DESC:FIELD OF INVENTION
The present invention relates to the field of sensors. More specifically, the invention relates to the quantitative and qualitative analysis of an analyte by a peroxidase chromogenic substrate reagent composition by colorimetry and fluorometry. The invention also relates to detection of pathogenic microorganisms in samples using the peroxidase chromogenic substrate reagent composition.

BACKGROUND OF INVENTION
Chromogenic assays yield a colored reaction product that absorbs light in the visible range. A consistent problem in chromogenic assays relying on peroxidic activity has been the tendency of hydroperoxides and chromogenic electron donors to spontaneously react before the addition of the catalytic enzyme. Because of this spontaneous reaction, the general practice is to mix the hydroperoxide and the chromogenic electron donor immediately before use. Broyles et al., Anal. Biochem., 94:211-219 (1979), reported that benzidine-H2 O2 solutions should be used within two hours of preparation, turning brown if stored overnight, even at 4° C. Liem et al., Anal. Biochem., 98:388-393 (1979), point out that TMB has good staining properties, but also that its solubility is low and that TMB is subject to oxidative decomposition. Brand et al., Biotechniques, 8:58-60 (1990) compared the stability of DAB and TMB using immunoblots and concluded that the TMB is functional for eight weeks with visible precipitates after 19 hours at room temperature and DAB is functional for four weeks with precipitates forming after 18 hours at room temperature. These precipitates are an indication of polymerization due to spontaneous oxidation. Thus, preparing a commercially viable product ready to use monoreagent (combining H2O2 along with chromogenic substrate form) with high sensitivity, long term storage stability is said to be a real technical challenge.
Different references have disclosed various technologies to stabilize substrate-chromogens for peroxidase, mainly by the use of: (1) cyclodextrins (gamma-cyclodextrin JP 06,165,696 and Beta-cyclodextrin WO 8,605,207); (2) surfactants (cationic JP 05,227,995 and non-ionic JP 62,294,099); (3) antibiotics (Bacitracin U.S. Pat. No. 5,206,150 and Penicillin U.S. Pat. No. 4,891,314); (4) polymers (U.S. Pat. No. 5,013,646 and U.S. Pat. No.4,615,972); and (5) chelators (EP 0271,713 and WO 9,002,339). It is evident that there remains a definite need for a versatile, environmentally safe and inexpensive technology to stabilize ready-to-use substrate-chromogen assays
Coliform bacteria originate as organisms in soil, vegetation and intestinal tract of warm blooded animals. E.coli (short for Escherichia coli),for example, are gram-negative anaerobic bacteria, found naturally in the lower intestine of warm blooded organisms and are excreted in their faeces. E. coli are not usually pathogenic, and have beneficial functions such as production of vitamin K and Vitamin B. They also prevent harmful bacteria from establishing themselves in the intestine. However, if ingested, the toxin that this bacteria produces can damage red blood cells, kidney and other organs. Current standard methods for detection of coliforms and E.colirely on growth of visible colonies which take a minimum of 18 hours. Hence there is a constant need for a rapid method of detection of E.coli which provides timely results of the bacterial concentration. The desired testing method should meet the following criteria: the method should be simple, sensitive, rapid, quantitative and easy to use and inexpensive. Also, the methods should abide by the guidelines provided by World health organization (WHO) for monitoring bacterial contamination in a public supply of water, or in food. The Current method of detection include polymerase chain reaction (PCR)-based method, enzyme-linked immunosorbent assay (ELISA) andimmunomagnetic separation method, microarray technology, fluorescent in situ hybridization and enzymatic methods. For example, well known method used in the industry to detect the presence of E.Coliis to add 4-methylumbelliferyl-D-glucuronide (MUG) to a growth medium to detect the bacterial enzyme glucuronidase (GUS), as GUS interacts with MUG to create a by product, 4-methylumbelliferone (4-MU), that emits a fluorescence and can be quantified and related to the number of cells. These methods are highly specific, but expensive and can only be performed in a laboratory and require well-trained staff. PCR-based assay and microarray technology run a high risk of inaccuracy owing to inhibition by components of the sample matrix, and ELISA requires extra labelled antibodies, while immunomagnetic separation method often requires a combination of other methods
Use of membrane filter technology to detect total and faecal coliforms necessitates concurrent or serial analyses using two different types of media incubated at two different temperatures. The combined procedures (total coliform test and either faecal coliform test or E. coli method) take about 28 to 48 hours. Several tests use liquid media in a Most Probable Number (MPN) test format that permits the statistical estimation, but not enumeration, of the target organisms. However, the MPN procedure tends to overestimate the numbers of organisms present which may result in apparent increased compliance violations and rejection of acceptable drinking water. Also, in many instances, the media are useful only for a limited range of samples. For example, it may be necessary to have a different medium for urine specimens than that for water and a third medium may be needed to test food.
US Patent number 4923804 discloses use of ß-glucuronides to test for E. coli and indoxyl-ß-D-glucuronide is a preferred agent. However, the medium used can only detect E. coli and does not provide for detection of total coliforms. Therefore, a second medium would be required to identify total coliforms. Also, the base medium contains glycerol as a nutrient and lacks an inducer and an inhibitor of gram negative bacteria that can give a false positive response. Glycerol in media also causes spreading of colonies making enumeration and discrimination difficult. The medium is incubated at an elevated temperature (44.5° C.) which could be detrimental to the recovery of injured microorganisms.
U.S. Pat. No. 4,591,554 discloses use of fluorescence analysis using umbelliferone derivatives, including phosphates and galactosides to test for E.coli. The media may also promote the growth of many other types of organisms such as gram positive bacteria, yeasts and fungi that may also be present in the samples due to the use of lactose as an inducer and absence of an inhibitor. An agar medium developed by Petzel and Hartman (Appl. Environ. Microbiology, Vol 24: 925–933) uses a selective medium for total coliform identification combined with detection of E. coli using 4-methylumbelliferyl-ß-D glucuronide. Drawback relating to this medium includes the inability to use standard diluents, a high false positive rate when high levels of Flavobacterium species or oxidase positive organisms are present in the water samples, and difficulty in distinguishing the natural fluorescence of Pseudomonas from the fluorescence produced during substrate breakdown. Furthermore, this medium cannot be used for precise enumeration of the target organisms because of the large number of other Gram negative bacteria that grow on it.
Another reference teaches a method to detect both total coliforms and E. coli. Total coliform colonies are identified by the production of blue color from the ß-galactosidase cleavage of the substrate X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), while E. coli colonies are detected by the fluorescence of 4-methylumbelliferone, produced by the cleavage of 4-methylumbelliferyl-ß-D-glucuronide by ß-glucuronidase (Manafi and Kneifel, Zbl. Hyg. 189: 225–234). The reference teaches the use of broth or agar containing 4-methylumbelliferyl-ß-D-glucuronide (MUG) and a galactoside with a non-fluorescing chromophore (X-Gal). It also teaches a 4-methylumbelliferyl-ß-D-galactoside (MUGal) with a glucuronide attached to a non-fluorescing chromophore, 4-nitrophenyl-ß-D-glucuronide (PNPG), in an agar medium containing bile salts to inhibit the growth of organisms that are not coliform bacteria. However, the results obtained are not accurate.
Commercially, fluorescence optical analysis based techniques and traditional plating techniques are currently being used to detect bacteria in the field. Although the techniques are rapid, they are too expensive and hence not feasible for bulk requirements.
Various kinds of biosensors have also been developed in recent times to detect the E. coli O157:H7, including microarray biosensor, Immunosensor, surface plasma on resonance (SPR) biosensor, waveguide biosensor and so on. But these test results cannot be easily seen by naked eyes due to professional instrument and operation limitations.
Therefore, the above mentioned techniques are either too expensive or time consuming and the accuracy of the detection methods may also vary. There is an ardent requirement for material and method for detection of pathogens, and other analytes. The present inventionaims to provide a low cost, highly sensitive, rapid detection based on colorimetric or fluorimetic based biosensor materialand method.
Summary of invention
Accordingly the present invention provides a peroxidase chromogenic substrate reagent composition comprising Citric acid ranging from 5mg to 380mg, buffer ranging from 1mg to 600mg, Ethylenediaminetetraacetic acid salt ranging from 1mg to 100mg, Polyvinylpyrrolidone ranging from 1mg to 400mg, Mannitol/Sorbitolranging from 1mg to 150mg, Polyethylene glycol-4K ranging from 1mg to 250mg, 2-hydroxypropyl-ß-cyclodextrin ranging from 1mg to 200mg, Sodium Perborate ranging from 1mg to 30mg, chromogenic substrate compound ranging from 1mg to 50mg, optionally D-Ribose/Xylose ranging from 1mg to 10mg, Dextran sulphate ranging from 1mg to 90mg, and dye ranging from 0.0001 mg - 0.0010mg; for analysing an analyte; a method of detection of an analyte, said method comprising acts of (a) filtering and/or optionally enriching the analyte with media and incubating, (b) filtering the media again by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer, and treating the filtered membrane with composition of present invention and maintaining for 30 min for the analysis by change in colour; a method of pathogenic detection, said method comprising acts of, a)enriching the specimen samples and incubating at a temperature of ranging from 30-40°C for a time about 3-10 hours, (b) filtering the incubated specimen by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer and (c) treating the filtered membrane with composition of present invention and maintaining for 30 min for the analysis based on colour change; a method for E. Coli detection, said method comprising acts of, (a) enriching the specimen with a media and incubating at a temperature ranging from 35°C-40°C for a duration ranging from 3-10 hours, (b) filtering the incubated media by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer, and (c) treating the filtered membrane with composition of present invention and incubating for 30 min for analysis based on colour change.
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: Malaria pf-HRP-2 ELISA at the end of incubation, (a) Malaria positive control :1- Composition of present invention , 2- TMB- Ultra Blue, 3-TMB prestained-Xt, 4-Kit-TMB; (b) Malaria negative control: 1- Composition of present invention, 2- TMB- Ultra Blue, 3-TMB prestained-Xt, 4-Kit-TMB.
Figure 2: Malaria pf-HRP-2 ELISA: Assay after stopping the reaction, (a) Malaria positive control :1- Composition of present invention, 2- TMB- Ultra Blue, 3-TMB prestained-Xt, 4-Kit-TMB; (b) Malaria negative control: 1- Composition of present invention, 2- TMB- Ultra Blue, 3-TMB prestained-Xt, 4-Kit-TMB.
Figure 3: HRP substrate Prestained composition & its method for E.coli detection (a), vacuum filtration, (b) spraying the composition on the membrane.
Figure 4:Fluorescence HRP assay using pre-stained reagent composition
Figure 5: Colorimetric HRP assay - Assay O.D at 450nm
Figure 6: Fluorescence based HRP assay using prestained reagent
Figure 7: Real time E.Coli detection using Prestained Chromogenic reagent
Figure 8: Specificity of Prestained composition for E.Coli detection
Figure 9: HRP assay using DOPR before &after stopping the reaction by adding stop reagent with HRP concentration in ng/mL.
Figure 10:HRP assay using ABTS monoreagent composition
Figure 11: HRP assay using o-PD monoreagent composition
Figure 12 : HRP assay using DAB monoreagent composition
Figure 13: HRP assay using 4-CNmonoreagent composition
Figure 14: HRP assay using AEC monoreagent composition
Figure-15: Estrogen receptor expression analysis in human breast biopsy samples and staining using TMB Monoreagent& its Observation at High Power using phase contrast microscope.

DESCRIPTION OF INVENTION
The embodiments herein and the various features of the proposed system are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. The illustrations used herein are intended to merely facilitate an understanding of ways in which the embodiments herein may be prescribed and further to enable those skilled in the art to practice the embodiment herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
Abbreviations
3,3',5,5'-tetramethylbenzidine (TMB), 3,3',5,5'-tetramethylbenzidine sulphate (TMB-S), 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB-HCl), 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid diammonium salt (ABTS) , 3,3'-DiAminoBenzide tetrahydrochloride (DAB), 3-Amino-9-ethylcarbazole (AEC), 4- Chloronapthol (4-CN), o-Phenylenediamine (o-PD), IHC-Immunohistochemistry
The present invention is in relation to a peroxidase chromogenic substrate reagent composition comprising Citric acid ranging from 5mg to 380mg, buffer ranging from 1mg to 600mg, Ethylenediaminetetraacetic acid salt ranging from 1mg to 100mg, Polyvinylpyrrolidone ranging from 1mg to 400mg, Mannitol/Sorbitolranging from 1mg to 150mg, Polyethylene glycol-4K ranging from 1mg to 250mg, 2-hydroxypropyl-ß-cyclodextrin ranging from 1mg to 200mg, Sodium Perborate ranging from 1mg to 30mg, chromogenic substrate compound ranging from 1mg to 50mg, optionally D-Ribose/Xylose ranging from 1mg to 10mg, Dextran sulphate ranging from 1mg to 90mg, and dye ranging from 0.0001 mg - 0.0010mg; for analysing an analyte.
In an embodiment of the present invention, the analyte is selected from a group comprising pathogens, carcinogenic cells, carcinogens, toxins and the like.
In an embodiment of the present invention the analysis is by colorimetric and/or fluorimetric analysis.
In an embodiment of the present invention, the analysis is by quantitative and qualitative analysis.
In an embodiment of the present invention, the method of analysis is selected from a group comprising enzyme-linked Immunosorbent assay, Incubation, Immunohistochemistry, Dot-ELISA, western blotting and the like.
In an embodiment of the present invention the chromogenic compound is selected from a group; 3,3',5,5'-tetramethylbenzidine, 3,3',5,5'-tetramethylbenzidine sulphate, 3,3',5,5'-tetramethylbenzidine dihydrochloride, 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid diammonium salt, o-phenylenediamine, 3,3'-DiAminoBenzide tetrahydrochloride, 3-Amino-9-ethylcarbazole,4- Chloronapthol, and the like.
In an embodiment of the present invention the peroxidase is selected from a group comprising Horseradish peroxidase (HRP), E.Coli/ pathogen detector in water and the like.
In an embodiment of the present invention the dye is selected from a group comprisingPhloxine B, sunset yellow, sunset orange, methyl orange, allura red and the like.
In an embodiment of the present invention the buffer is selected from a group of Trisodium citrate, imidazole, citrate-phosphate and the like.
In an embodiment of the present invention the composition is stable for 12 – 18 months at temperature 2 – 8 ° C; and 3- 6 months at 25-30 ° C.
The present invention is also in relation to a method of detection of an analyte, said method comprising acts of
(a) filtering and/or optionally enriching the analyte with media and incubating;
(b) filtering the media again by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer; and
(c) treating the filtered membrane with composition of claim 1 and maintaining for 30 min for the analysis by change in colour.
In an embodiment of the present invention, the analyte detection is at pH selected from a range of pH 4 – pH 6.
In an embodiment of the present invention, the colour change of the analyte is observed by light and electron microscopy, spectrophotometry, fluorometry, human eye and the like.
In an embodiment of the present invention, the analyte is selected from a group comprising pathogens, carcinogenic cells, carcinogens, toxins and the like
The present invention is also in relation to a method of pathogenic detection, said method comprising acts of;
(a) enriching the specimen samples and incubating at a temperature of ranging from 30-40°C for a time about 3-10 hours;
(b) filtering the incubated specimen by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer; and
(c) treating the filtered membrane with composition of claim 1 andmaintaining for 30 min for the analysis based on colour change.
The present invention is also in relation to a method for E. Coli detection, said method comprising acts of
a) enriching the specimen with a media and incubating at a temperature ranging from 35°C-40°C for a duration ranging from 3-10 hours;
b) filtering the incubated media by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer; and
c) treating the filtered membrane with composition of claim 1 and incubating for 30 min for analysis based on colour change.
In an embodiment of the present invention, the colony forming unit (CFU) is ranging from 1- 107 CFU and the time ranging from 10 hr to 0.15 hr respectively.
In an embodiment of the present invention the detection is at pH 4.0-4.5.
In an embodiment of the present invention composition for e-coli detection is Citric acid ranging from 5mg to 380mg, Trisodium citrate ranging from 1mg to 600mg, Ethylenediaminetetraacetic acid salt ranging from 1mg to 100mg, Polyvinylpyrrolidone ranging from 1mg to 400mg, Mannitol/Sorbitol 1mg to 150mg, Polyethylene glycol-4K ranging from 1mg to 250mg, 2-hydroxypropyl-ß-cyclodextrin ranging from 1mg to 200mg, Sodium Perborate ranging from 1mg to 30mg,Tartrazineranging from 70mg to 90mg, 3,3',5,5'-tetramethylbenzidine sulphate ranging from 1mg to 50mg,milliQ water and optionally D-Ribose/Xylose ranging from 1mg to 10mg, Dextran sulphate ranging from1mg to 90mg.
In an embodiment of the present invention, the peroxidise is endogeneous membrane peroxidise of ecoli.
The invention relates to the quantitative and qualitative analysis of an analyte by a peroxidase based analytical composition(also referred to as prestained composition) on the basis of colorimetry and fluorometry. The present invention involves peroxidase catalyzed composition for the analysis.
A wide variety of chromogenic peroxidase substrate can be suitably adopted based on in the disclosed composition for the analysis of specific analyte selected from a group comprising3,3',5,5'-tetramethylbenzidine (TMB), 3,3',5,5'-tetramethylbenzidine sulphate, 3,3',5,5'-tetramethylbenzidine dihydrochloride, 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid diammonium salt (ABTS) , 3,3'-DiAminoBenzide tetrahydrochloride (DAB), 3-Amino-9-ethylcarbazole (AEC), 4- Chloronapthol (4-CN), o-Phenylenediamine (o-PD)
The analyte can be selected from a group comprising bioanalytes, pathogens, biomarkers, , heavy metals and the like.
Composition of pre-stained peroxidase chromogenic substrate reagent

A peroxidase catalysed compositionis prepared by mixing suitable reagents in milliQ water. A typical composition in 50ml of milliQ water is given in Table 1. The pH is to be maintained in specific with respect to components of the composition used. The pH for this peroxidase catalysed composition can be selected from a range of pH 4 to pH 6.

Table 1: Provides a broad range of the components taken for the preparation of the
Pre-stained chromogenic substrate reagent in 50 mLmilliQ water.

Sl No Composition Quantity
1 Citric acid 5-380mg
2 Tri-Nacitrate 1-600mg
3 EDTA Na/K salt 1-100mg
4 PVP 1-400mg
5 Mannitol/Sorbitol 1-150mg
6 Chromogenic substrate 1-50mg
7 D-Ribose/Xylose (Optionally) 1-10mg
8 PEG-4K 1-250mg
9 HPßCD 1-200mg
11. SodiumPerborate(monohydrate) 1-30mg
12 Dye (Optionally) 0.1-15mg
13 Dextran sulphate (Optionally ) 1-90mg

Table 2: provides a concentration range of the components taken for the preparation of theperoxidase catalysed composition

Sl. No. Composition Concentration% w/v
1 TMB salt 0.03 to 0.1
2 Cyclodextrin derivative 0.01 to 0.5
3 Stabilizing agent(s) 0.01 to 1.0
4 Solubilising agent(s) 0.01 to 1.0
5 Peroxide 0.01 to 0.5
6 Precipitating agent 0.05 to 0.25
7 Inert soluble colouring agent 0.001 to 0.01

Peroxidase based reagent composition: storage stability & its performance
The compositions are prepared using reagents as shown in Table 1 and 2. The reagent is aliquoted in different batches of around 200 ml volume and the batches are stored at 20-30 0C and then periodically tested for its performance using analyte analysis. The stability data suggests that, this pre-stained chromogenic substrate reagent can remain stable in a brown bottle and it can perform effectively for maximum period of 6 months.

Examples:
1. Peroxidase TMB monoreagentcomposition for ELISA and its method
TMB-Di HClis mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 3. HRP peroxide isused for this composition. To that dissolved TMB solution, solubilizing and stabilizing agents such as PVP, PEG, Mannitol, and EDTA are added along with sodium perborate in a proportion given in Table 3.Thesubstratereagent composition is prepared by mixing chromogenic substance and other reagents in milliQ water. A typical composition in 75 ml of milliQ water is given in Table3. The pH is maintained at 4.5 to 4.9.
Table 3: Peroxidase composition for ELISA in 75 mL of MilliQ water
S.No No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.6
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 TMB-diHCl 0.05
7 PEG-4K 0.25
8 HPBCD 0.2
9 Na perborate(Tetrahydrate) 0.03
10 Acetonitril 20ml

The above composition has shown Batch-to-batch consistency in terms of its assay performance and also its property like blank O.D at 650nm < 0.075; long term storage stability etc. This composition is having a shelf life of minimum 12 months at 2-8°C storage and at least 3 months at 20-30°C storage condition. The composition & its process have the feasibility to scale up for Batch production.
Thecomposition is also modified/ spiked with suitable inert water solubledye in order to make it a colored/Prestainedcomposition, which is more suitable for ELISA techniques. The choice of the dye is preferably water based, completely inert, with absorbance not interfering in the measuring range of the analyte either at 450nm.The preferred dye is Phloxine B. The addition of Phloxine B to said composition makes it pink color.Thecompositionis prepared by mixing chromogenic substance and other reagents in milliQ water. A typical composition in 75ml of milliQ water is given in Table4.

Table 4: Peroxidase TMB monoreagentcomposition for ELISA with Phloxine B in 75 mL of MilliQ water

S. No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.6
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 TMB-diHCl 0.05
7 PEG-4K 0.25
8 HPBCD 0.2
9 Na perborate(Tetrahydrate) 0.03
10 Acetonitril 20mL
11 Phloxine B 0.0005

To assess the performance of the above aforesaid compositions,Table 3 and 4were tested in an actual ELISA test environment.Then the performances are compared among the assays performed using reagents available in the market with composition disclosed in the present invention. The tests are revealed that the performance of TMB monoreagent composition of present invention is on par or superior to the marketed kit reagent. Table5 and 6 show the results obtained from marketed TMB reagent (Malaria Elisa kit) and peroxidise catalysed composition of present invention as in Figure 1 and figure 2. Quantitative measurements are also evaluated. The optical density is superior for the TMB monoreagent developed according to present invention.

Table 5: Malaria pf-HRP-2 ELISA: Assay O.D at650nm
Label : Label 1
Mode : absorbance, wavelength: 650nm,
Temperature: 22.2° C
Sl.no Malaria Positive Control Malaria Negative Control
1 IISc TMB Blue Xt 1.5967 1.5576 0.0599 0.0602
2 TMB- Ultra Blue
(Present composition) 3.2771 3.2984 0.0686 0.061
3 TMB pre-stained-XT
(Present composition) 3.2298 3.1687 0.0845 0.0843
4 Kit-TMB 2.9887 2.9946 0.0576 0.0517
Table 6: Malaria pf-HRP-2 ELISA: Assay O.D at 450nm
Label : Label 1
Mode : absorbance, wavelength: 450nm,
Temperature: 22.2° C
Sl.no Malaria Positive Control Malaria Negative Control
1 IISc TMB Blue Xt Over Over 0.0841 0.0859
2 TMB- Ultra Blue
(Present composition) Over 3.6436 0.0924 0.0862
3 TMB pre-stained-XT
(Present composition) Over Over 0.1429 0.1431
4 Kit-TMB Over 3.6811 0.0661 0.0578

2. Prestained peroxidase reagent composition and method for E.Coli detection in water
The composition is prepared by converting tetramethylbenzidine to its sulfated salt (TMB-S). TMB-S is then mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 7. To the TMB solution thus obtained, solublizing and stabilizing agents such as PVP, PEG, Mannitol, Ribose and EDTA are added along with sodium perborate in a proportion given in Table 7 to obtain the composition in milliQ water. HRP peroxide is used for this composition. A typical composition in 100% of milliQ water is given in Table7. The pH is maintained at 4.0 to 4.5.
Table-7: HRP reagent composition in 100% MilliQ water.
S. No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.6
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 TMB-S 0.05
7 PEG-4K 0.25
8 HPßCD 0.3
9 Sodium perperborate
(Tetrahydrate) 0.03
10 Dextran Sulphate 0.1
11 Tartrazine 0.0008

In this method, initially 100ml water sample (with E.Coli 1cfu/ml) is collected from the source (pound,river,tank) and filtered using a funnel fitted with a gradient filter paper of different pore size ( 12µm & 5 µm ) placed over 100ml measuring cylinder . Water is filtered and collected in a 100ml measuring cylinder by gravitational flow and this process helps in removing completely the slurry and other interfering agents in the sample by the sieving effect of the filter paper.
This filtered 100ml water in a measuring cylinder is further transferred to a clean vial containing specific enrichment media for coliform bacterial growth. Four vials of 100ml water samples are prepared. The 4 x100ml water sample content is mixed and incubated inside a portable incubator maintained at 37oC for different durations namely 3hrs, 6hrs, 9hrs and 10hrs respectively. At the end respective incubation period, the vial containing 100ml water and media are filtered using a sterile filtration unit fitted with 0.45micron pore size membrane, by applying vacuum using hand held pump the content is filtered and subsequently membrane is washed with 10ml saline and 10ml 10mM acetate buffer pH 4.0. The filtered membrane is now gently removed using a sterile forceps and placed over a sterile plastic cover surface and then added 1ml of composition over the membrane and maintained for 30min. The green color appearing on the membrane after the addition and incubationwith the reagent indicates the presence of E.Coli in the sample (Figure 3 and Figure 4). In case of blank, the procedure followed will be similar to that of test and addition of prestained chromogenic substrate reagent and it gives only yellow color on the surface of membrane mimicking the composition reagent color. The green color seen on the membrane is due to endogenous membrane peroxidase activity of E.Coli with this reagent composition. Interestingly, this reagent is highly specific reacts only with live E.Coli cells to give colour change ( Figure- 8) and also with only E.Coli . Other gram positive/ negative bacteria’s did not react with this reagent, suggesting that this method is highly specific and moreover by using specific enrichment media during incubation period allowes only e.coli cells to grow thereby prevents false positive result using this reagent.

Table 8: Colorimetric HRP assay measurement at 450nm
Label : Label 1
Mode: Absorbance, wavelength: 450nm,
Temperature:22.1 °C
Sl.no HRP ng/mL DOPR DOPR
1 75 3.5090 3.8989
2 37.5 3.2205 3.3327
3 18.7 3.3076 3.2216
4 9.3 1.8988 1.8113
5 4.6 0.9057 0.9013
6 2.3 0.4912 0.4783
7 1.1 0.2795 0.3048
8 Blank 0.1912 0.1773

The present method detects E.Coli in 100ml water by real time method depending upon the titre of E.Coli present in the sample as shown in the (table-7). For E.g. If 1 cfu to 10cfu E.Coli present in 100ml water then it takes 10hrs for its detection by this method. This method has high specificity and detection only E.Coli as given in figure 5.
Table-9: Real time detection of E.Coli in water using peroxidase reagent method
E.Coli level in 100mL water Real time detection
1CFU to 10 CFU 10 hours
>10 CFU 9 hours
>1 x 103 CFU 6 hours
>1 x 105 CFU 3 hours
>1 x 107 CFU 10- 15 min
The prepared reagent is robust and can be stored at 25-30 °C (room temperature) for at least 6 months. This reagent also possesses fluorescence property as given in Figure 6, excitation max 280nm and emission max400nm.
3. ABTS monoreagent composition & its application in peroxidase based assays
ABTS is mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 10. To that dissolved ABTS solution, solubilizing and stabilizing agents such as PVP, PEG, Mannitol, and EDTA are added along with sodium perborate in a proportion given in Table 10.Thecomposition is prepared by mixing chromogenic substance and other reagents in milliQ water. A typical composition in 80ml of milliQ water is given in Table10. HRP peroxide is used for this composition. The pH is maintained at 4.5 to 5.0. HRP assay result using ABTS monoreagent composition is given in figure 10.
Table-10:- ABTS based composition in 80 mL of MilliQ water
S. No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.6
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 ABTS 0.05
7 PEG-4K 0.25
8 HPBCD 0.2
9 Na perborate (Tetrahydrate) 0.03
10 Acetonitrile 20mL

4. O-PD monoreagent composition and its application in peroxidase based assay
O-PD(o-Phenylenediamine) is mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 11. To that dissolved O-PD solution, solubilizing and stabilizing agents such as PVP, PEG, Mannitol, and EDTA are added along with sodium perborate in a proportion given in Table 11. HRP peroxide is used for this composition.Compositionis prepared by mixing chromogenic substance and other reagents in milliQ water. A typical composition in 80ml of milliQ water is given in Table 11. The pH is maintained at 4.5 to 5.0.
Table-11:- O-PD based composition in 80 mL of MilliQ water
S. No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.6
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 O-PD 0.05
7 PEG-4K 0.25
8 HPBCD 0.2
9 Na perborate (Tetrahydrate)

0.03
10 Acetonitrile 20ml

This O-PD monoreagent solution is modified/ spiked with suitable dye in order to make it a colored/Prestained TMB monoreagent, which is more suitable for ELISA techniques. The choice of the dye is preferably water based, completely inert, with absorbance not interfering in the measuring range of the analyte either at 450nm. HRP assay result using o-PD monoreagentcomposition is given in figure 11.

5. DOPR monoreagent composition and its application in peroxidase based assay
TMB is mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 13. To that dissolved TMB solution, solubilizing and stabilizing agents such as PVP, PEG, Mannitol, and EDTA are added along with sodium perborate in a proportion given in Table 13. HRP peroxide is used for this composition. The pH is maintained at 4.5 to 5.0.
This composition is called also referred to as dual optical peroxidase reagent (DOPR), as it gives visual Color change (Green) (figure 9)or change in fluorescence property by HRP in either kinetic assay mode or end point assay mode. Thepre-stained chromogenic substrate reagent is prepared by mixing chromogenic substance and other reagents in milliQ water. A typical composition in 62% of milliQ water is given in Table 13.
Table 13: DOPR composition in MilliQ water.
S. No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.60
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 TMB-diHCl 0.05
7 PEG-4K 0.25
8 HPBCD 0.2
9 Na perborate (Tetrahydrate) 0.03
10 Acetonitrile 33
11 Fluorescent dye (Tartrazine/Methyl orange /Fluorescein 0.0006

DOPR -Colorimetric HRP assay
HRP of known quantity weighted and dissolved in 0.1M phosphate buffer pH 6.5. Using this stock subsequent other working concentration of HRP are prepared using the above buffer. Initially, 100ul of the HRP ng/ml (75, 37.5, 18.75, 9.4, 4.7, 2.3, 1.2, 0.6) are taken in 96 wells (duplicates) and then followed by 50ul of DOPR reagent to these wells and incubated for 30min at temperature ranging from 20°C to 30°C. At the end of incubation time, the reaction is stopped by adding 0.25M H2SO4 (100ul) to the wells and O.D measured at 450nm.
DOPR- Fluorescence HRP assay
In case of Fluorescence based HRP assay, different HRP concentrations (as above) 100ul are taken in 96 well special plate in duplicates and its fluorescence (RFU) are measured using Tecan plate reader by Exitation-280nm and Emission-390nm, to get the initial value at 0 times. Subsequently, added 50ul DOPR reagent to the wells and mixed gently and then measured in a same manner every 5 minutes interval for 15 min duration.
This reagent is cost effective, robust, room temperature stable for at least 3-6 months suitable for any peroxidase assays including pseudoperoxidase like (Hb, HbA1c and the like).
6. AECmonoreagentcomposition & its application in peroxidase based assay
AEC(3-Amino-9-ethylcarbazole) is mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 14. To that dissolved AEC solution, solubilizing and stabilizing agents such as PVP, PEG, Mannitol, and EDTA are added along with sodium perborate in a proportion given in Table 14.HRP peroxide is used for this composition. A typical composition in 80ml of milliQwater is given in Table 14.The pH is maintained at 4.5 to 5.0.HRP assay result using AEC monoreagentcomposition is given Figure 14.
Table-14 : AEC based composition in 80 mL MilliQ water
S. No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.6
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 AEC 0.05
7 PEG-4K 0.25
8 HPBCD 0.2
9 Na perborate (Tetrahydrate) 0.03
10 Acetonitrile 20mL

7. 4-CN monoreagentcomposition & its application in peroxidase based assay
4-CN is mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 15. To that dissolved 4-CN solution, solubilizing and stabilizing agents such as PVP, PEG, Mannitol, and EDTA are added along with sodium perborate in a proportion given in Table 15.HRP peroxide is used for this composition.A typical composition in 80ml of milliQ water is given in Table 15.The pH is maintained at 4.5 to 5.0. HRP assay result using 4-CN monoreagent composition is given in figure 13.
Table-15: 4-Chloro-1-naphthol monoreagent composition in 80mL MilliQ water
S. No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.6
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 4-CN 0.05
7 PEG-4K 0.25
8 HPBCD 0.2
9 Na perborate (Tetrahydrate)
0.03
10 Acetonitrile 20mL

8. DAB monoreagentcomposition & its application in peroxidase based assay
DAB (3,3'-Diaminobenzidine) is mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 16. To that dissolved DAB solution, solubilising and stabilizing agents such as PVP, PEG, Mannitol, and EDTA are added along with sodium perborate in a proportion given in Table 16.HRP peroxide is used for this composition. A typical composition in 80ml of milliQ water is given in Table 16.The pH is maintained at 4.5 to 5.0. HRP assay result using DAB monoreagent composition is given in figure 12.
Table-16:- DAB monoreagent composition in 80 mL MilliQ water
S. No. Composition %
1 Citric acid 0.38
2 Tri-Na citrate 0.6
3 EDTA Na/K salt 0.1
4 PVP 0.5
5 Mannitol/Sorbitol 0.25
6 DAB (3,3'-Diaminobenzidine) 0.05
7 PEG-4K 0.25
8 HPBCD 0.2
9 Na perborate (Tetrahydrate) 0.03
10 Acetonitrile 20mL

9. A detailed experiment on E.Coli detection based on its membrane peroxidise activity using pre-stained chromogenic substrate reagent and various methods to be performed
The peroxidase catalysed, typically horseradish based peroxidase composition is used to detect E.coli cells directly up to 1x 105cfu/ml concentration in 100ml of the sample within 10 minutes. As the membrane endogenous peroxidase of E. coli reacts with the pre-stained chromogenic substrate reagent, it is noted that the intensity of the color change (Yellow-Light Green-Dark Green) is directly proportional to the E.colititer present in the sample. A visual color reader is used for semi quantitative analysis, while optical reader with a Smartphone App is also used for quantitative analysis of the results.

Composition of pre-stained chromogenic substrate reagent for E.Coli detection
Tetramethylbenzidine sulfated salt (TMB-S) is mixed with citrate buffer and hydroxylpropyl beta cyclodextrin (HPßCD) in a proportion given in Table 1, Table 2 and Table 17. To the solution thus obtained, solublizing and stabilizing agents such as PVP, PEG, Mannitol, Ribose and EDTA are added along with sodium perborate in a proportion given in Table 17, to obtain the pre-stained chromogenic substrate reagent. The pre-stained chromogenic substrate reagent is prepared by mixing chromogenic substance and other reagents in milliQ water. A typical composition in 1000ml of milliQ water is given in Table 17. The pH is maintained at 2.5 to 4.5.

Table-17: Specific composition of pre-stained chromogenic substrate reagent for E.Coli detection in 1000mL MilliQ water

Sl. No. Composition Qty
1 Citric acid 3.8g
2 Tri-Na citrate 6g
3 EDTANa/Ksalt 1g
4 PVP 5g
5 Mannitol/Sorbitol 2.5g
6 TMB-S 500mg
7 PEG-4K 2.5g
8 HPßCD 3g
9 Na perborate (Tetrahydrate) 300mg
10 Dextran Sulphate 1gm
12 Inert colouring agent
(Tartrazine) 80mg

The other salt forms of TMB which can be used in place of TMB-S are TMB-di HCl
(Tetramethyl Benzidine dihydrochloride hydrate) and TMB-PS (Tetramethyl Benzidine, 3-
sulfopropyl sodium salt). However, the preferred salt in the present invention is TMB-
S though other salts can also be used.
Preparation of the E.coli culture:
In the present invention, E.coli strain (ATCC 8739) is obtained from NCIM, Pune. The strain is cultured in a nutrient broth medium overnight at 37*C in a microbial incubator. 1x109 cfu/ml concentration of E.coli is prepared by following standard protocols. This is verified by standard pour plate assay (J.Olstadt, J et al., (2007), J of Water and health, 5,267). By serial dilution, further known titres of E.coli (108, 107, 106, 105, and 104) are obtained using saline.
Procedure for testing the presence of E.Coli in the testing sample using the peroxidase catalysed composition
100 ml of autoclaved sterile saline water is taken in a sterile container and spiked with known concentrations of E.Coli and mixed gently. This is added to filtration unit with cellulose acetate/nitrate mixture membrane filter paper (0.45µ pore size)-Millipore, vacuum is applied to preconcentrate cells on surface of membrane filter paper. 500ul of pre-stained chromogenic substrate reagent is added to the membrane. The membrane is kept at room temp for 10 min in dark. The detection limit for reagent is hence found to be 105 CFU/ml in 100 ml water. The intensity of the colour developed is found to be directly proportional to E.colititre in the water sample above 105 CFU/ml in 100ml sample.
Based on the method mentioned above, the E.coli detection limit is found to be 105 cfu/ml in 100ml water within 10 minutes (Figure 13b) i.ethe sensor can detect E.Coli of 1 cfu/ml in 100ml water within 6hr (Figure 13a shows detection at 8 minutes as well).

E.Coli detection using specific probes along with pre-stained chromogenic substrate reagent:
In this method, initially 100ml water sample is filtered using a 0.45micron filtration membrane to concentrate the bacterial cells and recovered using 1ml phosphate buffer pH 7.0. Later using specific capturing probes [Biotinylated antibodies/ Biotinylated Aptamers/ Biotinylated 4-mercaptophenylboronic acid], are added to the recovered bacterial cell and the contents are mixed and incubated at 25°C-30°C for 15-30min. Magnetic beads coated with streptavidin are added to the above contents and mixed and incubated for another 15-30min. At the end of incubation, the bound complex [E.Coli- biotin Ab/Aptamers/MPBA-Strep-MB] is separated by applying magnetic field. The supernatant is flushed out and the bound complex is transferred to a nitrocellulose membrane flow through assay strip. A drop of 2nd antibody-HRP conjugate reagent is added to the contents and the excess is flushed out with drop of wash buffer addition and a drop of pre-stained chromogenic substrate reagent is finally added which brings about a blue-green colour change at the surface of membrane. The intensity of the colour change is directly proportional to the E.Colititre in the sample. The developed colour in the nitrocellulose membrane can be read visually for semiquantitative results or quantitatively using a optical strip reader for quantitative results.
Pre-stained chromogenic substrate reagent stored at 20 °C to 30°C for four months as given in Figure 11 to six months in Figure 12 is able to detect E.coliin sample up to 105cfu/ml in100ml.

Live E.Coli cells detection specificity using composition of present invention
Pre-stained chromogenic substrate reagent is shown to detect only live E.coli cells. The figure 14 depicts comparison between known titre of E.Coli in water compared to an autoclaved version of the same sample.
To understand specificity of the method, the composition is tested for E.coli against three non-faecal coliform strains (i.e. Serratiamarcescens ATCC 14766, Enterobacter clocae ATCC 13047, Enterobacter cloacae NCIM 9145). It is observed that composition shows color change only for E.coli strains as in Figure 15, probably due to the endogenous peroxidase activity which is prominent in fecal coliforms only.

Real time E.Coli detection in water sample using the kit prototype
In this method, initially 100ml water sample (with E.Coli 1cfu/ml) is collected from the source (pound, river, tank) and filtered using a funnel fitted with a gradient filter paper of different pore size ( 12µm & 5µm ) placed over 100ml measuring cylinder . Water is filtered and collected in a 100ml measuring cylinder by gravitational flow and this process helps in removing completely the slurry and other interfering agents in the sample by the sieving effect of the filter paper as in figure 7.
The collected water containing only bacterial cells is now subjected to filtration using a 100ml syringe filter fitted with 0.45micron filtration membrane to concentrate the bacterial cells and later the filtered membrane is gently transferred to a clean vial containing LT Broth media [specific enrichment media for coliform bacterial growth] using a sterile forceps. The content is mixed and incubated inside a portable incubator maintained at 37oC for 6hrs. During this period, only E.Coli and coliform bacteria retained over the membrane will tend to grow and multiply according to its doubling time of 20sec, while other bacteria’s retained on the membrane cannot grow and tend to die immediately. Thus ensuring high specificity of E.Coli detection by using this procedure. At the end of the incubation time, the membrane is removed and placed over the plain surface. Now added a drop of prestained chromogenic substrate over the membrane and incubated for 10min. The blue-green colour appearing on the membrane indicated the presence of E.Coli. The present method detects E.Coliwith high specificity and detection sensitivity up to 1x105cfu/ml (within 6hrs) in real time. In case of blank, the procedure followed will be similar to that of test and composition of present invnetion to it gives only yellow colour on the surface of membrane mimicking the prestained reagent colour.

Rapid E.Coli detection in water sample using nanoparticles conjugate and prestained chromogenic substrate reagent
In order to improve further the detection limit of the existing method (1cfu/ml in 100ml detection within 6hrs), different strategies can be adapted known in the art, i.,e using Nanoparticles-MPBA conjugate. Thus, by adapting the same, the real time E.Coli detection within 2-3hrs in a sample is significantly improved, i.e., E.Coli with 1cfu/ml in 100ml can be detected within 2-3hrs using this method.
In this method, initially 100ml water sample (with E.Coli 1cfu/ml) is collected from the source (pound, river, tank) now subjected to filtration using a 100ml syringe filter fitted with 0.45micron filtration membrane to concentrate the bacterial cells and later the filtered membrane is gently transferred to a clean vial containing LT Broth media [specific enrichment media for coliform bacterial growth] using a sterile forceps. The content is mixed and incubated inside a portable incubator maintained at 37oC for 2 hrs. At the end of incubation period added magnetic nanoparticles-mercapophenylboronic acid [MNP-MPBA] conjugate suspended in suitable buffer, is mixed and kept over a magnetic bar to separate the bound [MNP-MPBA-E.Colicells] complex from the LTB media content. This process helps in separating only the live E.Coli cells from the other bacterial cells and dead cells and also LTB media. Supernatant containing LTB media, dead cells and other interfering agents are removed and to the separated complex [MNP-MPBA-E.Coli] is added peroxidase-MPBA conjugate or Pseudoperoxidase nanoparticles-MPBA conjugate and now incubated for additional 10min. Later the unbound free peroxidase-MPBA conjugate is separated by applying the magnetic field to the reaction vial. Finally added the prestained chromogenic substrate reagent to the complex [MNP-MPBA-E.Coli-MPBA-Peroxidase nanoparticles]. This brings about rapid colour change in the reaction vial. The intensity of the colour change is directly proportional to the E.Colititre in the sample.
Thus, the reagent of the present invention and the method thereof provides a sensitive, specific, cost effective, user friendly, robust and suitable for point of use testing of analyte.
,CLAIMS:We Claim:
1) A peroxidase chromogenic substrate reagent composition comprising Citric acid ranging from 5mg to 380mg, buffer ranging from 1mg to 600mg, Ethylenediaminetetraacetic acid salt ranging from 1mg to 100mg, Polyvinylpyrrolidone ranging from 1mg to 400mg, Mannitol/Sorbitol ranging from 1mg to 150mg, Polyethylene glycol-4K ranging from 1mg to 250mg, 2-hydroxypropyl-ß-cyclodextrin ranging from 1mg to 200mg, Sodium Perborate ranging from 1mg to 30mg, chromogenic substrate compound ranging from 1mg to 50mg, optionally D-Ribose/Xylose ranging from 1mg to 10mg, Dextran sulphate ranging from 1mg to 90mg, and dye ranging from 0.0001 mg - 0.0010mg; for analysing an analyte.
2) The composition as claimed in claim 1, wherein the analyte is selected from a group comprising pathogens, carcinogenic cells, carcinogens, toxins and the like.
3) The composition as claimed in claim 1, wherein the analysis is by colorimetric and/or fluorimetric analysis.
4) The composition as claimed in claim 1, wherein the analysis is by quantitative and qualitative analysis.
5) The composition as claimed in claim 4, wherein the method of analysis is selected from a group comprising enzyme-linked Immunosorbent assay, Incubation, Immunohistochemistry, Dot-ELISA, western blotting and the like.
6) The composition as claimed in claim 1, wherein the chromogenic compound is selected from a group; 3,3',5,5'-tetramethylbenzidine, 3,3',5,5'-tetramethylbenzidine sulphate, 3,3',5,5'-tetramethylbenzidine dihydrochloride, 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid diammonium salt, o-phenylenediamine, 3,3'-DiAminoBenzide tetrahydrochloride, 3-Amino-9-ethylcarbazole,4- Chloronapthol, and the like.
7) The composition as claimed in claim 1, wherein the peroxidase is selected from a group comprising Horseradish peroxidase (HRP), E.Coli/ pathogen detector in water and the like.
8) The composition as claimed in claim 1, wherein the dye is selected from a group comprisingPhloxine B, sunset yellow, sunset orange, methyl orange, allura red and the like.
9) The composition as claimed in claim 1; wherein the buffer is selected from a group of Trisodium citrate, imidazole, citrate-phosphate and the like.
10) The composition as claimed in claim 1; wherein the composition is stable for 12 – 18 months at temperature 2 – 8 ° C; and 3- 6 months at 25-30 ° C.
11) A method of detection of an analyte, said method comprising acts of
(a) filtering and/or optionally enriching the analyte with media and incubating;
(b) filtering the media again by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer; and
(c) treating the filtered membrane with composition of claim 1 and maintaining for 30 min for the analysis by change in colour.
12) The method of detection as claimed in claim 11; wherein the analyte detection is at pH selected from a range of pH 4 – pH 6.
13) The method of detection as claimed in claim 11, wherein the colour change of the analyte is observed by light and electron microscopy, spectrophotometry, fluorometry, human eye and the like.
14) The method of detection as claimed in claim 11, wherein the analyte is selected from a group comprising pathogens, carcinogenic cells, carcinogens, toxins and the like
15) A method of pathogenic detection, said method comprising acts of;
(a) enriching the specimen samples and incubating at a temperature of ranging from 30-40°C for a time about 3-10 hours;
(b) filtering the incubated specimen by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer; and
(c) treating the filtered membrane with composition of claim 1 andmaintaining for 30 min for the analysis based on colour change.
16) A method for E. Coli detection, said method comprising acts of
a) enriching the specimen with a media and incubating at a temperature ranging from 35°C-40°C for a duration ranging from 3-10 hours;
b) filtering the incubated media by 0.45micron pore size membrane and washing with saline and 10mM acetate buffer; and
c) treating the filtered membrane with composition of claim 1 and incubating for 30 min for analysis based on colour change.
17) The method of E. Coli detection as claimed in claim 16; wherein colony forming unit (CFU) is ranging from 1- 107 CFU and the time ranging from 10 hr to 0.15 hr respectively.
18) The method of E. Coli detection as claimed in claim 16; wherein the detection is at pH 4.0-4.5.
19) The method of E. Coli detection as claimed in claim 16; wherein composition of claim 1 is Citric acid ranging from 5mg to 380mg, Trisodium citrate ranging from 1mg to 600mg, Ethylenediaminetetraacetic acid salt ranging from 1mg to 100mg, Polyvinylpyrrolidone ranging from 1mg to 400mg, Mannitol/Sorbitol 1mg to 150mg, Polyethylene glycol-4K ranging from 1mg to 250mg, 2-hydroxypropyl-ß-cyclodextrin ranging from 1mg to 200mg, Sodium Perborate ranging from 1mg to 30mg,Tartrazineranging from 70mg to 90mg, 3,3',5,5'-tetramethylbenzidine sulphate ranging from 1mg to 50mg,milliQ water and optionally D-Ribose/Xylose ranging from 1mg to 10mg, Dextran sulphate ranging from1mg to 90mg.
20) The method as claimed in claim 16, wherein the peroxidise is endogeneous membrane peroxidise of ecoli.