Field of invention:
The present invention is in the field of bio-technology and more particularly relates to a bio-strip for semi-quantitative estimation of oxalate in urine.

Background of invention and relevant prior art:
Determination of oxalate in urine and blood is of great interest, as it directly indicates the concentration of oxalate in the human body. In humans, oxalate is a metabolic end product with no enzyme present to act on it and because no further degradation occurs, oxalate accumulates in urine. Due to enhanced endogenous synthesis and /or increased adsorption of oxalate from the diet, the oxalate accumulation leads to various ailments like hyperoxaluria, kidney stones, oxalosis etc. Because of the low solubility of oxalate, increased concentrations of oxalate in body fluids, including the urine, can lead to the deposition of calcium oxalate (oxalosis) in the kidney tissue (nephrocalcinosis) or urinary tract (urolithiasis, nephrolithiasis, kidney stones). Oxalosis occurs after the kidneys fail and the excess oxalate builds up in the blood, and then spreads to the eyes, bones, muscles, blood vessels, heart and other major organs. Severe hyperoxaluria if left untreated can lead to serious illness and even death. For these reasons, it is critical that hyperoxaluria should be diagnosed and treated as early as possible.
Thus it is apparent that measurement of oxalate in urine and plasma is very important for the diagnosis and medical management of innumerable disease conditions like primary and secondary hyperoxaluria, idiopathic calcium oxalate urinary stone diseases, nephrolithiasis, steatorrhoea, ileal disease, ethylene glycol poisoning, E-ferrol toxicity syndrome and so rapid, timely and efficient determination of oxalate in urine becomes imperative.
Various methods and bio-sensors are available for oxalate determination but they suffer from one or the other drawback such as complex procedures, requirement of laboratory facility, expensive equipment, skilled person for analysis & time consuming process.
Some common methods available for the measurement of oxalate in biological fluids include solvent extraction, direct precipitation, colorimetric, gas liquid chromatography (GLC), isotachophoresis, chemiluminiscent method, enzymatic colorimetric, enzymic UV spectrophotometric, enzyme electrode method, isotope dilution and mass spectrometry, ion chromatography, atomic absorption spectrophotometric, continuous flow method, high performance liquid chromatography (HPLC), zone electrophoresis and inorganic capillary electrophoresis, but they all require skilled labor and expensive equipment for efficient performance of tests.
Enzymic colorimetric method employing free (not immobilized) oxalate oxidase and chromogenic system comprising MBTH (3-Methyl-2-benzothiazolinone hydrazine) /4-aminophenazone, DMAB (3-dimethyl aminobenzoic acid)/Phenol and peroxidase in PB buffer of pH 7.0 is commercially available for routine analysis but this method is time consuming and a skilled person is required to conduct such tests.
Bio-sensors for detection of oxalate in various samples, comprising oxalate oxidase from various sources such as barley; banana etc. are known wherein the enzyme has been immobilized on different substrates, but they are essentially different from the present invention and suffer the disadvantage of being less rapid and inconvenient to a unskilled user.
Inamdar et al, 1986, describes dual enzyme membrane strips comprised of 2 enzymes oxalate oxidase and peroxidase entrapped in acrylamide membrane and use of such strips in determination of urinary oxalate. A crude preparation of 45-60% acetone cut obtained from banana fruit peel (Musa paradisiaca; French plantain) homogenate was used as a source of oxalate oxidase, which decomposed oxalate into CO2 and H2O2. The oxalate content of a given urine sample was determined by introducing the enzyme membrane strip into an aliquot of buffered urine containing a suitable chromogen and then measuring the color developed due to the interaction of peroxidase with the H2O2 and the chromogen. Urine samples were pretreated with sodium nitrite to eliminate the interference of ascorbic acid in the assay. The process of the assay described is more complicated and the sample analysis takes more than an hour and can be conducted only in a laboratory. Moreover this is a dual enzyme strip and as such is different in construction from the present invention and more costly because of quantum and number of components involved.
Reusable strip of oxalate oxidase for determination of urinary oxalate were made in the laboratory of inventors (Madanpotra et al, 2004; Bharat et al, 2005; Sharma et al, 2007; Godara et al, 2008) where enzyme oxalate oxidase is obtained from partially purified barley seedlings/sorghum leaves/ Amaranthus leaf which has been immobilized covalently on alkylamine/ arylamine glass beads affixed on a plastic strip and their application in quantitative method for discrete analysis of oxalate in urine has been studied. These methods were based on measurement of H2O2 generated from urinary oxalate by strip bound oxalate oxidase by Trinder’s color reaction. However, the high cost of alkylamine/ arylamine glass beads made these strips expensive and their fixation onto plastic strip was fleeting, thus reducing the stability. Moreover the added disadvantage was that the sample analysis was possible only in laboratory by skilled person and it took considerable time i.e. 15 to 25 min.
Patent application number 2393/DEL/2004 from the laboratory of inventors discloses the process of covalent immobilization of protein (oxalate oxidase) onto PVC support. The invention discloses the use of PVC strip and tube bound sorghum oxalate oxidase for urinary oxalate determination. Patent application number 2394/DEL/2004 from the same laboratory describes the process of covalent immobilization of protein (sorghum oxalate oxidase) onto eggshell membrane. These strips are quite different in construction from the present invention and are less sensitive.
US Patent 424423 describes an altogether different concept of entrapment of oxalate-reducing enzymes onto polymeric matrices for lowering free oxalate concentrations in fluids. The invention is concerned with an efficient means of lowering free oxalate in biological fluid by using oxalate-reducing enzymes entrapped on to polymeric matrices.
US Patent 4539118 discloses a technique for separating oxalate from urine and other biological fluids. The method involved mixing of the biological fluid with an adsorbent to separate oxalate from the fluids, washing the adsorbent-oxalate complex and releasing oxalate by reacting the complex with alkali.
US Patent 5604111 concerns with the use of formyl-CoA transferase enzyme together with oxalyl-CoA decarboxylase for detection and measurement of oxalate in biological samples. The use of the enzyme system resulted in the conversion of oxalate into carbon dioxide and formate. Because the production of formate is directly correlated to the concentration of oxalate present in a sample, the determination of the resulting formate concentration provided an accurate, sensitive and rapid means for detecting even low levels of oxalate. Thus the inventive concept of this patent is quite different from that of present invention.
US Patent 6177478 discloses an altogether different approach for reducing oxalate levels in a patient that includes administering to the patient a therapeutically effective amount of non-absorbable amine polymers such as a polymer characterized by a repeat unit having the formula: ##STR1##and salts and copolymers thereof.
US Patent 6835554 describes a method and corresponding kit for determining the redox status of an organism. The method comprised performing a colorimetric assay of a urine sample by mixing the sample with at least one member of a group of disclosed reagents and an acid and upon stable color formation, comparing the resulting color to a color scale to indirectly determine the oxidative load, or stress, experienced by that organism. The corresponding kit comprises at least one of the reagents described above, an acid, and a color scale.
Most of prior art describing quantitative oxalate determination had one or more limitations such as requirement of laboratory, skilled person for analysis & time consuming process, therefore it is not possible to test the urine at person’s bedside easily and quickly. Thus the present invention achieves what was unachieved in prior art and to the best of our knowledge there is no other method available for determination of oxalate in urine, which can be conveniently used by a person without the help of a laboratory and which provides instant result.

Objects of the invention:
The object of the present invention is to provide instant analysis of oxalate in urine which can be conducted in absence of a laboratory facility and expensive equipment.
One of the most important objective of the present invention is to provide a simple, disposable bio-strip for estimation of oxalate in urine, that is easy to use and can be operated by a person of ordinary skill, without the use of any special equipment at a person’s bed-side.
It is another object of the present invention to provide a bio-strip that is of low cost, thereby making it economically available to the purchasing public.
It is yet another object of the present invention to provide a convenient method for estimation (semi-quantitative) of oxalate in urine, which is less complex and can be executed by a person of ordinary skill.
Another important object of the present invention is to provide common people with an economical kit comprising of the aforementioned bio-strip and a color chart for rapid estimation (semi-quantitative) of oxalate in urine.

Summary of Invention:
The aim of the present invention is to provide a disposable bio-strip and a rapid, convenient method for analysis (semi-quantitative) of urinary oxalate.
To make the bio-strip for instant detection of urinary oxalate, first the enzyme oxalate oxidase was purified from leaves of 10-day old grain sorghum seedling plants by ammonium sulphate fractiation (0-80%), Sephadex G-100 gel filtration and ion exchange chromatography on DEAE- Sephacel. The purified enzyme showed 97-fold purification with 12.6 % yield (Table-2). The enzyme was cross-linked with 1% glutaraldehyde and then adsorbed onto CA pads affixed on a ‘hard art’ paper strip along with buffer and O-dianisidine. As soon as a drop of oxalate solution was put on the bio-strip, it showed yellow color with O-dianisidine as chromogen. The intensity of color was highest at 0.4 M ionic strength and pH 5.0 of reaction buffer and the result was achieved within 45 seconds. The intensity of the color increased as the oxalate concentration was increased from 20mg/100ml to 200mg/100ml, which served the base of semi-quantitative determination of oxalate. The strip was used for instant semi-quantitative determination of oxalate in urine at the bedside of patients.
The method utilizing the aforesaid bio-strip for instant analysis of urinary oxalate comprises the steps of: (a) dipping and moving a disposable bio-strip in urine sample to be tested, (b) removing the bio-strip from the sample, (c) air-drying the bio-strip dipped in test sample, (d) observing the color change of the bio-strip and (d) comparing the color of bio-strip with a standard reference chart as herein described.
The present method of semi-quantitative analysis of urinary oxalate has the advantage that it can be used by the concerned person with great ease and provides instant and reliable result. The strip is expected to be helpful in estimation of oxalate in urine which differentiates normal urine from hyperoxaluric urine. Table 1 presents a comparative account of the advantages of the bio-strip of the present invention with known bio-strips.

Table 1. A comparative table on the advantages of the bio-strip of the present invention with known bio-strips.

Author, Year Type of oxalate oxidase Support used for immobilization Type of immobili-zation Analysis time Storage stability in cold (in days) Type of oxalate analysis Whether analysis possible without laboratory/by patient
Inamdar et al, 1989 Purified Banana peel oxalate oxidase Acrylamide membrane Entrapment 60 minutes ND Quantitative No
Madanpotra et al, 2004 Partially purified Barley oxalate oxidase Alkylamine glass beads affixed on a plastic strip Covalent 22 minutes 20 Quantitative No
Bharat et al, 2005 Partially purified Sorghum leaf oxalate oxidase Alkylamine glass beads affixed on a plastic strip Covalent 15 minutes 14 Quantitative No
Sharma et al, 2007 Partially purified Amaranthus oxalate oxidase Alkylamine glass beads affixed on a plastic strip Covalent 25 minutes 40 Quantitative No
Godara et al, 2008 Partially purified Amaranthus oxalate oxidase Arylamine glass beads affixed on a plastic strip Covalent 25 minutes 60 Quantitative No
Present Purified Sorghum leaf oxalate oxidase Cellulose Acetate strip (CA) Adsorption 45
seconds 90 Semi-quantitative
Yes

Brief description of the accompanying drawings:

Figure 1: Bio-strip of cellulose acetate containing oxalate oxidase enzyme, reaction buffer and O-dianisidine as the chromogen.

Figure 2: Reference chart depicting universal color codes.

Figure 3: Preferable embodiment of the reference chart to be supplied with kit.
Figure 4: Effect of oxalate concentration on color intensity of the bio-strip.

Detailed Description of the invention with reference to figures/examples:
The present invention is described with reference to the tables/ figures /examples etc. and specific embodiments and this description is not meant to be construed in a limiting sense. Various alternate embodiments of the invention will become apparent to persons skilled in the art upon reference to the description of the invention. It is therefore contemplated that such alternative embodiments form part of the present invention.
Accordingly, the present invention aims at providing a disposable bio-strip for rapid estimation of oxalate in urine that comprises of enzyme oxalate oxidase cross-linked with glutaraldehyde and a chromogen which are immobilized and adsorbed onto a membrane pad affixed on to a supporting strip.
The present invention also provides a convenient method for estimation of oxalate in urine, which involves the use of the said bio-strip and thus is rapid, less complex and can be executed by a person of ordinary skill at a person’s bed-side.

Plan of work for the present invention:
1. Extraction and purification of oxalate oxidase from sorghum leaves.
2. Preparation of disposable bio-strip for oxalate determination.
3. Standardization of optimal assay conditions for strip.
4. Analytical application of bio-strip for semi-quantitative determination of urinary oxalate.
5. Study of effects of various urinary substances on the performance of the bio-strip.

A rapid and convenient method of semi-quantitative analysis of urinary oxalate has been developed using a novel bio-strip through this invention. The method is based on the development of color due to following chemical reaction:-

+ O2 H2O2 + 2CO2

Oxalate oxidase
H2O2 + Chromogen Colour
(O-dianisidine) (Yellow color)

The color is formed due to interaction of H2O2 with chromogen, which is generated from oxalate by strip bound oxalate oxidase.
The method described in the present invention for determination of urinary oxalate, involves the use of the bio-strip and includes the steps of: (a) dipping and moving the disposable bio-strip in urine sample to be tested, (b) removing the bio-strip from the sample, (c) air-drying the bio-strip dipped in test sample, (d) observing the color change of the bio-strip and (d) comparing the color of bio-strip with a standard reference chart.
Each disposable test strip comprises of enzyme oxalate oxidase, cross-linked with glutaraldehyde, a reaction buffer and a chromogen; immobilized and adsorbed onto a membrane pad affixed on to a supporting strip. The color-change reagent comprises an oxalate oxidase enzyme and a hydrogen donor indicator or chromogen that changes color when oxidized.
The oxalate oxidase may be one of Sorghum grain oxalate oxidase, Sorghum leaf oxalate oxidase, Barley oxalate oxidase, Banana peel oxalate oxidase, Pseudomonas oxalate oxidase, Wheat germin/oxalate oxidase, strawberry fruit oxalate oxidase, beet stem oxalate oxidase, moss oxalate oxidase, Amaranthus leaf oxalate oxidase.
The reaction buffer can be selected from a group comprising of sodium succinate buffer, sodium citrate buffer and TRIS HCl buffer.
The membrane pad can be selected from a group comprising cellulose acetate (CA) pad, PVC, PVA, PEG, silica glass matrix, egg shell, pig intestine membrane, nylon tube.
The chromogen can be selected from a group comprising O-dianisidine, O-Dianisidine dihydrochloride.
The material of supporting strip can be selected from a group comprising of hard art paper, plastic strip. Hard art paper is a high-quality type of paper, usually having a smooth coating of china clay or similar substance on it which makes it resistant to water.
As a preferred embodiment, each disposable test bio-strip (Figure1) comprises of enzyme oxalate oxidase obtained from sorghum leaves, cross-linked with glutaraldehyde, sodium succinate reaction buffer and O-dianisidine as the chromogen; immobilized and adsorbed onto a cellulose acetate (CA) membrane, affixed with a non-reactive fixative such as “Araldite” on to a supporting strip made of hard art paper.
A disposable bio-strip for estimation of oxalate in urine, said bio-strip comprising of 20 µl to 100 µl of enzyme oxalate oxidase cross-linked with 10 µl to 50 µl glutaraldehyde, 20 µl to 100 µl of 0.4 M reaction buffer and 20 µl to 100 µl of chromogen; immobilized and adsorbed onto a membrane affixed on to the supporting strip.
The present invention also provides a process of preparation of the bio-strip for estimation of oxalate in urine.
The formation of bio-strips includes the following steps – (a) cutting the supporting strips of desired size, (b) cutting the membrane pads (preferably of cellulose acetate) of appropriate size, (c) applying a thin, uniform layer of the fixative on one end of the supporting strip and then affixing the membrane pad with the fixative on to the supporting strip, (d) crosslinking enzyme oxalate oxidase with glutaraldehyde and then immobilizing the same onto membrane pad by adsorption and for this purpose the membrane was saturated with the reaction buffer that contains glutaraldehyde and then the oxalate oxidase enzyme and the chromogen were adsorbed on to the membrane pad one by one and (e) drying the strip at 40oC for 3 hrs or at room temperature for a longer time.
The present invention also provides a kit comprising a disposable bio-strip and a reference chart , for estimation of oxalate in urine.
The present invention further provides a semi-quantitative method of estimation of oxalate by detecting the color change of the test bio-strip, represented in terms of universal color codes as described in description and figures.
O-dianisidine was chosen as the preferred chromogen as its use resulted in rapid chromogenic reactions and a stable bio-strip.
The reference color chart for estimation of oxalate concentration by bio-strip is as presented in Figure 2 of the present specification. The color chart depicts the colors in terms of corresponding universal color codes where the original brilliant greenish yellow color is depicted as universal color code 10 Y 9/2 and the changed color of the test strip after the reaction that corresponds to various concentrations of oxalate is depicted in various shades of yellow (vivid yellow to deep yellow) as universal color codes 5 Y 8/12, 5 Y 7/10 , 2.5 Y 7/10 , 5 Y 6/10 , 2.5 Y 6/10 and 5 Y 7/14 respectively. The normal permissible (20 mg/l) of oxalate in urine is depicted in terms of vivid yellow color (universal color code 5 Y 8/12 ) and any color in darker shade depicted by range of 5 Y 7/10 to 5 Y 7/14 as depicted in figure 2, denotes the presence of high amount of oxalate in urine. So it is easy to differentiate a normal urine sample from a hyperoxaluric urine sample, by just comparing the colors of the test strip after reaction with the sample with the reference color chart.
The reference chart if and when to be supplied with the kit could be modified for the ease of consumers and could preferably or optionally depict only the colors brilliant greenish yellow and various shades of yellow from vivid yellow to strong yellow to deep yellow and mention the corresponding oxalate concentrations (in normal and hyperoxaluric range) as presented in Figure 3. The chart to be supplied with the kit could either be made as a small strip of hard art paper resistant to water or as a plastic strip. This chart could optionally be affixed on the cover of kit to reduce the costs, in case the cover of kit is made of water resistant stuff. Thus even a common man would be able to perform the test, compare visually with the reference chart (Figure 3) and come to a conclusion as to whether the urine is normal or hyperoxaluric and what is the approximate concentration of oxalate in the sample.
In accordance with the foregoing and other objects, the present invention also provides a test kit comprising a plurality of individually-sealed test strips, each for detecting the presence and intensity of oxalate in urine. The corresponding kit is packaged with bio-strips and a reference color chart such as described above, to help the users determine the urine oxalate concentration at bed side.
The oxalate determining bio-strip of the present invention is fast and easy to use, producing reliable results in just 45 seconds. This test detects the presence of oxalate in urine and can provide an approximation of urine oxalate concentration from 20mg/1litre to 200mg/1litre. It can also detect oxalate in other fluids such as blood serum etc.
Storage stability:
The bio-strip showed no loss of its activity after its storage at 4ºC for three months. At room temperature, the bio-strip is stable for one week.

Application of bio-strip in differential semi-quantitative analysis of oxalate in various urine samples:
The bio-strip was able to differentiate between normal and hyperoxaluric urine samples on the basis of color developed within 45 seconds at room temperature, respectively. The chromogen showed strong/deep yellow color with hyperoxaluric samples compared to that vivid yellow color of normal sample. Thus the bio-strip was able to differentiate between normal and artificially made hyperoxularic urine samples.

Examples:
This invention is described in greater detail with reference to the following examples. These examples provide a better understanding of the methodology and product of the invention, but it should be noted that the present invention is not limited to the below mentioned examples only and that various other modifications and implementations are possible.

Example 1. Extraction and Purification of oxalate oxidase
a. Ten day old grain sorghum was raised in laboratory as described by Pundir & Nath, 1984.
b. Extraction of oxalate oxidase from 10 day old leaves was carried out as described by Satyapal and Pundir, 1993.
c. Assay of oxalate oxidase was done as described by Satypal & Pundir, 1993.
d. An oxalate oxidase was purified from leaves of 10-day old seedling plants of sorghum using ammonium sulphate fractiation (0-80%), Sephadex G-100 gel filtration and ion exchange chromatography on DEAE- Sephacel with a 97-fold purification and 12.7 % yield (Satypal & Pundir, 1993). The purified enzyme had a specific activity of 322 units/mg as can be seen in Table 2 which presents the stages of purification of oxalate oxidase from leaves of 10-days old grain sorghum.

Table 2: Purification of oxalate oxidase from leaves of 10-days old grain sorghum

Purification step
Total volume (ml) Activity (µmolH2O2/min/ml)
Protein (mg/ml)
Specific Activity (units/mg)
Purification fold
Yield (%)

Crude enzyme
800
14.00
4.20
3.30
1.0
100

Ammonium sulphate precipitation (0-80%)
35
68.88
3.80
18.12
5.49
21.5

Sephadex G-100
45
44.96
0.88
51.09
15.48
18.1

DEAE-Sephacel
20
71. 00
0.22
322.0
97.0
12.7
The starting material was 250 gm leaves (Fr wt)

Example 2. Preparation of disposable biostrip

a. Cutting of supporting strips:
Supporting strips of 13×1 cm size were cut from ‘hard art paper’ with scissors.
b. Cutting of cellulose acetate (CA) pads:
The pads of 1×0.7 cm were cut from cellulose acetate strips (14×0.7 cm.) with the help of scissors.
c. Affixation of CA pads onto supporting strip:
A thin layer of 0.2 mm thickness of a fixative namely ‘Araldite’ was applied uniformly on one end of the supporting strip up to a height of 0.4 cm. with the help of a brush and then the CA pad was affixed on this fixative layer.
d. Immobilization of reaction buffer, chromogen and oxalate oxidase on CA strip :
Sorghum oxalate oxidase was crosslinked with glutaraldehyde and then immobilized onto CA strip by adsorption. For this purpose, 100µl of sodium succinate reaction buffer, 20µl of 1% glutaraldehyde in reaction buffer, 50µl enzyme and 20µl O-dianisidine were placed on the CA pad after adsorption of each, one by one. Then the strip was dried at 40oC for 3 hrs.

Example 3. Testing of biostrip for oxalate oxidase activity:
0.1 ml of the aqueous oxalic acid (0.2mg/ml) was placed on the biostrip and waited till the color was noticed on strip. It was compared with its respective blank strip, which lacks the enzyme/substrate. The time required for development of color was also noted.

Example 4. Standardization of working of disposable biostrip :
Following kinetic properties of immobilized oxalate oxidase immobilized on CA pad were studied to standardize the optimal working conditions of bio-strip.
4.1. Ionic strength:
The activity of immobilized oxalate oxidase or the color intensity of bio-strip was observed at solutions of various ionic strength/ concentrations i.e. 0.1, 0.2, 0.3, 0.4, and 0.5 M reaction buffer. The intensity of the color increased as the ionic strength or concentration of reaction buffer was increased, the highest being at 0.4 M, after which no increase in color intensity was seen. Hence in the subsequent experiments, the ionic strength of the reaction buffer was 0.4 M.
4.2. Optimum pH:
The activity of immobilized oxalate oxidase or the color intensity of bio-strip was observed at pH 4.0, 4.5, 5.0, 5.5 and 6.0 using 0.4 M reaction buffer. The highest intensity of color was seen at pH 5.0. Hence in all the subsequent experiments, the pH of reaction mixture was at pH 5.0.
4.3. Effect of chromogen concentration:
Various amounts of the chromogen (20, 40, 60, 80 and 100 µl) were tested and the apt concentration of (O-dianisidine) required for the preparation of bio-strip, was found to be 20µl (2mg/ml). Hence in all the subsequent experiments, the volume of chromogen used was 20µl (2mg/ml).
4.4 Time of Incubation:
As soon as the drop of oxalate was put on the biostrip, it starts developing color. The biostrip showed highest intensity of pinkish brown color in 45 seconds. Hence in all the subsequent experiments, the color of strip was noted after 45 seconds.
4.5. Effect of oxalate concentration:
The effect of oxalate concentration on CA pad bound enzyme on the varying amount of oxalate ranging from 20mg/L to 200mg/L (20, 40, 80, 120, 160 and 200) was tested. The lowest intensity of biostrip color was obtained at 20mg/L, while the highest intensity of biostrip color was obtained at 200mg/L (Figure 4). Yellowish color was developed in presence of oxalate in urine when O-toluidine was used. The intensity of color of the test was compared with their respective blank and noted in terms of + marks. This rating by + signs was based on intensity of color as shown in Table 3 and used as standard for semi-quantitative determination of oxalate.

Example 5. Semi-quantitative analysis of urinary oxalate:
The biostrip showed pink color within 30 sec indicating the presence of oxalate in the urine. The color intensity of the test strip corresponded to respective oxalate concentrations (20mg/L – 200mg/L) which is represented by ‘+’ signs as depicted in Figure 2 and 4. Thus the bio-strip was able to differentiate between normal and hyperoxaluric urine samples. 20mg/L of oxalate is the limit for a normal sample while any concentration more than that i.e. 40mg/L to 200mg/L represents a hyperoxaluric sample.

Example 6. Comparison of urinary oxalate analysis by present method with
standard enzymic colourimetric method
The difference between the color shown by the present biostrip with normal and clinical sample shown by the present bio-strip was confirmed by the quantitative analysis of both normal and clinical sample by standard enzymic colorimetric method as the latter shown oxalate concentration at 30mg/L in normal and 58mg/L and 89mg/L in clinical samples.

Advantages of the present invention:
The bio-strip of the present invention can be used by a person at his/her bedside without the help of a laboratory and skilled person to differentiate a normal urine sample from a hyperoxaluric sample with convenience and in very short time. Moreover, the bio-strip showed no loss of its activity after its storage at 4ºC for three months.
References:
1. Godara S and Pundir C S (2008). Urinary & serum oxalate determination by oxalate oxidase immobilized onto affixed arylamine glass beads. Ind J Med Res 127: 370-376
2. Inamdar KV, Tarachand U and Raghavan K G (1986). Immune complex of banana oxalate oxidase: use in quantification of urinary oxalate. Anal Lett 22, 841-851.
3. Madanpotra S, Chaudhary R, Singh S & Pundir C S (2004) Preparation of reusable strip of barley oxalate oxidase for determination of urinary oxalate. Ind J Chem Technol 11, 495-499
4. Bharat Naveen & Pundir C S (2005). Determination of urinary oxalate with a reusable sorghum leaf oxalate oxidase strip. Asian J Phys 14(1&2) 103-109
5. Sharma N, Sharma M, Kumar V and Pundir C S (2007). Measurement of urine and plasma oxalate with reusable strip of amaranthus leaf oxalate oxidase. Ind J Pharma Sc 69 (5): 669-673
6. Pundir CS and Nath R (1984). Occurrence of an oxalate oxidase in sorghum leaves. Phytochem. 23: 1871-1974.
7. Satypal and C.S. Pundir (1993). Purification and properties of an oxalate oxidase from leaves of grain sorghum hybrid CSH-5. Biochim Biophys Acta., 1161, 1-5.

We claim
1. A disposable bio-strip for estimation of oxalate in urine, said bio-strip comprising of 20 µl to 100 µl of enzyme oxalate oxidase cross-linked with 10 µl to 50 µl glutaraldehyde, 20 µl to 100 µl of 0.4 M reaction buffer and 20 µl to 100 µl of chromogen; immobilized and adsorbed onto a membrane affixed on to the supporting strip.
2. A method of estimation of oxalate in urine with the bio-strip as claimed in claim 1, the said method comprising the steps of: (a) dipping and moving the disposable bio-strip in urine sample to be tested, (b) removing the bio-strip from the sample, (c) air-drying the bio-strip dipped in test sample, (d) observing the color change of the bio-strip and (d) comparing the color of bio-strip with a standard reference chart as herein described.
3. A bio-strip as claimed in claim 1, wherein the enzyme oxalate oxidase is the sorghum leaf oxalate oxidase.
4. A bio-strip as claimed in claim 1 or 3, wherein preferably 50 µl of sorghum oxalate acetate enzyme is used.
5. A bio-strip as claimed in claim 1, wherein preferably 20 µl of glutaraldehyde is used.
6. A bio-strip as claimed in claim 1, wherein the reaction buffer is selected from a group comprising of sodium succinate buffer, sodium citrate buffer and TRIS HCl buffer.
7. A bio-strip as claimed in claim 1 or 6, wherein the reaction buffer is preferably sodium succinate buffer.
8. A bio-strip as claimed in claim 1 or 7, wherein preferably 100 µl of 0.4 M sodium succinate buffer is used.
9. A bio-strip as claimed in claim 1, wherein preferably 20 µl of 1% glutaraldehyde is used.
10. A bio-strip as claimed in claim 1, wherein the chromogen is selected from a group comprising O-dianisidine, O-Dianisidine dihydrochloride.
11. A bio-strip as claimed in claim 1 or 10, wherein the chromogen is preferably O-dianisidine, wherein preferably 20 µl of O-dianisidine is used.
12. The bio-strip as claimed in claim 1, wherein the material of supporting strip is selected from a group comprising hard art paper, plastic strip.
13. The bio-strip as claimed in claim 12, wherein the supporting strip is preferably of hard art paper, as herein described.
14. The bio-strip as claimed in claim 13, wherein the supporting strip is preferably of 13 x 1 centimeter in size.
15. The bio-strip as claimed in claim 1, wherein the membrane is selected from a group comprising cellulose acetate (CA) pad, PVC, PVA, PEG, silica glass matrix, egg shell, pig intestine membrane, nylon tube.
16. The bio-strip as claimed in claim 1 or 15, wherein the membrane is preferably (CA) cellulose acetate pad.
17. The bio-strip as claimed in claim 16, wherein the CA pad is preferably of 1 x 0.7 centimeter in size.
18. The method as claimed in claim 2, wherein the estimation is semi¬-quantitative estimation.
19. The method as claimed in claim 2, wherein the semi-quantitative estimation is done by detecting the color change of the test bio-strip from vivid yellow to deep yellow, represented in terms of universal color codes as herein described.
20. A process of preparation of the bio-strip as claimed in claim 1, comprising the following steps:-
a. affixing the membrane onto the supporting strip,
b. saturating the affixed membrane with a reaction buffer,
c. treating membrane of step ‘b’ with glutaraldehyde,
d. immobilizing purified oxalate oxidase onto treated membrane of step “c”, by cross-linking the enzyme oxalate oxidase with glutaraldehyde,
e. treating the membrane of step ''d’ to chromogen to obtain the said bio-strip and
f. drying the bio-strip as obtained above.
21. The process of preparation of the bio-strip as claimed in claim 20, wherein the sorghum leaf oxalate oxidase is immobilized by cross linking with glutaraldehyde on cellulose acetate (CA) pad saturated with sodium succinate buffer and subjected to treatment with O-dianisidine.
22. The process of preparation of the bio-strip as claimed in claim 20, wherein preferably 100 µl of 0.4 M sodium succinate is used as the reaction buffer.
23. The process of preparation of the bio-strip as claimed in claim 20 or 21, wherein preferably 20 µl of 1% of glutaraldehyde is used for immobilization.
24. The process of preparation of the bio-strip as claimed in claim 20 or 21, wherein preferably 50 µl of sorghum oxalate acetate enzyme is used.
25. The process of preparation of the bio-strip as claimed in claim 20 or 21, wherein preferably 20 µl of O-dianisidine is used as the chromogen.
26. A kit comprising a disposable bio-strip and a reference chart as claimed in claim 1 or 2, for estimation of oxalate in urine.