DESC:Field of invention

The present invention relates to the field of biosensors. The invention is about a device (A) for quantitative analysis of a bio-analyte. The quantification of the bio-analyte involves the usage of a novel pre-stained composition forbio-analyte processing,which instantly gives the color change required for the quantification of bio-analyte. The device (A) is compatible with various bio-analyte processing methods,by strip or by assay. The scope of biosensor is wide as it can analyze analyte on the basis of colorimetry, fluorescence, chemiluminescence and the like. The invention provides economical and convenient device and method for Point-of -Care diagnosis.

Background of invention

Point of care (POC) diagnostics is gaining importance in the role of public health in monitoring health conditions, especially for long term diseases like, diabetics, hypocholesterolemia and the like, which requires frequent monitoring of health conditions. Point of care diagnostics is very convenient for patients who find it difficult to visit laboratories frequently. The integration of Smart phone based point of care technology in Healthcare has gained additional attention in modern diagnostics, either alone or in conjunction with add-on devices, for data collection, analysis, display, and transmission, making them popular in POC diagnostics.

Smartphone-based devices and applications (SBDAs) are the most promising and effective means of delivering mobile health diagnosis with cost effectiveness and remote sensing. Several SBDAs have been commercialized for the personalized monitoring and/or management of basic physiological parameters, such as blood pressure, weight, body analysis, pulse rate, electrocardiograph, blood glucose, blood glucose saturation, sleeping and physical activity. With advances in Bluetooth technology, software, cloud computing and remote sensing, SBDAs provide real-time on-site analysis and telemedicine opportunities in remote areas. This scenario is of utmost importance for developing countries, where the number of Smartphone users is about 70% of 6.8 billion cell phone subscribers worldwide with limited access to basic healthcare service. The technology platform facilitates patient-doctor communication and the patients to effectively manage and keep track of their medical conditions. Besides tremendous healthcare cost savings, SBDAs are very critical for the monitoring and effective management of emerging epidemics and food contamination outbreaks.

According to disclosure in patent document, US 8947656 B2, Optical filters are needed and is adaptable to only one particular smartphone accesses due to limitation of the optical device and its design. In the non- patent literature document, Biotechnology.J, Shreedhar Gautam et al., Smartphone-based portable wireless optical system for the detection of target analytes; 2017 states the usage of Smartphone-based Portable and Wireless Optical System in which gold nanoparticles (GNP) and an enzyme, horse radish peroxidase (HRP), to generate colorimetric signals in response to two model target molecules, melamine and hydrogen peroxide are used. The colorimetric signal produced by the presence of the target molecules is converted to an electrical signal by the inbuilt electronic circuit of the device. The converted electrical signal is then measured wirelessly via multimeter in the smartphone which processes the data and displays the results. This process is expensive, complicated and inconvenient for the patients to perform these tests as Point-of-care.

In Anal. Chem., 2014, 86 (19), pp 9554–9562, an Android application for measurement of nitrite concentration and pH determination in combination with a low-cost paper-based microfluidic device is presented. The application uses seven sensing areas, containing the corresponding immobilized reagents, to produce selective color changes when a sample solution is placed in the sampling area. The complications in the process makes the analysis tedious and requires an experienced analyst to operate and analyze.

The point of care can be done for variegated bio-analyte samples such as for blood, urine, veterinary diagnostics and the like. Most trivial and important bio-analyte sample is Blood.HemoglobinA1c (HbA1c) is a specific type of glycated hemoglobin wherein glucose is attached to the amino terminal valine residue of one or both of the hemoglobin A beta chains. By measuring glycated hemoglobin (HbA1c), clinicians are able to get an overall picture of what the average blood sugar levels have been over a period of weeks/months. Glycated hemoglobin reflects an average plasma glucose level over the past 8 to 12 weeks and quantitative assessment of the glycated hemoglobin provides a clue to assess the stages of secondary complications associated with diabetes namely diabetic retinopathy, diabetic neuropathy and cardiovascular complications.

US 7943385 discloses utilization of one or two different types of oxidizing agents which selectively oxidize low-molecular weight reducing substances and high-molecular weight (mainly hemoglobin) reducing substances in blood samples, coupled with enzymatic reactions catalyzed by proteases, fructosyl amino acid oxidase. The limitationsrelatingto this method are, there is no point of care HbA1c assay kit available in the market based on this and moreover, it is suitable for laboratory purpose only and it requires cold storage and test is expensive due to the raw materials like enzymes and chromogenic dyes used in this method.

US Patent 6562581 discloses the quantitative determination of HbA1c based on its Pseudoperoxidase activity using organic dye of two reagent systems such as mono- and diamines, phenols, polyphenols, leucodyes, and other compounds along with inorganic (e.g., hydrogen peroxide) or organic peroxides. In this method, the % HbA1c is analysed directly without additional total Hb analysis, using spectrophotometric method or reflectance method. In this method APBA is coupled to Biodyne C membrane bydicyclohexylcarbodiimide (DCC) method. The major drawback in this method is related to the chromogenic substrate used for detecting HbA1c, has to be prepared only at the time of analysis, by mixingchromogenic substrate along with hydrogen peroxide at appropriate ratio and more over visual semiquantitative analysis of HbA1c levels are not shown in this method.

The long-term diseases requires frequent monitoring of the medical conditions of the patients. The reliable and economical point-of-care diagnosis will be beneficial to a large pool of patients to keep a record of the medical analysis and communicate the results to a medical practitioner conveniently. Thus, there is an immense need of economical, robust and reliable point-of-care diagnosis.

Summary of invention:
Accordingly, the present invention relates to the quantitative determination of variegated bio-analytes A device (A) for quantitative analysis of a bio-analyte comprising-
an optical reader system (2) comprising a light source (3), a Wi-Fi camera(4) connected to a power source (5) which is optionally focused through lens (7) to capture and transmit real-time images of a reaction of the bio-analyte with a composition comprising Citric acid, Acetic acid , Tri-sodium citrate, TMB-S, EDTANa salt, PVP, HPBCD, N,N-diethylhydroxylamine, Mannitol/Sorbitol, Sodium-perboratemonohydrate, D-Ribose, Tartrazine, Dextran Sulphate and MilliQ water; an opaque box (1) with a slit to house the optical reader system (2); and
a communicable instrument (6) programmed and integrated to the optical reader system (2) to process, save, exhibit and transfer the result of analysis; a method of quantitative analysis of a bio-analyte using a device (A), said method comprising acts of a) pre-wetting composition Citric acid, Acetic acid , Tri-sodium citrate, TMB-S, EDTANa salt, PVP, HPBCD, N,N-diethylhydroxylamine, Mannitol/Sorbitol, Sodium-perboratemonohydrate, D-Ribose, Tartrazine, Dextran Sulphate and MilliQ water, b) reacting bio-analyte sample with the composition, c) exposing the reacted sample to the optical reader system (2) of the device (A), d) capturing real time images of the reaction, and e) communicating therealtime images to the communicable instrument (6) programmed to process, save, exhibit and transfer the result of analysis; and a kit comprising device A, bio sensorstrips and reagents for quantitative analysis of a bioanalyte.

Brief description of figures
The features of the present invention can be understood in detail with the aid of appended figures. It is to be noted however, that the appended figures illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention.
Figure 1: schematic illustration of Biosensor or Device (A).
Figure 2:illustrates the design and compatibility of the Optical Reader System (B) in analyzing (a) Bio-analyte processed in a strip (b) Bio-analyte processed in assay cassettes.
Figure3:illustrates coupling of 3-aminophenylboronic acid(3-APBA) to the glycatedhemoglobin.
Figure 4: illustrates 3-APBA bound glycatedhemoglobin when treated with pre-stainedTMB composition produces a blue -green color
Figure 5: shows the steps taken to perform the Dip stick- Point of Care HbA1c assay using pre-stained TMB composition.
Figure 6: illustrates Magnetic nanoparticles based HbA1cassay using pre-stained TMB composition.
Figure 7: shows Microtiter well based HbA1c assay using pre-stained TMB reagent composition.
Figure 8: illustrates Dip stick based Point of Care HbA1c assay using its multicalibrators and its calibration graph, using its Optical strip reader.
Figure 9: shows dip stick point of care HbA1c assay-Visual color chart for semiquantitative analysis.
Figure 10:provides a schematic representation of Dip stick point of Care HbA1c strip design.
Figure 11:provides a Dip stick HbA1c assay for visual detection of HbA1cusing patient blood samples by using HbA1cmulticalibrator.
Figure 12(a-c): provide Dip stick HbA1c assay using patient blood samples of known HbA1c values.
Figure 13: explains point of care dip stick HbA1c assay kit prototype.
Figure 14: schematic diagram of Dual reflectance/fluorescence detection unit.
Figure 15: Fluoorescence HRP assay using prestained TMB reagent.

Detailed description of invention
The foregoing description of the embodiments of the invention has been presented for the purpose of illustration. It is not intended to be exhaustive or to limit the invention to the precise form disclosed as many modifications and variations are possible in light of this disclosure for a person skilled in the art in view of the figures, description and claims. It may further be noted that as used herein and in the appended claims, the singular “a” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by person skilled in the art.

The present invention is in relation to a device (A) for quantitative analysis of a bio-analyte comprising-
an optical reader system (2) comprising a light source (3), a Wi-Fi camera(4) connected to a power source (5) which is optionally focused through lens (7) to capture and transmit real-time images of a reaction of the bio-analyte with a composition comprising Citric acid, Acetic acid, Tri-sodium citrate, TMB-S, EDTANa salt, PVP, HPBCD, N,N-diethylhydroxylamine, Mannitol/Sorbitol, Sodium-perboratemonohydrate, D-Ribose, Tartrazine, Dextran Sulphate and MilliQ water;an opaque box (1) with a slit to house the optical reader system (2);and
a communicable instrument (6) programmed and integrated to the optical reader system (2) to process, save, exhibit and transfer the result of analysis.
In an embodiment of the invention, the bio-analyte is quantitatively analysed by color, fluorescence or chemiluminescence of the real time images of the reaction.
In an embodiment of the invention, the optical reader system (2) is placed in the opaque box (1) with Wi-Fi camera (4)facing upwards if Flow through assay/dip stick biosensor stripsused for analysis of bio-analyte.
In an embodiment of the invention, the optical reader system (2) is placed in the opaque box (1) with Wi-Fi camera (4) facing downwards if bio-sensor assay strips of lateral flow assay cassettes comprising the composition are used for the analysis of bio-analyte.
In an embodiment of the invention, the communicable instrument (6) is a mobile phone or a computer system programmed to analyse by color, fluorescence or chemiluminescence of the real time images of the reaction.
In an embodiment of the invention, the composition of in 100ml milliQ water comprises Citric acid (0.01-1%), Acetic acid (0.0001-0.01%), Tri-sodiumcitrate (0.01-1%), TMB-S (0.01-0.5%), EDTANa salt (0.01-1%), PVP (0.01-1%), HPBCD(0.01-0.5%), N,N-diethylhydroxylamine (0.0001-0.01%), Mannitol/Sorbitol (0.01-1%), Sodium-perboratemonohydrate (0.01-0.5%), D-Ribose (0.01-1%), Tartrazine (0.1-1%), Dextran Sulphate (0.01-1%) .
In an embodiment of the invention, the bio-analyte is selected from a group comprising Hormones, Antigens, Antibodies, Toxins, Enzymes, Metabolites, Heavy metals, Biomarkers, Receptors, Tumour markers, Cytokines and the like; preferably glycated haemoglobin, haemoglobin, albumin, creatinin, myloperoxidase and the like.
In an embodiment of the invention, the bio-analyte is obtained from a group of samples comprising blood, urine, serum, saliva, soil, milk, water and the like.
In an embodiment of the invention, the device is customised as dual optical reader system suitable for fluorescence and reflectance biosensor.
In an embodiment of the invention, flow through, dip stick, and lateral flow assay is analysed
The present invention is also in relation to a method of quantitative analysis of a bio-analyte using a device (A), said method comprising acts of
a) pre-wetting composition Citric acid, Acetic acid , Tri-sodium citrate, TMB-S, EDTANa salt, PVP, HPBCD, N,N-diethylhydroxylamine, Mannitol/Sorbitol, Sodium-perboratemonohydrate, D-Ribose, Tartrazine, Dextran Sulphate and MilliQ water;
b) reacting bio-analyte sample with the composition;
c) exposing the reacted sample to the optical reader system (2) of the device (A);
d) capturing real time images of the reaction; and
e) communicating therealtime images to the communicable instrument (6) programmed to process, save, exhibit and transfer the result of analysis.
In an embodiment of the invention, the analysis is by kinetic or end point assay mode.
In an embodiment of the invention, composition is pre-wetted in strip analysis.
In an embodiment of the invention, optical reader system (2) is inside the opaque box (1) of device (A) with Wi-Fi camera (4) facing upwards; if the analysis is by dip stick strip analysis.
In an embodiment of the invention, optical reader system (2) is inside the opaque box(1) of device (A) with Wi-Fi camera (4)facing downwards; if the analysis is by lateral flow analysis.
In an embodiment of the invention, analysis is related to color, fluorescence and chemiluminescence of the reaction of the bio-analyte with the composition.
In an embodiment of the invention, the real time images in kinetic assay mode or end point assay mode are captured between 30s to 120s after the initiation of the reaction of the bio-analyte with the composition.
In an embodiment of the invention, the method is for quantitative analysis of H1bAc/ blood glucose.
The present invention is also in relation to a kit comprising device A, biosensor strips and reagents for quantitative analysis of a bioanalyte.

The present invention relates to a device (A)( refer to Figure 1) which is a portable biosensor used for quantitative analysis of bio-analyte.The biosensor or a device (A) comprises an Optical Reader System (B)integrated with a communicable device (6) such as smartphone or a computer.The quantification of the bio-analyte involves the usage of a novel pre-stained composition forbio-analyte processing,which instantly gives the color change required for the quantification of bio-analyte.The device is useful in human healthcare diagnostics, animal disease diagnostics, plant pathogen diagnostics, environment monitoring, and food quality evaluation and the like. The invented biosensor has diverse application in the field of Biotechnology (biomarkers), human diagnostics, environmental science, food and plant pathogen detection, veterinary diagnostics and the like.Various Bio-analytes such as Hormones, Antigens, Antibodies, Toxins, Enzymes, Metabolites, Heavy metals, Biomarkers, Receptors, Tumor markers, Cytokines and the like can be detected using this device and method. The bio-analytes from different bio-analyte sample can be analyzed using the invented device and method, such as blood samples, urine, serum, saliva, soil, milk, water and the like. The device (A) is not limited as a colorimetric biosensor but can be used wherein fluorescence, chemiluminescence, reflectance and the like are measured.

In the present invention, a low-cost portable device (A), with anoptical reader system (B)(SmartBioSense) which is housed inside an opaque box (1) is designed. The optical reader systemcomprises a Wi-Fi camera (4) connected to a power source (5) with a power cable (8); an LED light source(3), a result reading window (2) and a lens (7) to improve the image pixel quality.The communicable device with the RGB color reader App is integrated with the image processing program, which processes real-time images taken by camera(4) of the optical reader system.
The configuration of the camera (4), LED light source (3), a result reading window (2) and the Lens (7) in this Optical reader system (SmartBioSense)(B)can be customized according to the nature of the Biosensor used. For instance, for a Biosensor strip such as blood glucose or HbA1c dip stick, or urine dip strips and the like, the Optical Reader system(B) is designed with Wi-Fi camera (4) facing top towards the slit window, above which the biosensor strip will be kept during analysis.
This methodology overcomes the limitations of environmental influence during analyte detection with its Biosensor using Smartphone(6). Moreover, portability of device with the Optical Reader System (SmartBioSense) (B) and its inbuilt application program is compatible to any communicable brands available in the market. In addition, this App tend to run the RGB Image of the Biosensor assay strip and show the result instantly making it user friendly and reliable.
The device works when power battery or source (5) of optical reader system (B) is switched ON and the Smartphone App for the Bio-analyte is selected to establish Wi-Fi connectivity. Once it is connected, the Bio-analyte is kept either inside or outside the Optical reader system (B) during analysis, allowing the Wi-Fi camera (4) to focus through the Lens (7) and read the RGB color change in the biosensor strip from the result reading window (2), either in kinetic or end point assay mode, and instantly communicate in a real time manner to Smartphoneand further processes and analyze by Smartphone App for specific Bio-analyte (Algorithm, for RGB to CIE-LAB conversion based equation) results in specific units. The results can be stored for later use and communicated to a clinician for review and suggestions.

The synergistic composition(also referred as pre-stained composition) comprises reagents as discussed in table 1-2. The important reagent in the composition is Tetramethylbenzidine (TMB), wherein TMB solution is prepared by converting tetramethylbenzidine to its sulfated salt (TMB-S). Typicaly the composition comprises Citric acid, Acetic acid , Tri-sodium citrate, TMB-S, EDTANa salt, PVP, HPBCD, N,N-diethylhydroxylamine, Mannitol/Sorbitol, Sodium-perboratemonohydrate, D-Ribose, Tartrazine, Dextran Sulphate and MilliQ water. The pH of the solution is maintained at 2.5 to 3.5.

Table 1: The pre-stained composition’s wide workable range in 100ml milliQ water.
Sl.No. Composition Quantity in percentage
1 TMB-S 0.01-0.5
2 HPBCD 0.01-0.5
3 EDTA-Na salt 0.01-1
4 PVP 0.01-1
5 Mannitol 0.01-1
6 Citric acid 0.01 -1
7 Tri-Nacitrate 0.01-1
8 N,Ndiethylhydroxylamine 0.0001-0.01
9 Sodium-perboratemonohydrate 0.01-0.5
10 Acetic acid 0.0001-0.01
11 D-Ribose 0.01-1
12 Tartrazine 0.1-1
13 Dextran sulphate 0.01-1

Table 2: A typical sample 100ml of the composition in milliQ water with specific quantities of the reagents-
Sl.No. Composition Quantity percentage
1 Citric acid 0.1532
2 Acetic acid 0.001
3 Tri-Na-citrate 0.24
4 TMB-S 0.055
5 EDTA Na salt 0.025
6 PVP 0.0125
7 HPBCD 0.075
8 N,Ndiethylhydroxylamine 0.00025
9 Mannitol/Sorbitol 0.1875
10 Sodium-perboratemonohydrate 0.03
11 D-Ribose 0.025
12 Tartrazine 0.008
13 Dextran sulphate 0.10

The addition of Tartrazine to TMB composition makes the reagent composition yellow in color. This pre-stained TMB composition prepared as above, possess pH around 3.0 ±0.5 and can be used for detecting glycated hemoglobin in the blood sample.

The reagent composition possesses dual optical properties namely colorimetric as well as fluorescence property. With this compositions, bioanalytes with Pseudoperoxidase activity such as (Hb, HbA1c, Albumin, Creatinin, Myloperoxidase and the like or with peroxidase activity ( E.Coli,) and the like can be analyzed. This composition can be used in any format, lateral flow assay format, dip stick format (HbA1c) and the like.

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 and above), accuracy, precision, linearity (up to 400mg/dl), reproducibility, batch to-batch consistency and long shelf life (upto 12 months) at 20-30 °C (room temperature) storage.

The device (A) is designed in a way making it compatible with various biosensor strips. For instance, this can be refabricated to accommodate lateral flow immunoassay strips to analyze the results quantitatively either by colorimetric/fluorescence/chemiluminescence and the like.

The device(A)can analyze the bio-analyte in the form of a dip stick biosensor strip as well as assay cassettes strip. In case of dip stick biosensor strip, the Wi-Fi camera (4) faces upwards (Figure 2(a)) towards the result window in the Optical Reader System (B) for the analysis. A Lateral flow assay Biosensor cassette strip are used for testing, in such case the optical reader system (B) is designed with Wi-Fi camera (4) facing downwards (Figure 2(b)) towards the result window of the Lateral flow assay. The Lateral flow assay both control and test line images will be captured, analyzed and displayed with appropriate units, using Optical Reader system (B).

Calibration graphscorresponding to bio-analyte will be fed into the application system with Calibrators /or known Bio-analyte sample values and tested using Biosensor strips in the optical strip reader (2) integrated with smartphone (6). The obtained the calibration graph and its equation is incorporated into the Smartphone App program for unknown sample analysis.

The Smartphone (6) App analyze the RGB color change of the assay or strip, which is further converted to either Hue Value by using HSV formula or CIE-LAB (a color space specified by International Commission on Illumination) value in which, B value is used to generate calibration curve using its standards and subsequent usage of Algorithm to give unknown analyte results.
The quantitative analysis is initiated by switching ONthe power source(5) of Optical reader system (B) and the Smartphone (6) App, and thereby selecting the Blood glucose assay in the mobile display. Once the Optical Reader system (B) is connected to smartphone(6) by Wi-Fi, the sensor strip is kept over the opening slit of the Optical reader system (B) and a drop of prewetting buffer is added followed by 5µl blood sample is added to the strip and the composition in the strip and the blood sample react to form color change. The Smartphone key in the application for Wi-Fi camera (4) with timer is turned ON which capture the Images of the strip at the end of 30 seconds and the captured RGB Images are further processed and analyzed for specific Blood glucose values in mg/dl using the Algorithm, for RGB to CIE-LAB conversion based equation. The Smartphone (6) App analyze the RGB color change of the assay strip, which is further converted to either Hue Value by using HSV formula or CIE-LAB (a color space specified by International Commission on Illumination) value in which, B value is used to generate calibration curve using its standards and subsequent usage of Algorithm to give unknown analyte results.
A detailed Analysis of HbA1c using the invented device as a HbA1c Biosensor
For exemplary purpose, the usage of the device is described for HbA1c analysis. During HbA1c analysis using strips,HbA1c assay (without total hemoglobin level analysis in a sample), is carried out and compared the results with the gold standard HPLC method. HbA1c analysis involves a color change in the assay strip and this color change isanalysed using a visual color reader for semi quantitative HbA1c values in range or using the device of present inventionintegrated to a Smartphone for quantitative HbA1c values. In addition, the App can highlight the severity / level of the secondary complications associated with diabetes and helps the clinicians to treat the patients for better diabetic management. Also, point-of-care-test (POCT) direct HbA1c Biosensor has many following features: Low cost disposable strips, robust & room temperature storage kit for 6 months, Rapid detection of results (within 5minutes), Assay linearity ranges from 4% to 15% and more suitable for low resource settings & primary health centers across the country.

The whole blood is treated with lysate buffer pH 8-9, containing saponin, which not only favors rapid lysis of RBC to release HbAoand HbA1c in the buffer but also tends to inhibit HbAopseudoperoxidase activity. Subsequent treatment with boronic acid affinity ligand matrix either through paper dipstick format or magnetic bead format brings about separation of HbA1c from the lysate. The formation of the cyclic ester between dihydroxyborylgroup of the boronic acid derivative and the 1,2-cis-diol group presented in the carbohydrate moiety of HbA1C molecule proceeds at high pH values. The preferred pH range of the reaction is 7.5-10.0. A number of buffer solutions can be used, ranging from phosphate, glycine, ammonium acetate, CAPS, taurine, and others capable of maintaining a suitable pH of the reaction mixture, more preferably boric acid-borax buffer. At this alkali pH range, only HbA1c binds to 3-aminophenylboronic acid (3-APBA),while other analytes in the lysate including HbAo or glycated albumin does not bind to it.The separated HbA1c, bound to APBA (Figure 3) possesses pseudoperoxidase activity.

On addition of pre-stained TMB composition, a gradient of yellow-green-blue color change is observed (Figure 4). The intensity of the color change or gradient is directly proportional to the amount of HbA1c present in the blood sample. The method allows for measurement of HbA1c level in the blood sample, either by semiquantitative level by using visual color chart or quantitatively by Red Green Blue image analysis using a direct Smartphone and its App/ or optical strip color reader along with smart phone and its App.

In an embodiment of present invention, rapid glycatedhemoglobin analysis can be achieved through various methods such as flow through assay format or by dip stick assay format, magnetic bead assay format,Microtiter plate format, by using the pre-stainedTMB composition.
Experimental
Example 1: Dip stick Point of Care HbA1c assay using pre-stained TMB composition
In the dip stick assay format, (Figure 5) whole blood ranging from about 2µl -5µl is added to a lysis buffer, mixed and allowed to stand for 30-60 seconds, and then a dip stick is inserted to the lysate and allowed to stand for 30-60 seconds. The dip stick is then removed and rinsed with wash buffer twice and blotted using a tissue paper. Finally a drop of pre-stained TMB composition (10µl-15µl/drop) is added to the reaction membrane of dip stick, which brings about different shades of blue-green color depending upon the level of HbA1c bound to the membrane. The color developed on the reaction membrane can be read by using a visual HbA1c color reader for semiquantitative HbA1c level after 60 seconds or based on RGB image analysis using a Smartphone/Optical reader and its App for quantitative result. The image processing in a HbA1c is given in Flow Chart 1.These images are instantly analyzed further by the smart phone RGB reader App, to give a quantitative HbA1c/Blood glucose results instantly within few seconds. An example for quantitative analysis of assay with graph and values are given in figure 8. Initially, by using multi calibrators of the analytes (HbA1c/Blood Glucose) respective calibration graphs are obtained using the optical strip reader and communicable device. By using its respective calibration graph, the unknown sample HbA1c/Blood glucose values are obtained.

Flow Chart1: Sequential steps (Algorithm) to convert the color image of the reaction biosensor Strip into a quantitative analyte value in a HbA1c Biosensor

Example 2: Magnetic nanoparticles based HbA1c assay using pre-stained TMB reagent composition
In a magnetic bead assay format, (figure 6) when whole blood ranging from about2ul -5ul is added to the lysis buffer, mixed and allowed to stand for 30-60 seconds, followed by addition of magnetic bead APBA conjugate reagent, around 50ul to 100ul and allowed to mix for 30-60 seconds. Then by keeping the lysate tube over the magnet, brings about sedimentation of its beads, the supernatants are discarded. The sedimented beads are washed and added pre-stained TMB reagent 100ul /testis and incubated for 60-120 sec. This brings about different bluish green color depending upon the level of HbA1c bound to the beads. The bluish-green color developed on the wells can be read based on RGB image analysis using a Smartphone App/Optical reader for quantitative result.

Example 3: Microtiter well based HbA1c assay using pre-stained TMB reagent composition

In a 96 Microtiter plate format, (Figure 7) APBA are immobilized covalently on the surface of the microtiter wells and blocked with suitable agents. Blood lysate prepared [by method previously mentioned] is added to the 96 microtiter wells and incubated for 60 seconds. After incubation period, the supernatant is decanted and the wells are flushed by washing twice using buffer and finally pre-stained TMB composition 100ul /test is added and incubated for 60-120 sec. The bluish-green color developed on the wells can be read based on RGB image analysis using a Smartphone color reader App for quantitative result.

Example 4-Dip stick HbA1c point of care assay using smartphone directly
In this method, dip stick HbA1c assay is done as shown in example 1. During analysis, the HbA1c strip after addition of pre-stained TMB composition is kept over the unique color charts, which tend to adapt and nullify the environmental lighting during testing. By focusing Smartphone camera directly over the unique color chart (11) having the dip stick HbA1c strip (10), the color change of the biosensor strip is captured in real time and its RGB image are instantly processed by its App and results are shown (Figure 9). In this method, one can quantify HbA1c level using smart phone directly without an optical strip reader.

Example 5-Semiquantitative point of care HbA1c assay using its visual color reader
In this method, dip stick HbA1c assay is done as shown in example-1. During analysis, the HbA1c strip, after the addition of Pre-stained TMB reagent is kept over the Visual color chart to interpret a precise HbA1c range within a given time (Figure- 11,12 a,12 b,12 c). Addition of pre-stained TMB to the dip stick membrane brings about color change with time. The intensity of the color change is directly proportional to HbA1c level. The interpretation has to be done within 2 minutes at ambient lighting condition. The Visual HbA1c color chart for semiquantitative results has been developed using the commercial HbA1c multicalibrators. Gradient of blue-green color indicates respective semiquantitative HbA1c level namely [ Light green shade on the dip stick indicates HbA1c level as 4 to 6%; dark green shade on the dip stick indicates HbA1c level as 6-8% and bluish green as 8 to 15%].

Example 6-Dip stick HbA1c strip design.
In the dip stick method, a Biodyne C(Nylon membrane from PALL Lifescience,USA) membrane is coated with 3-aminophenylboronic acid (3-APBA), by using EDC-NHS covalent coupling method. The prepared boronate affinity membranes are cut into appropriate specific dimensions 0.9cmx 0.5cm and attached to a thick backing material like PVC sheet or OHP sheet. The surface of the membrane is covered with a nylon mesh of 100 micron pore size. (Figure 10) The fabrication allows the membrane to be attached to the backing material as well as it helps in bringing about wicking (spreading) effect of pre-stained TMB reagent onto the membrane.

Example7 -Dip stick HbA1c assay kit stability analysis
The kit comprising, lysisbuffer,wash buffer, pre-stained TMB reagent and HbA1c dip stick membranes are prepared fresh and are analyzed using the calibrators as shown in example-3 and further stored ata temperature ranging from about 20°C to about 30°C for longer duration. Periodically, the kit content isanalyzed using the known HbA1c samples as well its calibrators using Visual color reader for semiquantitative manner. The data obtained from these studies suggest that, the Dip stick HbA1c kit content in the present format can be stored at room tempt at least 6 months and performs efficiently on par with the freshly prepared batch reagents (Figure13).

Example 8 - Point of Care dip stick HbA1c assay kit prototype
The invention also provides for a kit for point of care HbA1c test, comprising the following a)HbA1c dip stick storage vials, b) Pre-stained TMB ,c) Lysis buffer d) Wash buffer, e) Visual color reader (Semiquantitative reader) and f) device A for quantitative detection of glycated hemoglobin.

Example 9- Optical Strip Reader integrated to Smart phone for quantitative dual reflectance/fluorescence Biosensor
Figure 14 provides the diagram of the modified device (A), which is customized as a dual biosensor for analysis by both reflectance and fluorescence. The device can detect both reflected image as well as fluorescence signal from the biosensor strip. For reflectance operation, the illumination LED( white or yellow) (18) is used to uniformly illuminate the sensor. The reflected light is focused on the camera (12) through the beam splitter and lens (14). The filter (13) blocks any UV light. For fluorescence operation, the ultraviolet LED (17) is used to excite the sensor through the beam splitter (15). The emitted fluorescence ( green or red) is focused on the camera through the beam splitter. The filter blocks the excitation of UV light.

Example 10: Dual Optical Property of prestained TMB reagent composition

Novel Prestained yellow color TMB liquid monoreagent (precipitate form) brings different shades of color precipitate [ranging from yellow -Light green-Dark Green- Bluish Green-Blue] shades of colors, during reaction and thus helps to distinguish visually the analyte levels by using its visual colour reader. This finds application in biotechnology like healthcare HbA1c biosensor, as well as Pseudoperoxidase based assays; Immunohistochemistry, Food pathogenesis like E.Coli detection, Western blotting, microarray and Dot-ELISA in research etc.

In addition to this, the same composition also possesses fluorescence property with excitation @ 280nm and emission at 400nm. The working principle using HRP assay, in which the RFU tend to decrease with time according to the HRP level i.e if more HRP is present in the sample, it brings rapid decrease in RFU value; while minimal HRP brings slow decrease in RFU value with time ( Figure 15).

The prestaining of TMB reagent composition in liquid form is achievable by mixing water soluble organic dyes or food additive colours at appropriate ratio namely Tartrazine, Erythrocone B, Allura red Ac, Brilliant Blue FCF and the like either alone or in combination alsong with its unique composition.

Thus, the present invention provides an economical and robust method for quantitative determination of a bioanalyte, including glycatedhemoglobinwith a simple portable device. The device helps in point of care diagnostics with simplicity and accuracy.
,CLAIMS:WE CLAIM:
1. A device (A) for quantitative analysis of a bio-analyte comprising-
an optical reader system (2) comprising a light source (3), a Wi-Fi camera(4) connected to a power source (5) which is optionally focused through lens (7) to capture and transmit real-time images of a reaction of the bio-analyte with a composition comprising Citric acid, Acetic acid, Tri-sodium citrate, TMB-S, EDTANa salt, PVP, HPBCD, N,N-diethylhydroxylamine, Mannitol/Sorbitol, Sodium-perboratemonohydrate, D-Ribose, Tartrazine, Dextran Sulphate and MilliQ water;an opaque box (1) with a slit to house the optical reader system (2);and
a communicable instrument (6) programmed and integrated to the optical reader system (2) to process, save, exhibit and transfer the result of analysis.
2. The device (A)as claimed in claim 1, wherein the bio-analyte is quantitatively analysed by color, fluorescence or chemiluminescence of the real time images of the reaction.
3. The device (A) as claimed in claim 1, wherein the optical reader system (2) is placed in the opaque box (1) with Wi-Fi camera (4)facing upwards if Flow through assay/dip stick biosensor stripsused for analysis of bio-analyte.
4. The device (A) as claimed in claim 1, wherein the optical reader system (2) is placed in the opaque box (1) with Wi-Fi camera (4) facing downwards if bio-sensor assay strips of lateral flow assay cassettes comprising the composition are used for the analysis of bio-analyte.
5. The device (A) as claimed in claim 1, wherein the communicable instrument (6) is a mobile phone or a computer system programmed to analyse by color, fluorescence or chemiluminescence of the real time images of the reaction.
6. The device (A) as claimed in claim 1, wherein the composition of in 100ml milliQ water comprises Citric acid (0.01-1%), Acetic acid (0.0001-0.01%), Tri-sodium citrate (0.01-1%), TMB-S (0.01-0.5%), EDTANa salt (0.01-1%), PVP (0.01-1%), HPBCD(0.01-0.5%), N,N-diethylhydroxylamine (0.0001-0.01%), Mannitol/Sorbitol (0.01-1%), Sodium-perborate monohydrate (0.01-0.5%), D-Ribose (0.01-1%), Tartrazine (0.1-1%), Dextran Sulphate (0.01-1%) .
7. The device (A) as claimed in Claim 1, wherein the bio-analyte is selected from a group comprising Hormones, Antigens, Antibodies, Toxins, Enzymes, Metabolites, Heavy metals, Biomarkers, Receptors, Tumour markers, Cytokines and the like; preferably glycated haemoglobin, haemoglobin, albumin, creatinin, myloperoxidase and the like.
8. The device (A) as claimed in Claim 1; wherein the bio-analyte is obtained from a group of samples comprising blood, urine, serum, saliva, soil, milk, water and the like.
9. The device (A) as claimed in claim 1, wherein the device is customised as dual optical reader system suitable for fluorescence and reflectance biosensor.
10. The device (A) as claimed in claim 1, wherein flow through, dip stick, and lateral flow assay is analysed.
11. A method of quantitative analysis of a bio-analyte using a device (A), said method comprising acts of
a) pre-wetting composition Citric acid, Acetic acid , Tri-sodium citrate, TMB-S, EDTANa salt, PVP, HPBCD, N,N-diethylhydroxylamine, Mannitol/Sorbitol, Sodium-perboratemonohydrate, D-Ribose, Tartrazine, Dextran Sulphate and MilliQ water;
b) reacting bio-analyte sample with the composition;
c) exposing the reacted sample to the optical reader system (2) of the device (A);
d) capturing real time images of the reaction; and
e) communicating therealtime images to the communicable instrument (6) programmed to process, save, exhibit and transfer the result of analysis.
12. The method as claimed in claim 11, wherein the analysis is by kinetic or end point assay mode.
13. The method as claimed in claim 11, wherein composition is pre-wetted in strip analysis.
14. The method as claimed in claim 11, wherein optical reader system (2) is inside the opaque box (1) of device (A) with Wi-Fi camera (4) facing upwards; if the analysis is by dip stick strip analysis.
15. The method as claimed in claim 11, wherein optical reader system (2) is inside the opaque box(1) of device (A) with Wi-Fi camera (4)facing downwards; if the analysis is by lateral flow analysis.
16. The method as claimed in claim 11, wherein the analysis is related to color, fluorescence and chemiluminescence of the reaction of the bio-analyte with the composition.
17. The method as claimed in claim 11, wherein the real time images are captured in kinetic assay mode or end point assay mode between 30s to 120s after the initiation of the reaction of the bio-analyte with the composition.
18. The method as claimed in claim 11, wherein the method is for quantitative analysis of HbA1c/ blood glucose.

19. A kit comprising device A, biosensor strips and reagents for quantitative analysis of a bioanalyte.