WO2016030711A1

WO2016030711A1 - Stable phosphatase substrate formulation and uses thereof

Info

Publication number: WO2016030711A1
Application number: PCT/IB2014/064039
Authority: WO
Inventors: Vijaywanth MATHUR; Dhanada DESHPANDE; Jignesh SAVANI; Prakash BAPODARA
Original assignee: Diasys Diagnostics India Private Limited
Priority date: 2014-08-25
Filing date: 2014-08-25
Publication date: 2016-03-03

Abstract

The present disclosure relates to an alkaline phosphatase (ALP) substrate formulation comprising: (a) at least one substrate; (b) at least one buffer; (c) at least one ALP activator; (d) at least one surfactant; (e) at least one thermal stabilizer and (f) at least one oxygen scavenger. The ALP substrate formulation as disclosed herein is stable for prolonged period of time and useful in increasing sensitivity of the electrochemical immunoassay. The present disclosure also relates to a method of preparation of the ALP substrate formulation, and a package comprising the ALP substrate formulation.

Description

STABLE PHOSPHATASE SUBSTRATE FORMULATION AND USES THEREOF

TECHNICAL FIELD

The present disclosure relates to an electrochemical enzyme substrate formulation and its use in electrochemical immunosensors. More particularly, the present disclosure relates to an alkaline phosphatase (ALP) substrate formulation, which is stable for prolonged period of time. The said formulation is useful in wide range of biosensors/immunosensors utilizing electrochemical immunoassay approach.

BACKGROUND OF THE INVENTION

Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. Immunological methods have become important tools useful for detecting antigens, for example, peptides, proteins, nucleic acids and biological cells. A wide variety of methods have been developed for the detection of antigens. Among them, Western Blot, Dot Blot, ELISA and Immunohistology are the four most commonly used methods.

Enzymatic reactions are widely used in immunoassays as clinical diagnostics. Enzyme- Linked Immunosorbent Assay (ELISA) involves a reaction between a substrate and an enzyme. In principle, an enzyme is coupled to an antibody against the antigen which is to be determined. The assay is usually performed in trays of polystyrene or polypropylene to which the antigen is immobilised. The enzyme is chemically coupled to the antibody and incubated with a substrate solution containing a suitable buffer, a chromogenic substrate. The enzyme converts the colourless substrate (chromogen) to a coloured product on reaction of the enzyme with the substrate.

Alkaline phosphatase (ALP) is broadly used as antibody labeled enzyme in detection of broad spectrum of analytes. ALP is highly accepted as its linear reaction rate increases the sensitivity over increased incubation time and also due to availability of wide range of substrates. Over the last few years, efforts have been made to improve the enzymes used in ELISA. Enzymes have been modified so as to increase their stability, handling ability and to permit their re-use. Relatively insignificant efforts have however been made to elaborate the substrate used in ELISA, despite the fact that great problems arise when the substrate is dissolved in a suitable solvent system. Also, the substrates used in ELISA are inherently unstable due to exposure to light and they oxidize upon exposure to atmospheric oxygen. ALP substrates are particularly unstable in solution changing colour to brown.

To overcome the above problems, currently ALP substrate formulations are being developed that comprise of stabilizers that enhance the stability for long term storage and reactivity, that results in easy detection of low quantities of analytes. Stable alkaline phosphatase substrate formulations have been prepared that can be stored at 37 °C for a maximum period of one week without impairment of sensitivity. But when kept for a longer duration of time, the sensitivity of the ALP substrate formulations start deteriorating.

Therefore, there is a need to develop an improved ALP substrate formulation with an improved manufacturing process, and improved packaging method to tackle the instability and handling ability of ALP substrate in solution. In addition to enhancing stability of ALP substrate in solution, it is also desirable to provide new improved ALP substrate formulation that increases the sensitivity of the formulation for longer period of time and improves precision of the assay.

The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure relates to an alkaline phosphatase (ALP) substrate formulation, which exhibits excellent stability and sensitivity for electrochemical immunoassay (ELISA) application.

In one aspect, the present disclosure provides an alkaline phosphatase (ALP) substrate formulation comprising: (a) at least one substrate; (b) at least one buffer; (c) at least one ALP activator; (d) at least one surfactant; (e) at least one thermal stabilizer; and (f) at least one oxygen scavenger. In another aspect, the present disclosure provides a method of preparing the ALP substrate formulation as disclosed herein, comprising the steps of:

(i) mixing at least one buffer, at least one ALP activator and at least one surfactant to form a substrate buffer;

(ii) heating the substrate buffer and then purging it with a mixture of helium and nitrogen to form a purged buffer;

(iii) adding at least one oxygen scavenger and at least one ALP substrate to the purged buffer and dissolving added solid components; and

(iv) adding at least one thermal stabilizer to the mixture obtained in step (iii), then mixing to form the ALP substrate formulation.

In another aspect, the present disclosure provides a package comprising the ALP substrate formulation as disclosed herein, an aluminium foil, a desiccant pouch and an oxygen scavenger sachet.

In one aspect of the present disclosure, the ALP substrate formulation is useful in ELISA for measuring clinical diagnostic parameters in a biological sample of a subject, preferably a mammal including human.

Further aspects of the invention will be apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates peak currents as a function of the concentration of HbAlc between 0 and 17.3 % with the ALP substrate formulation containinglO mM 4-aminophenyl phosphate in substrate buffer.

Figure 2 illustrates peak currents as a function of the concentration of HbAlc between 0 and 15.2 % with the ALP substrate formulation containing 15 mM 4-aminophenyl phosphate in substrate buffer.

Figure 3 illustrates peak currents as a function of the concentration of HbAlc between 0 and 11.6 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer. Figure 4 illustrates peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 niM 4-aminophenyl phosphate in substrate buffer and 1 niM sodium sulfite.

Figure 5 illustrates peak currents as a function of the concentration of HbAlc between 0 and 14.3 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 2 mM sodium sulfite.

Figure 6 illustrates peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 3 mM sodium sulfite. Figure 7 illustrates peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 1.5 mM sodium sulfite.

Figure 8 illustrates peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 1.5 mM sodium sulfite.

Figure 9 illustrates peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 15% ethylene glycol, 85% substrate buffer and 1.5 mM sodium sulfite.

Figure 10 illustrates peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl in 25% ethylene glycol, 75% substrate buffer and 1.5 mM sodium sulfite.

Figure 11 illustrates peak currents as a function of the concentration of HbAlc between 0 and 15 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 3 mM sodium bisulfite. Figure 12 illustrates peak currents as a function of the concentration of HbAlc between 0 and 13.05 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 1.5mM sodium bisulfite.

Figure 13: ALP substrate stability graph. DETAILED DESCRIPTION OF THE INVENTION

Unless the context requires otherwise, throughout the specification and claims which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to." Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

The headings and abstract of the disclosure provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

Reference will now be made in detail to the exemplary embodiments of the present disclosure, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.

In describing the embodiments of the invention, specific terminology is resorted for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that each specific term includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

Embodiments of the present disclosure relate to an alkaline phosphatase (ALP) substrate formulation that is stable for prolonged period of time and useful in wide range of biosensors/immunosensors utilizing electrochemical immunoassay approach.

In an embodiment, the present disclosure provides an ALP substrate formulation comprising: (a) at least one substrate; (b) at least one buffer; (c) at least one ALP activator; (d) at least one surfactant; (e) at least one thermal stabilizer and (f) at least one oxygen scavenger.

In an embodiment of the present disclosure, the substrate is electrochemically active.

In an embodiment of the present disclosure, the substrate is selected from the group consisting of 4-aminophenyl phosphate (4-APP), 2-phospho-L-ascorbic acid, hydroquinone diphosphate, ferrocene derivatives, o-phenylenediamine dihydrochloride, osmium complex, β-naphthyl phosphate, phenyl phosphate and 3,3', 5,5'- tetramethylbenzidine (TMB).

In one exemplary embodiment of the present disclosure, the substrate is 4-aminophenyl phosphate (4-APP). In another exemplary embodiment of the present disclosure, the substrate is 4- aminophenyl phosphate (4-APP) monosodium salt hydrate.

In another exemplary embodiment, the concentration of substrate present in the ALP substrate formulation as disclosed herein ranges from 5 mM to 50 mM.

In another exemplary embodiment, the concentration of substrate present in the ALP substrate formulation as disclosed herein ranges from 10 mM to 30 mM.

In another embodiment of the present disclosure, the buffer is selected from the group consisting of diethanolamine (DEA) buffer, tris buffer, phosphate buffer, triethanolamine (TEA), 2-amino-2-methyl-l-propanol (AMP), N-bis(2-hydroxyethyl)-2-taurine (BHET), (cyclohexylamino)-l -propanesulfonic acid (CAPSO), (cyclohexylamino)-l -ethanesulfonic acid (CHES), 2-hydroxyethyl-piperazinyl-N-2-ethanesulfonic acid (HEPES), (N- morpholinyl)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazinyl-N,N-bis(2-ethanesulfonic acid) (PIPES), piperazinyl-N,N-bis(2-hydroxy ethanesulfonic acid) (POPSO), tris(hydroxymethyl)methyl-3-amino propanesulfonic acid (TAPSO), tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-2-hydroxyethyl- piperazinyl-N-3-propanesulfonic acid (HEPES) and citrate acetate buffer.

In one exemplary embodiment of the present disclosure, the buffer is selected from diethanolamine (DEA) buffer or tris buffer.

The buffer plays a role of accepting a phosphate molecule, and thus it increases the reaction rate and the sensitivity of the assay. In an embodiment of the present disclosure, the ALP activator is selected from the group consisting of soluble salts of magnesium, sodium, calcium and potassium or a combination thereof.

In an exemplary embodiment of the present disclosure, the ALP activator is selected from the group consisting of magnesium chloride, sodium chloride and potassium chloride or a combination thereof.

In an embodiment of the present disclosure, the surfactant is selected from the group consisting of TWEEN® 20, TRITON® X-100, polysorbate, poloxyethylene ether, polyethylene glycol ether, sorbitan polyoxyethylene ether fatty acid esters, alkylphenol polyoxyethylene ethers, sorbitan polyoxyethylene ether lauric acid ester, sorbitan polyoxyethylene ether oleic acid ester and octylphenol polyoxyethylene ether or a combination thereof.

In one exemplary embodiment of the present disclosure, the surfactant is selected from TWEEN® 20, TRITON® X-100 or a combination thereof. The surfactant is required to evenly distribute the product formed after the reaction of enzyme and substrate.

In an embodiment of the present disclosure, the thermal stabilizer can be ethylene glycol.

In an embodiment of the present disclosure, the thermal stabilizer can also act as a preservative.

In an embodiment of the present disclosure, ethylene glycol acts both as a thermal stabilizer and as a preservative.

In an embodiment of the present disclosure, the oxygen scavenger is selected from the group consisting of sodium sulfite, sodium bisulfite, sodium thiosulfate, sodium lignosulfate, ammonium bisulfite, hydroquinone, diethylhydroxyethanol, diethylhydroxylamine, methylethylketoxime, ascorbic acid, erythorbic acid and sodium erythorbate.

In an exemplary embodiment of the present disclosure, the oxygen scavenger is selected from sodium sulfite or sodium bisulfite. The oxygen scavenger prevents oxidation of the ALP substrate formulation. In an exemplary embodiment, the concentration of the oxygen scavenger present in the ALP substrate formulation as disclosed herein ranges from 0.5 mM to 7 mM.

In another exemplary embodiment, the concentration of the oxygen scavenger present in the ALP substrate formulation as disclosed herein ranges from 1 mM to 5 mM.

In an embodiment of the present disclosure, pH of the ALP substrate formulation ranges from 9 to 11.

In an embodiment, the present disclosure provides a method of preparing an alkaline phosphatase (ALP) substrate formulation, comprising the steps of:

According to the present disclosure, the heating and then the purging of the substrate buffer with the mixture of helium and nitrogen gases is done to remove dissolved oxygen from the mixture. This helps to prevent the substrate oxidation that generally occurs after a certain period of time. All the components of the ALP substrate formulation as disclosed herein play a vital role in assay performance.

In an embodiment, the method of preparation as disclosed herein, the solid components, such as oxygen scavenger, ALP substrate can be dissolved by sonication.

The method of preparation of the ALP substrate formulation as disclosed herein is preferably carried out in an amber coloured bottle.

The ALP substrate formulation as disclosed herein can be filled in cartridges under inert atmosphere to prevent oxidation. In an embodiment, the present disclosure provides a package comprising the ALP substrate formulation as disclosed herein, an aluminium foil, a desiccant pouch and an oxygen scavenger sachet.

In an embodiment, the thickness of the aluminium foil can range from 0.5 mils to 2 mils.

In an embodiment, the capacity of the oxygen scavenger sachet can range from 50 cc to 200 cc.

The ALP substrate formulation of the present disclosure can be used in a wide range of biosensors/immunosensors utilizing electrochemical immunoassay approach. In an exemplary embodiment of the present disclosure, the ALP substrate formulation is stable for at least 12 months at 2-8 °C, based on extrapolated data from the accelerated substrate shelf life study.

In another exemplary embodiment of the present disclosure, the ALP substrate formulation is stable for at least 45 days at 37 °C. The ALP substrate formulation as disclosed herein is substantially stable and easily dispersible in solution form. The ALP substrate formulation of the present disclosure not only increases the sensitivity of the assay but also helps in uniform dispersion of electrochemical products formed, such as aminophenol, ascorbic acid and hydroquinone.

One should appreciate that although the present disclosure has been explained with reference to 4-aminophenyl phosphate (4-APP) substrate formulation in the examples described below, any other electrochemically active substrate can be used in biosensors incorporating enzyme labeled secondary antibody, all of which are completely covered within the scope of the instant disclosure.

One should further appreciate that although the present disclosure has been explained with reference to electrochemical immunosensor for HbAlc, any other test for disease markers such as cardiac, thyroid markers on a biological sample can be implemented, all of which are completely covered within the scope of the instant disclosure.

The present disclosure is illustrated with working examples, which is intended to illustrate the working of the invention and not intended to take restrictively to imply any limitations on the scope of the present invention. The following abbreviations or terms are used herein:

ELISA : Enzyme-Linked Immunosorbent Assay

ALP : Alkaline phosphatase

4-APP : 4-Aminophenyl phosphate

HbAlc : Glycated haemoglobin

NaCl : Sodium chloride

MgCl₂ _: Magnesium chloride

mM : Millimolar

: Microlitre

uA : Microamperes

°C : Degree Celsius

EXAMPLES

Example 1: Preparation of substrate buffer 0.1 M diethanolamine buffer was dissolved in de-ionized water and mix thoroughly on magnetic stirrer. The pH of the buffer is checked and maintained at 9.8. After the pH adjustment 2 mM magnesium chloride (MgCl₂), 1 M sodium chloride (NaCl) and 0.1% Tween® 20 were added to the solution. The final volume is made as per requirement.

Example 2: Preparation of 4-Aminophenyl phosphate (4-APP) formulation

The substrate buffer (prepared as described in Example 1) was heated at 50 °C. The substrate buffer was then purged with a mixture of 20 % helium and 80 % nitrogen for 1 hour maintaining the temperature at 50 °C. The purged buffer solution was cooled to 2-8 °C till the solution attained a temperature of 20 °C. Aliquot of the buffer solution, so as to constitute 75 % of the total volume of the substrate was prepared. To this buffer solution, 1.5 mM sodium bisulfite powder and 20 mM 4-aminophenyl phosphate were added. This buffer solution was then sonicated for 20 minutes to completely dissolve the added solid components. Then ethylene glycol was added to constitute 25 % of the total volume of substrate to be prepared and mixed gently to avoid bubbles. The entire process was carried out in amber coloured bottle.

Example 3: Study of ALP substrate formulation at different concentrations of ALP substrate, 4-aminophenyl phosphate in HbAlc electrochemical immunoassay

Table 1; Assay of ALP substrate formulation containing 10 mM 4-aminophenyl phosphate in substrate buffer

Figure 1 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 17.3 % with the ALP substrate formulation containinglO mM 4-aminophenyl phosphate in substrate buffer.

Table 2: Assay of ALP substrate formulation containing 15 mM 4-amino phenyl phosphate in substrate buffer

Figure 2 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 15.2 % with the ALP substrate formulation containing 15 mM 4-aminophenyl phosphate in substrate buffer. Table 3: Assay of ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer

Figure 3 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 11.6 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer.

Conclusion: Figure 3 shows a linear increase in peak current with increase in percentages of HbAlc, thus showing that 20 mM 4-aminophenyl phosphate yields better results.

Example 4: Study of ALP substrate formulation at different concentrations of oxygen scavenger HbAlc electrochemical immunoassay

Table 4: Assay of ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 1 mM sodium sulfite

Figure 4 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 1 mM sodium sulfite.

Table 5: Assay of ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 2 mM sodium sulfite Level Cone. Of HbAlc (%) Current (uA)

Blank 0 34.6

LI 4.99 136.7

L2 7.68 105.8

L3 12.1 129.6

L4 14.3 116.9

Figure 5 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 14.3 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 2 mM sodium sulfite. Table 6: Assay of ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 3 mM sodium sulfite

Figure 6 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 3 mM sodium sulfite.

Table 7: Assay of ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 1.5 mM sodium sulfite

Figure 7 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in substrate buffer and 1.5 mM sodium sulfite.

Conclusion: Figure 7 shows a linear increase in peak current with increase in percentages of HbAlc, thus showing that 1.5 mM sodium sulfite produce better results than other concentration of sodium sulfite.

Example 5: Study of ALP substrate formulation at different concentrations of thermal stabilizer and preservative; and substrate buffer in HbAlc electrochemical immunoassay

Table 8: Assay of ALP substrate formulation containing 20 mM of 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 1.5 mM of sodium sulfite

Figure 8 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 1.5 mM sodium sulfite.

Table 9: Assay of ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 15% ethylene glycol, 85% substrate buffer and 1.5 mM sodium sulfite Level Cone. Of HbAlc (%) Current (uA)

LI 4.99 134.12

L2 7.68 150.5

L3 12.1 160.6

L4+L4 14.3 170.82

L4 16.5 206.52

Figure 9 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl in 15% of ethylene glycol, 85% substrate buffer and 1.5 mM sodium sulfite.

Table 10: Assay of ALP substrate formulation containing 20 mM 4-aminophenyl in 25% ethylene glycol, 75% substrate buffer and 1.5 mM sodium sulfite

Figure 10 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 16.5 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 25% ethylene glycol, 75 % substrate buffer and 1.5 mM sodium sulfite.

Conclusion: Figure 10 shows a linear increase in peak current with increase in percentages of HbAlc, thus showing that 25% ethylene glycol yielded better results than other concentration.

Example 6: Study of ALP substrate formulation using sodium bisulfite as oxygen scavenger at different concentrations in HbAlc electrochemical immunoassay Table 11; Assay of ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 3 mM sodium bisulfite

Figure 11 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 15 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 3 mM sodium bisulfite.

Table 12; Assay of ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 1.5 mM sodium bisulfite

Figure 12 illustrates an analytical curve showing peak currents as a function of the concentration of HbAlc between 0 and 13.05 % with the ALP substrate formulation containing 20 mM 4-aminophenyl phosphate in 10% ethylene glycol, 90% substrate buffer and 1.5 mM sodium bisulfite.

Conclusion; Figure 12 shows a linear increase in peak current with increase in percentages of HbAlc, thus showing that 1.5 mM sodium bisulfite yielded better results than other concentration. Example 7: Composition of ALP substrate formulation

Example 8: Method of analysis

The substrate prepared was filled in the substrate chamber of the cartridge. The cartridge was then inserted into the device and the sample was loaded in the sample channel. The sample incubation was followed by detection antibody incubation. Finally the substrate follows through the channel and a fixed volume of the substrate is incubated over the sensor for a specific time. The substrate generates an electrochemical product on catalysis by the enzyme. The current generated by the product was measured through a Chronoamperometry technique. The strength of the current was directly proportional to the concentration of the analyte.

Example 9: Study of ALP substrate stability

The stability of the prepared ALP substrate was checked over a period of 45 days. This substrate solution was filled in cartridges and the cartridges were stored in aluminium foil having a desiccant and an oxygen scavenger sachet at 37 °C for the said period. A small aliquot of the solution from respective cartridge at a particular day was analysed.

The test is carried out as follows:

After the substrate is prepared, a qualification test is carried out as follows:

• 50 μΐ_^ of substrate was taken and incubated over a carbon nanotubes coated sensor for 30 seconds and then Chronoamperometric analysis is done and currents at 0.2 seconds are noted. This is Zero day reading.

• Similarly small aliquots were taken on respective days and qualification is carried out. No. Of Days Blank @ 0.25 sec

0 20.65

2 18.24

21 14.03

30 11.36

45 11.99

Example 10: Extended ALP substrate stability studies

Accelerated stability study was performed with 4-Aminophenyl phosphate formulation. 1 mL aliquots of freshly prepared ALP substrate formulation was packed under nitrogen in cartridges and kept at 37 °C for ~ 45 days. It was observed that the formulation was visually colourless and stable for 45 days at 37 °C as verified also from the baseline blank data of the shelf-life ALP substrate. An increasing trend in the blank reading of the ALP substrate indicates degradation of the ALP substrate.

Claims

An alkaline phosphatase (ALP) substrate formulation comprising (a) at least one substrate; (b) at least one buffer; (c) at least one ALP activator; (d) at least one surfactant; (e) at least one thermal stabilizer; and (f) at least one oxygen scavenger.

The alkaline phosphatase substrate formulation according to claim 1, wherein the substrate is selected from the group consisting of 4-aminophenyl phosphate, 2-phospho- L-ascorbic acid, hydroquinone diphosphate, ferrocene derivatives, o-phenylenediamine dihydrochloride, osmium complex, β-naphthyl phosphate, phenyl phosphate and 3,3',5,5'- tetramethylbenzidine.

The alkaline phosphatase substrate formulation according to claim 1, wherein the substrate is 4-aminophenyl phosphate (4-APP).

The alkaline phosphatase substrate formulation according to claim 1, wherein the substrate is 4-aminophenyl phosphate (4-APP) monosodium salt hydrate.

The alkaline phosphatase substrate formulation according to claim 1, wherein the buffer is selected from the group consisting of diethanolamine (DEA) buffer, tris buffer, phosphate buffer, triethanolamine, 2-amino-2-methyl-l-propanol, N-bis(2- hydroxyethyl)-2-taurine, (cyclohexylamino)- 1-propanesulfonic acid,

(cyclohexylamino)- 1-ethanesulfonic acid, 2-hydroxyethyl-piperazinyl-N-2- ethanesulfonic acid, (N-morpholinyl)ethanesulfonic acid, 3-(N- morpholino)propanesulfonic acid, piperazinyl-N,N-bis(2-ethanesulfonic acid), piperazinyl-N,N-bis(2-hydroxy ethanesulfonic acid), tris(hydroxymethyl)methyl-3- amino propanesulfonic acid, Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and N-2-hydroxyethyl-piperazinyl-N-3 -propanesulfonic acid and citrate acetate buffer.

The alkaline phosphatase substrate formulation according to claim 1, wherein the buffer is selected from diethanolamine (DEA) buffer or tris buffer.

7. The alkaline phosphatase substrate formulation according to claim 1, wherein the ALP activator is selected from the group consisting of soluble salts of magnesium, sodium, calcium and potassium or a combination thereof.

8. The alkaline phosphatase substrate formulation according to claim 1, wherein the ALP activator is selected from the group consisting of magnesium chloride, sodium chloride, calcium chloride and potassium chloride or a combination thereof.

9. The alkaline phosphatase substrate formulation according to claim 1, wherein the surfactant is selected from the group consisting of TWEEN® 20, TRITON® X-100, polysorbate, poloxyethylene ether, polyethylene glycol ether, sorbitan polyoxyethylene ether fatty acid esters, alkylphenol polyoxyethylene ethers, sorbitan polyoxyethylene ether lauric acid ester, sorbitan polyoxyethylene ether oleic acid ester and octylphenol polyoxyethylene ether or a combination thereof.

10. The alkaline phosphatise substrate formulation according to claim 1, wherein the surfactant is selected from TWEEN® 20, TRITON® X-100 or a combination thereof.

11. The alkaline phosphatase substrate formulation according to claim 1, wherein the thermal stabilizer is ethylene glycol.

12. The alkaline phosphatase substrate formulation according to claim 1, wherein the thermal stabilizer acts as a preservative.

13. The alkaline phosphatase substrate formulation according to claim 1, wherein the oxygen scavenger is selected from the group consisting of sodium sulfite, sodium bisulfite, sodium thiosulfate, sodium lignosulfate, ammonium bisulfite, hydroquinone, diethylhydroxyethanol, diethylhydroxylamine, methylethylketoxime, ascorbic acid, erythorbic acid and sodium erythorbate.

14. The alkaline phosphatase substrate formulation according to claim 1, wherein the oxygen scavenger is selected from sodium sulfite or sodium bisulfite.

15. The alkaline phosphatase substrate formulation according to claim 1, wherein pH of the ALP substrate formulation ranges from 9 to 11.

16. A method for preparing the alkaline phosphatase substrate formulation according to claim 1 comprising the steps of:

17. The method according to claim 16, wherein the substrate buffer is purged with a mixture of 20% helium and 80% nitrogen for at least 1 hour.

18. The method according to claim 16, wherein the ALP substrate formulation is filled in cartridges under inert atmosphere.

19. A package, wherein the package comprises the alkaline phosphatase substrate formulation of claim 1 filled in the cartridges, an aluminium foil, a desiccant pouch and an oxygen scavenger sachet.