and bring about satisfactory plasma separation. PMVMA or PVA used in the buffer composition brings about RBC aggregation on the surface of plasma separating membrane. Imidazole and EDTA prevents lysis of cellular components of blood and binds to free Hb present in the blood sample preventing their interference in the test. Further, Imidazole, by binding to free Hb, prevents variation in colour development (during reaction) and hence, reduces variability in the test outcome (measurement of blood glucose concentration) due to varying hematocrite between different blood samples. Further, as the overall buffer composition is water based, lysis of RBCs is prevented. Improvement in the plasma separation efficiency of conventional membranes (e.g. LF-1 made of glass fiber), as illustrated in FIG. 2A, and low cost commonly available membranes (e.g. nylon/cotton, nylon/paper etc.), as illustrated in FIG. 2B and FIG. 2C, can easily be observed following the pre-wetting (addition of the buffer composition before addition of the sample/blood) of plasma separating membrane using the buffer composition mentioned above in comparison to plasma separation without addition of the buffer composition.
[0099] The reaction membrane 106can be constructed from any material known to a person skilled in the art including any commercial nylon membrane make, silk, polyester, polyethylene, polypropylene and the like which can be treated and/or coated with the reagent composition as fully described below. In an aspect, use of Biodyne as a reaction membrane is configured. In accordance with an embodiment to the present disclosure, the reaction membrane can be treated with a reagent composition that can include one or a combination of components selected from a group consisting of: buffer(s) like Phosphate-citrate buffer containing hydroxypropylbetacyclodextrin, Imidazole acetate buffer, Tris buffer, MES, Phosphate, Acetate buffer or any other buffers known to a person skilled in the art, dye(s) , like Tartrazine, chromogenic substrate like TMB (3,3',5,5'-Tetramethylbenzidine) or any other dyes known to a person skilled in the art, polymeric substance(s) like PMVMA, PVP, PEG or any other polymeric substances known to a person skilled in the art, enzyme(s) like Glucose Oxidase (GOD), Peroxidase (HRP) or any other enzymes known to a person skilled in the art, which can react with the analyte under test to generate intermediate(s), which on reaction with chromogenic substrate produce a measurable colour change, water, and an enzyme mix composition. The enzyme mix composition can include one or more components selected from buffer(s) such as sodium acetate, phosphate buffer, or any other buffer known to a person skilled in the art, polyhydric alcohol such as Ethylene glycol, propylene glycol, glycerol or any other polyhydric alcohols known to a person skilled in the art, non-ionic surfactants such as Tween 20, Triton x 100 or any other non-ionic surfactants (detergents) known to a person skilled in the art, and a biomolecule stabilizer composition. In an aspect, the biomolecule stabilizer composition can include one or a combination of components selected from buffer(s) such as Tris buffer, MES, HEPS, Phosphate buffer, Acetate buffer, Citrate buffer, or any other buffers known to a person skilled in the art, polymeric substance(s) such as Gelatin, BSA or any other polymeric substances known to a person skilled in the art, EDTA-Sodium or Potassium salt, Tri-Sodium citrate orcitric acid, amino acid(s) like DL-Aspartate, DL-Glutamate, DL-Methionine, DL-Lysin, L-Lysine, L-Arginine, L-Histidine, or any other amino-acids known to a person skilled in the art, sugar(s) such as sorbitol, mannitol, trehalose, xylose, ribose or any other sugar known to a person skilled in the art, preservative(s) such as Thimersol, Azide, providone or any other preservatives known to a person skilled in the art, Ammonium sulphate, polyhydric alcohol(s) such as propylene glycol, ethylene glycol, or any other polyhydric alcohols known to a person skilled in the art, polymeric substance(s)such as PMVMA,PVP,PVA,PEG,BSA or any other polymeric substances known to a person skilled in the art.
[00100] In an implementation, the reagent composition that can be used to treat or coat the reaction membrane includes one or a combination of components selected from Phosphate-citrate buffer (0.01M - 0.5M, pH 5.0- 6.0) containing hydroxypropylbetacyclodextrin (0.1ml- 5ml), PVP (1mg- 50mg), PEG-4K (1mg- 50mg), Tartrazine (0.01mg- 10mg), TMB (0.01mg- 10mg), PMVMA (0.01mg- 10mg), Glucose Oxidase (GOD) (5U - 2000U), Peroxidase (HRP) (5U- 1500U), Milli Q (0.25ml-5ml), Imidazole acetate buffer (0.01M-0.5M, pH 5.5-6.9) or Tris buffer or MES or Phosphate or Acetate buffer (0.005ml-1ml) and an enzyme mix composition (0.01ml- 10ml). The enzyme mix composition includes one or more of following components: sodium acetate or phosphate buffer (0.01M - 0.5M, pH 5.0 - 6.5) (0.25ml-2ml), Ethylene glycol or propylene glycol or glycerol (0.005ml-0.1ml), Tween 20 or Triton x 100 or non-ionic detergent (0.005ml-0.1ml) and a biomolecule stabilizer composition (0.005ml-0.1ml). The biomolecule stabilizer composition includes one or more of following components: Tris buffer or MES or HEPS or Phosphate or Acetate or Citrate buffer (10mM -100mM, pH 6.0-6.9) (1ml-9ml), Gelatin or BSA (0.01gm-2gm), EDTA-Sodium or Potassium salt (2mg-25mg), Tri-Sodium citrate orcitric acid (2mg-50mg), DL-Aspartate or DL-Glutamate (0.1mg-5mg), DL-Methionine or DL-Lysin (0.1mg-10mg), L-Lysine or L-Arginine or L-Histidine (0.1mg-10mg), Sorbitol or Mannitol (0.1mg-100mg), Trehalose or Xylose or Ribose (0.1mg-100mg), Thimersol or Azide or providone (1mg-100mg), Ammonium sulphate (1mg-500mg), Propylene glycol or Ethylene glycol(0.5ml-5ml) and PMVMA or PVP or PVA or PEG or BSA (0.05gm-1gm).In an aspect, the reagent composition that can be used treat or coat the reaction membrane can include one or more of following components: Phosphate-citrate buffer (0.01M - 0.5M, pH 5.0- 6.0) containing hydroxypropylbetacyclodextrin (0.5ml- 2ml with 0.1%-0.75% cyclodextrin), PVP (10mg- 30mg), PEG-4K (10mg- 30mg), Tartrazine (0.05mg- 5mg), TMB (0.05mg- 5mg), PMVMA (0.05mg- 5mg), Glucose Oxidase (GOD) (5U - 200U), Peroxidase (HRP) (5U- 150U), Milli Q (0.5ml-1.5ml), Imidazole acetate buffer (0.01M-0.5M, pH 5.5-6.9) or Tris buffer or MES or Phosphate or Acetate buffer (0.01ml-0.5ml) and an enzyme mix composition (0.1ml- 1.5ml). The enzyme mix composition includes one or more of following components: sodium acetate or phosphate buffer (0.01M - 0.5M, pH 5.0 - 6.5) (0.5ml-0.95ml), Ethylene glycol or propylene glycol or glycerol (0.01ml-0.03ml), Tween 20 or Triton x 100 or non-ionic detergent (0.01ml-0.03ml) and a biomolecule stabilizer composition (0.01ml-0.03ml). The biomolecule stabilizer composition includes one or more of following components: Tris buffer or MES or HEPS or Phosphate or Acetate or Citrate buffer (10mM -100mM, pH 6.0-6.9) (2ml-7ml), Gelatin or BSA (0.01gm-0.75gm), EDTA-Sodium or Potassium salt (5mg-15mg), Tri-Sodium citrate orcitric acid (5mg-15mg), DL-Aspartate or DL-Glutamate (0.2mg-1.5mg), DL-Methionine or DL-Lysin (0.5mg-2.5mg), L-Lysine or L-Arginine or L-Histidine (0.2mg-2.5mg), Sorbitol or Mannitol (0.5mg-20mg), Trehalose or Xylose or Ribose (0.5mg-20mg), Thimersol or Azide or providone (5mg-50mg), Ammonium sulphate (10mg-320mg), Propylene glycol or Ethylene glycol (0.5ml-2.5ml) and PMVMA or PVP or PVA or PEG or BSA (0.1gm-0.5gm). The reagent composition can be prepared by following any of the methods known to a person skilled in the art. The reagent membrane can be treated and/or coated with the reagent composition by following any of the methods known to a person skilled in the art.
[00101] In aspect, reagent composition of the present disclosure can include a Biomolecule Stabilizer that can further include one or a combination of a buffer, a chelating agent, an organic solvent, a protein, a preservative, and a salt substance. In another aspect, the buffer can be any or a combination of Tris buffer, Imidazole, Sodium acetate, Phosphate, trisodium citrate, and citrate, and wherein the chelating agent can be any or a combination of EDTA and Tri-Sodium citrate, and wherein the organic solvent can be any or a combination of ethylene glycol, glycerol, Propylene glycol, and wherein the protein can be any or a combination of Gelatin and BSA, and wherein the preservative can be any or combination of thimerosol, azide, and providone, and wherein the salt substance is Ammonium sulphate.
[00102] The advantageous reaction membrane 106realized according to an embodiment to the present disclosure can include analyte detection reagents that include various polymers, amino acids, and sugars as mentioned above. In dry state, these components act as Stablizing agents to chromogenic substrate(s) and detection enzyme(s). The advantageous effect of the reagent composition can be observed from plots as illustrated in FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D. The reaction membrane coated/treated with the reagent composition, according to embodiments to the present disclosure, on accelerated stability testing (as illustrated in FIG. 3A and FIG. 3B) shows significant retention of activity (70% - 80%) over a period of time of 200-250 days. The reaction membrane coated/treated with the reagent composition, according to embodiments to the present disclosure, on room temperature stability testing (as illustrated in FIG. 3C and FIG. 3D) shows significant retention of activity (70% - 80%) over a period of time of 400-450 days. Further, the sere agents block pores of the reaction membrane preventing entry of small size cells including platelets and the like present in the sample to the reaction site. During the pre-wetting step (addition of buffer composition to the plasma separating membrane before addition of the sample), a small amount of buffer composition leaches from the plasma separating membrane to reach the reaction membrane where, it solubilizes sugars, amino acids and polymers present over the reaction membrane and hence, helps in rapid absorption of the plasma alone without interference from blood cell(s). Further, small hydrophilic molecules such as glucose present in the sample dissolve rapidly in the buffer composition to enter freely and rapidly to reaction side of the membrane (towards the reaction viewing window) to further react with reagent(s) to produce colour, without interference from blood cells.
[00103] In accordance with an embodiment to the present disclosure, method of fabrication of glucose testing strip can include steps of cutting a sheet of suitable dimensions to obtain top and bottom support strips with perforations of appropriate diameter to realize top and bottom support strips with blood sample receiving window and reaction viewing window respectively; affixing a plasma separating membrane to the top support strip; obtaining a reaction membrane pre-treated or pre-coated with the reagent composition; affixing the reaction membrane to the plasma separating membrane; affixing a spreading screen with appropriate pore size (open area) to the bottom surface of reaction membrane; and affixing a bottom support strip with reaction viewing window to the reaction membrane to realize a glucose testing strip.
[00104] In accordance with an embodiment to the present disclosure and as illustrated in FIG. 4, a kit for semi-quantitative estimation of glucose present in a sample or a blood sample includes: a glucose assay strip 102 and a colour chart410. As illustrated in FIG. 5A, FIG. 5B and FIG. 5C, addition of a drop of the pre-wetting buffer composition (8-10µl drop size) followed by addition of a blood sample to the strip, by using blood sample cups of predetermined volume (3-5µl), allows for achieving rapid & efficient plasma separation without any RBC lysis. Eventually, glucose present in the separated plasma is acted upon by the specific enzymatic reagent (Glucose oxidase/Peroxidase) along with chromogenic substrate (TMB) and dye (tartrazine) coated on the reaction membrane, to bring out a visual gradient colour change (Initial yellow to green colour shades). The intensity of the green colour shades produced (different green shades: yellowish green/light green/dark green/bluish green) at a given time interval (30-60sec) on the reaction membrane (as seen in the result window of the strip) is directly proportional to the glucose level present in the blood sample. The colour produced in the result window of the strip can be read visually using a colour chart as illustrated in FIG. 5D.
[00105] In accordance with an embodiment to the present disclosure, a method of semi-quantitative estimation of glucose present in a sample or a blood sample includes the steps of: addition of pre-wetting buffer composition (preferably 8-10µl) to the blood sample receiving window of glucose assay strip; addition of a sample or a blood sample (preferably 3-5µl) by using blood sample cups of predetermined volume (3-5µl) to the blood sample window of glucose assay strip; allowing sample or plasma separated from the blood sample to react for a pre-determined period at the reaction membrane; comparing the colour obtained at the reaction viewing window to a control or standard; and estimating the amount of glucose in the blood sample based on the comparison step. The correlation or semi-quantitative estimation can be done by the naked eye by comparing the colour obtained at the reaction viewing window to colour charts of varied and defined colour intensities at various concentrations of glucose.
[00106] In accordance with an embodiment to the present disclosure and as illustrated in FIG. 6A, FIG. 6B and FIG. 6C, a kit for quantitative estimation of glucose present in a sample or a blood sample can include a glucose assay strip 102, a platform 610, optionally an illuminating device 620 (e.g. LED light source), and a computing device630 with image capturing capabilities. A Smartphone, tablet, camera, computer, PDA or any other computing device known to a person skilled in the art can be used as a computing device. Further, an application/program/software can be developed and installed on any of the computing device(s) known to a person skilled in the art to confer image processing and glucose estimation capabilities. Alternatively, a separate image capturing device can be used which can be operatively coupled to a computing device to input image(s) captured by an image capturing device to a computing device for further processing. Addition of a drop of the pre-wetting buffer composition followed by addition of a sample or a blood sample to the glucose assay strip 102allows for a reaction between separated glucose present in the sample and reagents present in the reaction membrane resulting in a colour change which can be observed at the reaction viewing window 116. For observation of a color change from the reaction viewing window, a platform can be provided with a colour observation window over which the glucose assay strip can be placed so that the reaction viewing window of the strip is in vertical alignment with the colour observation window. An illuminating device (e.g. LED light source) 620 can optionally be placed at an appropriate position, preferably beneath the platform610, to illuminate at least a part of the reaction viewing window of the strip. A computing device with the image capturing capabilities or a separate image capturing device can be provided at an appropriate position, preferably beneath the platform 610, to capture image(s) of the reaction viewing window (colour) to observe the colour change. In case a separate image capturing device and a separate computing device are used, the image(s) captured by the image capturing device can then be fed to the computing device for further processing of the images. The computing device converts RGB values (colour) to HSV values (HSV colour space) as known to a person skilled in the art, wherein the HSV space has three variables for expressing a colour, which are hue (H), saturation (S), and brightness (V) instead of R, G, and B. Even though H, S, and V are needed to fully express a colour, only H is needed to discriminate the colours of specific frequencies because H represents the primary colours or combinations of them and distinguishes one colour family from another. Since the colorimetric assays measure only the types of colour (not the spectrum intensities of wavelengths), H values extracted from RGB pixels can be used to correlate the colour on strip to pH value. The H value can be calculated through known algorithms with appropriate equations (Anal. Chem. 2000, 72(7), 282 A-288 A).The computing device correlates and output the amount of glucose present in the sample based on the calculated H values using a calibration graph(s) or correlation table(s) or correlation chart(s)stored in the computing device.
[00107] In accordance with an embodiment to the present disclosure, a method of quantitative estimation of glucose present in a sample or a blood sample can include the steps of addition of a drop of the pre-wetting buffer composition (8-10µl drop size) using a dropper to the blood sample receiving window of glucose assay strip; addition of a sample or a blood sample (3-5µl drop size) by using blood sample cups of predetermined volume (3-5µl) to the blood sample window of glucose assay strip; allowing the sample to react for a pre-determined period at the reaction membrane; capturing image(s) of at least a part of the reaction viewing window of the strip; processing images to extract Hue (H) values corresponding to the colour observed at the reaction viewing window; comparing the Hue (H) values to a control or standard; and estimating the amount of glucose in the sample based on the comparison step. An exemplary method of quantitative estimation of glucose present in a sample or a blood sample is illustrated in FIG. 7A, FIG. 7B and FIG. 7C. Plot showing linearity of the blood glucose as measured by the kit according to an embodiment to the present disclosure and as measured by accucheck is illustrated in FIG. 7D.
[00108] In accordance with an embodiment to the present disclosure, a kit for semi-quantitative estimation of HbA1C and estimation of associated risk profile for complications related to increased HbA1c in a blood sample can include a glucose assay strip 102 (not shown) and a colour chart800 (as illustrated in FIG. 8). Addition of a drop of the pre-wetting buffer composition (8-10µl drop size) followed by addition of a blood sample to the strip by using blood sample cups of predetermined volume (3-5µl), allows for achieving rapid & efficient plasma separation without any RBC lysis. Eventually, glucose present in the separated plasma can be acted upon by the specific enzymatic reagent (Glucose oxidase/Peroxidase) along with chromogenic substrate (TMB) coated on the reaction membrane, to bring out a colour change (Initial yellow to green colour shades). The intensity of the green colour shades produced (different green shades: yellowish green /light green/ dark green/ bluish green) at a given time interval (30-60sec) on the reaction membrane (as seen in the result window of the strip) is directly proportional to the glucose level present in the blood sample. The colour produced in the result window of the strip can be read visually using a colour chart to estimate amount of glucose present in the sample. Since, the level of HbA1C is directly proportion to the amount of fasting glucose present in the blood sample, HbA1C can be calculated based on the correlation charts indicating value(s) of HbA1c corresponding to the observed colour at the reaction viewing window. Mathematical formula(s) used for creation of correlation chart(s) or table(s) are as known to a person skilled in the art (Wilhelm T, Anton L, Michaela R (2008) Methods Inf M ed; 47(4): 346-55; Bhavna et al, 2011 Volume: 2: Issue-2: April-June, p58-61). Results of optimization and comparative study of positive correlation equation between fasting blood glucose (FBS) and HbA1c values from > 800 different patient samples (Male, female, normal and diabetic) is illustrated in FIG. 9.
[00109] In accordance with an embodiment to the present disclosure, a method of semi-quantitative estimation of HbA1c in a blood sample can include the steps of addition of pre-wetting buffer composition (preferably 8-10µl) to the blood sample receiving window of glucose assay strip; addition of a sample or a blood sample (preferably 3-5µl) by using blood sample cups of predetermined volume (3-5µl) to the blood sample window of glucose assay strip; allowing sample or plasma separated from the blood sample to react for a pre-determined period at the reaction membrane; comparing the colour obtained at the reaction viewing window to a control or standard; and estimating the amount of HbA1c in the blood sample based on the comparison step. The correlation or semi-quantitative estimation can be done by the naked eye by comparing the colour obtained at the reaction viewing window to colour charts of varied and defined colour intensities at various concentrations of HbA1c.
[00110] While the foregoing describes various embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof. The scope of the invention is determined by the claims that follow. The invention is not limited to the described embodiments, versions or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.

EXAMPLES
[00111] Example1: Pre-wetting and plasma recovery study using conventional expensive membrane and low cost commonly available plasma separation membrane(s)
In the study, conventional blood separation matrix namely LF-1 (Glass fiber), and low cost plasma separation membranes (Nylon, Cotton fiber, Paper etc.) were used. Different hematocrite level EDTA containing whole blood samples were used for the study. Initially, pre-wetting buffer 20µl/cm2 was added followed by addition of blood sample (20 µl) and the time taken for clear plasma front to cover a distance was measured. Using calibration graphs for each of the respective membranes, plasma yield was calculated. Study highlights that LF-1 membrane provides highest yield in plasma recovery (80- 95%) for different hematocrite levels by pre-wetting method compared to Nylon/paper or nylon/cotton combination matrix (38-44%). Results of the study are as illustrated in FIG. 10A.
[00112] Example 2: Study of Pre-wetting on time of plasma separation for conventional expensive membrane and low cost commonly available plasma separation membrane(s)
In the study, conventional blood separation matrix namely LF-1 (Glass fiber), and novel plasma separation membranes (Nylon, Cotton fiber, Paper etc.) were used. Different hematocrite level EDTA containing whole samples were used for the study. Initially, pre-wetting buffer 10µl/cm2 was added followed by addition of a blood sample (5 µl) and the time taken for clear plasma to appear was noted. In the study, commonly available plasma separation matrix (Nylon/Cotton or Nylon/Paper) provides rapid plasma separation for different hematocrite samples (4-13 sec) compared to LF-1 that takes at least 10-18 seconds. Results of the study are as illustrated in FIG. 10B.
[00113] Example 3: Pre-wetting based plasma separation and its usefulness in point of care blood glucose flow-through assay
In the study, plasma separation matrix LF-1, Cotton and Paper were used to study its application in point of care blood glucose assay in a flow through assay format. Pre-wetting buffer was added to the plasma separation membrane followed by addition of 5ul of blood droplet using blood sample cups of predetermined volume (3-5µl). This brings about plasma separation which subsequently is acted upon by GOD/POD, along with TMB chromogenic substrate coated on nylon membrane (reaction membrane) containing polymers, to give green colour. In this experiment, physical examination of reaction membrane reveals that there was no sign of hemolysis or presence of RBCs (evident from microscopic observation). The sensitivity of the assay is not affected by pre-wetting step. Moreover, pre-wetting based plasma separation using either LF-1 or Cotton/Paper plasma separation matrix allows for detection of blood glucose ranging from 15mg/dl to 400mg/dl. Results of the study are as illustrated in FIG. 10C. Following observations were made in the study: Method is compatible with both conventional (glass fiber, polysulfone etc.) and low-cost commonly available membranes (paper, cotton fiber, cellulose acetate, nylon etc.); Pre-wetting step enables rapid and efficient plasma separation within 5-20 seconds irrespective of the plasma separating membrane used; haemolysis free plasma separation can be achieved; testing requires only 3-5 µl sample; the method is compatible with colorimetric point of care diagnostic assays in flow through/lateral flow assays.

[00114] Example 4(a): Pre-wetting buffer composition
The buffer composition includes Imidazole-Acetate buffer prepared using Milli Q water (0.01M-0.1M, pH 6.5), EDTA di-sodium salt (0.01-0.1 %), PMVMA (0.6-0.9 %), DHA (1:10- 100K dilution) and Methyl Orange (0.008-0.1mg/ml).
[00115] Example 4(b): Blood glucose assay strip - reaction membrane formulation
Reaction membrane (Biodyne) was coated with a reagent composition which includes: Phosphate-citrate buffer (0.01M - 0.5M, pH 5.0- 6.5) containing hydroxypropylbetacyclodextrin (with 0.1-0.5%), PVP (10mg-50mg), PEG-4K (10mg-50mg), Tartrazine (0.05mg-5mg), TMB (0.05mg-2mg), PMVMA (0.05mg-2mg), Glucose Oxidase (GOD) (10U-200U), Peroxidase (HRP) (7U-200U), Milli Q (0.5ml-2ml), Imidazole acetate buffer (0.01M-0.5M, pH 5.5-6.9, 0.01ml-2ml) and an enzyme mix composition (0.1ml-0.5ml). The enzyme mix composition includes: sodium acetate buffer (0.01M - 0.5M, pH 5.0-6.5, 0.5ml), Ethylene glycol (0.01ml), Tween 20 (0.01-0.5ml) and a biomolecule stabilizer composition (0.01ml). The biomolecule stabilizer composition includes: Tris buffer (20mM, pH 6.0-7.5, 2 ml-10ml), Gelatin (0.01gm-0.2gm), EDTA-Sodium (5mg-15mg), Tri-Sodium citrate (5mg-15mg), DL-Aspartate (0.2 mg-10mg), DL-Methionine (0.5 mg-10mg), L-Lysine (0.2 mg-5mg), Sorbitol (0.5 mg-15mg), Trehalose (0.5 mg-5mg), Thimersol (5 mg-30mg), BSA (0.2gm-0.5gm),Ammonium sulphate (10 mg-200mg), Propylene glycol (0.5ml-2.5ml) or PMVMA (0.1 gm). The dried reaction membrane can be used for glucose testing strip fabrication. The optimized reaction membrane formulation and process allows for good assay sensitivity (minimum 15mg/dl), accuracy, precision, linearity (up to 400mg/dl), reproducibility, batch to-batch consistency and long shelf life (minimum 12 months)at room temperature storage.
[00116] Example 5 (a): Blood glucose assay strip design
The glucose testing strip suitable for point of care test is a single use disposable strip with dimensions of 4.9cm ( L) x 6mm (W). Thin glazy white Paper sheet of 0.4mm thickness was used for preparation of support strips. Top strip includes a sample window with diameter of 4mm at the centre portion of the strip. Bottom strip includes a reaction viewing window with diameter of 4mm at the centre portion of the strip. Nylon mesh (hydrophobic, 80 micron pore size) was used as spreading screen. Plasma separating membrane made of glass fiber (LF-1) or cotton fiber or paper was used for different experiments.
[00117] Example 5 (b): Blood glucose assay strip fabrication
A blood glucose assay strip was fabricated according to an embodiment to the present disclosure as illustrated in FIG. 11Ato FIG. 11G. White glazy hard board paper sheets of A3 size of thickness of 1.25mm- 1.35 mm were cut into A4 size, using scissors. The A4 sheet was cut exactly to obtain a dimension of 4.9cm length & 21cm width. Later by using a paper cutting machine, exactly 4mm diameter perforations were obtained at the centre of these sheets. This centre hole becomes a reaction viewing window in the strip. The perforated paper sheet was laminated with 100% transparent clean OHP sheet by using a pressure sensitive double adhesive tape (3M tape) on its one side (Posterior). On the other side (Anterior) of this sheet, a nylon mesh (Hydrophobic) of 80 micron pore size (open area) was attached, by using a double adhesive tape. The above processed paper sheet is now ready to be used for assembling the reaction membrane. The reaction membrane pre-coated with the reagent (and stored in a desiccator until ready to be used for assembling) was cut to dimensions of either 5cm or 10cm length and 0.9cm width and placed exactly above the bottom support sheet. Over the reaction membrane, a blood separation membrane, either glass fiber or cotton fiber or paper of similar dimension (either 5cm or 10cm length and 0.9cm width) was placed. Nylon mesh is placed exactly above the plasma separating membrane. The pre cut- perforated paper sheet (top support strip) of 4.9cm length x 21 cm width with a sample window hole in the centre is coated with double adhesive (at both the ends of the sheet except at the centre portion) and placed exactly over the pre-aligned stack of membranes and gently pressed in order to obtain sandwich format membranes between the paper sheets (upper and lower support strips).The assembled membrane structure can now be cut using a scissor/cutting machine to obtain individual strips. Each strip is around 4.9cm (L) and6mm (W). The obtained strips in small batches can be either used for biochemical assay testing immediately or it can be stored in vials/desiccators at room temperature for long term use.
[00118] Example 6: Visual blood gluco dot–point of care blood glucose assay kit
Visual blood gluco dot strip is fabricated as explained above. In this example, LF-1 matrix was used as a plasma separation membrane while reaction membrane formulation was same as explained above. Pre-wetting buffer was prepared as mentioned above. Visual colour reader values in different ranges (15-30, 30-70, 70-100, 100-140, 140-200, 200-400, >400mg/dl) were optimized using known glucose samples. To evaluate the performance of Visual blood gluco dot–point of care blood glucose assay kit both internally & externally the following criteria were analyzed namely Blood sample volume, test sensitivity, linearity, batch-to-batch consistency, room temperature storage stability, accuracy, precision, result readout time, any interference , hematocrite effect, etc. For reference purpose, the branded Glucometer (Accucheck/Free lifestyle) was used. In the study, following observations were made: the glucose assay strip according to embodiments to the present disclosure shows better range of predictive values/analysis by means of visual method and either of capillary blood or venous blood (in EDTA vial) can be used for testing, it requires only 3-5µl blood volume/assay, it was found to be suitable for blood samples with different hematocrite values ranging from 20% to 60%, it was found to be suitable for low resource settings and does not require glucometer or its calibrators, suitable for either Fasting/Random or Post Prandial blood glucose tests at home/clinic, ideal for prognosis & diagnosis of glycaemic conditions in health camps, repeatability of the assay, better assay sensitivity and Linearity noted- detects 15mg/dl (Low) to >400mg/dl (High), visual result readout time is within 30-60seconds, room temperature storage kit with minimum shelf life of 12 months at 40C to 350C.
[00119] Example 7: Visual HbA1c dot predict –Indirect HbA1c semi-quantitative range analysis based on Fasting blood glucose analysis
In this study, cotton was used as a blood separation membrane in addition to the reaction membrane as mentioned above. Pre-wetting buffer was prepared as mentioned above. Visual HbA1c colour reader and its assay was optimized using positive correlation equation between fasting blood glucose sample and HbA1c values from > 800 different patient samples (Male, female, normal, diabetic). Following observations were made in the study: it provides visual read-out for semi-quantitative estimation of HbA1c, it enables prediction of both HbA1c and associated complications; it does not require HbA1c calibrators, it provides detection range of4.0% to 15 % HbA1c level; it is found to be suitable for low resource settings; and it requires only a drop of sample/capillary blood (3-5 µl).
[00120] Example 8: Point of care Blood Glucose assay using a smartphone and optical reader (the platform)
Rectangular optical reader device is fabricated using a thick acrylic sheet, which provides suitable environment for a smartphone (with associated software/application installed in it) to be placed inside it. In addition to this, the device also provides a suitable platform for placing the blood glucose assay strip over it at one corner end. Near the reaction viewing window of the strip, there is a small opening in the optical reader device through which the smartphone camera can capture the strip colour change during the course of assay. The LED light source of the reader device is illuminated by connecting it to a 10mA, 5V power supply. Following observations were made in the study: it enables quantitative blood glucose estimation; directly correlating to semi-quantitative HbA1c level/diabetes complication forecasting using fasting blood glucose analysis c an be performed using the kit and following the methods; low cost commonly available membranes can be used in the method; testing requires only 3-5 µl of blood; blood samples with 20% to 60 % hematocrite levels can be used for the study; the method and kit shows linearity up to 500 mg/dl; kits and strips are stable and have shelf life of more than one year (12 months); the method and kits are sensitive to glucose concentration as low as 30 mg/dl; validation studies using accucheck glucometer for more than 700 samples shows batch to batch consistency.
[00121] Example 9: Point of care Blood Glucose assay reaction membrane composition & its batch performance study - long term room temperature storage
The reaction membranes were prepared in different batches (GS-01, GS-02, GS-03 & GS-04) at different durations, as mentioned below in the Table 1, using disclosed formulations and processes. These reaction membranes were individually cut into appropriate dimensions, wrapped with aluminium foil and stored in a desiccators or storage vial at room temperature (25ºC to 35ºC).
Table 1
Batch GS-01 GS-02 GS-03 GS-04
Date of Fabrication 10-07-2013 18-02-2014 24-04-2014 16-07-2014

At the time of blood glucose analysis, single reaction membranes from each batch were taken out from the vial and fabricated into a strip using blood separation membrane (conventional or low-cost membranes according to the present disclosure). In cases where reaction membranes were used along with conventional blood separation membrane, the blood glucose strips were labelled as Visual Blood Gluco Dot (Version-1).In cases where reaction membranes were used along with low-cost membranes as blood separation membrane, these glucose strips were labelled as Smart Gluco Sense (Version-2). The whole blood samples were collected in EDTA coated tubes of different hematocrite levels and random samples from different types of patients were used for the analysis. For reference purpose, Accucheck kit was used while, Visual Blood Gluco Dot strips and Smart Gluco Sense strips were used as test. The blood glucose assay was done following a method that involved sequential steps of: initial pre-wetting of plasma separation membrane with a drop of pre-wetting buffer composition; addition of whole blood sample (3-5µl) via an inverted cup to the strip; and reading the colour change on other side of the strip visually using its colour reader (colour chart) within 30 to 60 seconds. This method gives semi-quantitative glucose values. The results of the study are as illustrated in FIG. 12A and FIG. 12B.Following observations were made in the study: the real time storage stability of the reaction membrane at room temperature for different batches prepared in-house (GS-01 to GS-04) remain robust and perform at par with the reference (Accucheck) values i.e. within the expected precise glucose values in the reader scale; the blood glucose reaction membrane composition and its process provides batch to batch consistency and performs to its efficiency all the time compared to Accucheck readings i.e. within the precise expected glucose values in the reader scale; the reaction membrane compatibility and its performance in Version-1 as well as Version-2 are more precise and efficient; the shelf life of Visual Blood Gluco dot strip (Version-1) and Smart Gluco dot strip (Version-2) is more than 12 months from the date of fabrication; and frequent opening and closing of the storage vials during analysis do not affect stability of these reaction membranes.
[00122] Example 10: Visual blood gluco dot- point of care blood glucose assay kit performance analysis during long term storage at room temperature
To study performance of Visual Blood glucose assay kit, both version-1 and version-2 were subjected to real time storage stability at room temperature. In cases where reaction membranes were used along with conventional blood separation membrane, the blood glucose strips were labelled as Visual Blood Gluco Dot (Version-1). In cases where reaction membranes were used along with low-cost membranes as blood separation membrane, these glucose strips were labelled as Smart Gluco Sense (Version-2). Both Version 1 and Version-2 were prepared in different batches and stored in their vials at room temperature (20ºC to 35ºC). Respective batches of pre-wetting buffers were also prepared and stored in eppendorf tubes. Fresh whole blood samples were collected in EDTA coated tubes of different hematocrite levels, random blood samples obtained from local diagnostic labs were used for the analysis. Both Version-1 and Version-2 assay kits were used as test (T) and Accucheck kit was used as reference (R).The blood glucose assay test method includes sequential steps of: initial pre-wetting of plasma separation membrane with a drop of buffer composition; addition of whole blood (3-5µl) using an inverted cup to the strip; and reading the colour change on the other side of strip visually using its colour chart within 30-60 seconds. This method gives semi-quantitative glucose values. As illustrated in Table 2 and Table 3 below and in Figure 13A to 13B, blood glucose assay performance using Visual Blood glucose assay kit (Version-1) suggest that it has a long shelf life of more than 12 months when stored at room temperature (20ºC to 35ºC).

Table 2
Visual Blood Gluco Dot
(Version 1) V1A
(16-07-2014) V1B
(19-03-2015)
Stability duration (as on 31-08-2015) 13 months 5 months
Stability duration (as on 16-12-2015) 17 months 9 months
Table 3
Smart Gluco Sense
(Version 2) V2A
(09-03-2015) V2C
(14-08-2015)
Stability duration (as on 31-08-2015) 6 months <1 month
Stability duration (as on 16-12-2015) 9 months 4 months
Visual Blood glucose assay strips (Version-1) are more robust and not affected by frequent opening and closing of storage vials, as evident from the data. Visual Blood glucose assay strips (Version-1) stability data also emphasises batch to batch consistency. The data on blood glucose assay performance using Smart Gluco Scan assay kit (Version-2), suggest that it has a long shelf life of minimum 9 months when stored at room temperature and expected to perform efficiently for more than 12 months from the data of manufacturing. This can be explained from the fact that, reaction membrane composition, real time storage stability data suggests that it is robust and performs efficiently for more than 16 months at room temperature storage in its vial. The reaction membrane with the same composition has been used in both the versions (Version-1 & 2) strips as well. Moreover, for blood separation purpose, in version-2 (Smart Gluco Scan strips) only untreated/inert cotton fiber are used along with the reaction membrane. Thus, one can say that Smart Gluco Scan assay kit is robust and has a long shelf life of more than 12 months at room temperature storage. Smart Gluco Scan assay strips (Version-2) stability data also emphasises batch to batch consistency. Smart Gluco Scan assay strips (Version-2) are more robust and does not affected by frequent opening and closing of its storage vials, as evident from the data.

[00123] Example 11: Pre-wetting based plasma separation and blood glucose assay
Example-11A Pre-wetting buffer & its matrix [Glass fiber + Nylon] in Visual Blood glucose assay – [Version-1]
Pre-wetting buffer composition: 10mM Imidazole-Acetate with 0.01% EDTA, pH 6.5
Plasma separation matrix: Glass fibre (LF-1)
Reaction membrane: Nylon membrane [0.45 micron]
Procedure: The addition of a single drop of this pre-wetting buffer (<10µl) to the biosensor reaction strip followed by drop of blood sample (< 5µl), addition using a inverted cup brings about rapid, efficient non-hemolytic plasma separation and thus colour formation at the nylon membrane, which can be read using its colour reader within 30-60seconds. Results of the study are as depicted in Figure 14A.
Example-11B Pre-wetting buffer & its matrix (cotton fiber +Nylon) in visual blood glucose assay- [Version-2]
Pre-wetting buffer composition: 500mM Imidazole-Acetate with 0.01% EDTA, PMVMA, DHA and methyl orange, pH 6.5
Plasma separation matrix: Cotton fiber
Reaction membrane: Nylon membrane [0.45 micron]
Procedure: The addition of a single drop of this Pre-wetting buffer (<10µl) to the biosensor reaction strip followed by drop of blood sample (< 5µl), addition using a inverted cup brings about rapid, efficient non-hemolytic plasma separation and thus colour formation at the nylon membrane, which can be read using its colour reader within 30-60 seconds. Results of the study are as depicted in Figure 14B.
Example-11C Pre-wetting buffer & its matrix (Whatman filter paper + Nylon) in Visual Blood glucose assay- [Version-3]
Pre-wetting buffer composition: 100mM PBS with 0.01% EDTA
Plasma separation matrix: Cellulose acetate [Whatman#1 ]
Reaction membrane: Nylon membrane [0.45 micron]
Procedure: The addition of a single drop of this pre-wetting buffer (<10µl) to the biosensor reaction strip followed by drop of blood sample (< 5µl), addition using a inverted cup, brings about rapid, efficient non-hemolytic plasma separation and thus colour formation at the nylon membrane, which can be read using its colour reader within 30-60 seconds. Results of the study are as depicted in Figure 14C.

Example-11D Pre-wetting buffer & its matrix (Polysulfone membrane + Nylon) in visual blood glucose assay
Pre-wetting buffer composition: 100mMES with 0.01% EDTA
Plasma separation matrix: Polysulfone membrane- [Vivid-Pall]
Reaction membrane: Nylon membrane [0.45 micron]
Procedure: The addition of a single drop of this pre-wetting buffer (<10µl) to the biosensor reaction strip followed by drop of blood sample (< 5µl), addition using a inverted cup brings about rapid, efficient non-hemolytic plasma separation and thus colour formation at the nylon membrane, which can be read using its colour reader within 30-60 seconds. Results of the study are as depicted in Figure 14D.
Example-11E Pre-wetting buffer & its matrix (Hydrophilic membrane + Nylon) in visual blood glucose assay
Pre-wetting buffer composition: MilliQ with 0.01% EDTA
Plasma separation matrix: Hydrophilic nylon mesh [Satti]
Reaction membrane: Nylon membrane [0.45 micron]
Procedure: The addition of a single drop of this pre-wetting buffer (<10µl) to the biosensor reaction strip followed by drop of blood sample (< 5µl), addition using a inverted cup brings about rapid, efficient non-hemolytic plasma separation and thus colour formation at the nylon membrane, which can be read using its colour reader within 30-60seconds. Results of the study are as depicted in Figure 14E.

ADVANTAGES OF THE PRESENT INVENTION
[00124] The present disclosure overcomes disadvantages associated with conventional glucometers and glucose testing strips.
[00125] The present disclosure provides a glucose testing/biosensor strip that uses commonly available low cost membranes for achieving desired plasma separation.
[00126] The present disclosure provides a buffer composition that can be used to pre-wet membranes to improve their plasma separation efficiency& rapidly using a 3-5µl of blood.
[00127] The present disclosure provides a glucose/analyte testing/biosensor strip with enhanced stability and longer shelf life (more than 12 months at room temperature).
[00128] The present disclosure provides a glucose/analyte testing/biosensor strip comprising only two membranes, one membrane for achieving plasma separation and other as a reaction membrane.
[00129] The present disclosure provides kits and method(s) that enables semi-quantitative estimation of blood glucose over a wide range (15-30 mg/dl, 30-70 mg/dl, 70-100 mg/dl, 100-140 mg/dl, 140-200 mg/dl, 200-400 mg/dl, >400 mg/dl).
[00130] The present disclosure provides kits and method(s) that does not require glucometer or its calibrators.
[00131] The present disclosure provides kit(s) and method(s) that show linearity of glucose detection over a wide range (15-400mg/dl).
[00132] The present disclosure provides kit(s) and method(s) need very small volume of sample (3-5µl).
[00133] The present disclosure provides affordable and single use disposable biosensor strips.
[00134] The present disclosure provides kit(s) and method(s) are suitable for blood samples with wide range of hematocrite values (20% to 60%).
[00135] The present disclosure provides biosensor strips, which have shelf life of more than 12 months.
[00136] The present disclosure provides a smartphone, optical reader and associated software for quantitative estimation of blood glucose, which can provide one or a combination of semi-quantitative or quantitative measurement of blood analytes in 30-60 seconds.
[00137] The present disclosure provides kit(s)and methods with the capability of detection of HbA1c levels and associated secondary complications based on the FBS analysis.