Title of Invention

"A PROCESS FOR THE PREPARATION OF APO-ASCROBIC ACID OXIDASE IMMOBILIZED MEMBRANE ELECTRODE FOR THE DETERMINATION OF COPPER IONS"

Abstract

A process for the preparation of Apo-ascrobic acid oxidase immobilized membrane electrode for the determination of copper ions The invention relates to a process for the preparation of an enzyme electrode for the determination of copper ions by immobilizing the enzyme in a membrane by mixing a solution of ascorbic acid oxidase and lysozyme and glutaraldehyde with a membrane, allowing the said enzyme immobilized membrane to dry for a period of 1-2 hours, washing the above said enzyme membrane with phosphate buffer at a pH of 5.6 to remove the excess of glutaraldehyde, sandwiching the above said enzyme membrane between a Teflon and cellophane membrane and placing it on the Clark dissolved oxygen electrode to obtain the enzyme membrane electrode, immersing the above said immobilized enzyme membrane electrode in a buffer solution containing guanidine chloride and EDTA to prepare the desired Apo-ascrobic acid oxidase immobilized membrane electrode.

Full Text

'the present invention is related to an analytical device based on an Immobilized Ascorbic acid oxidize enzyme and amperometric biosensor for estimation copper (II) ions in solutions,. An alternative method of Cu(ll) estimation is to use an immobilized matalloenzyme which need Cu(ll) ions in its molecular configuration and this ions can be effectively leached out. Metalloenzymes essentially need metal ions as a cofactor in their active site for catalytic activity. Removing the metal ions from the enzyme by using strong chelating agents gives an 'apoenzyme' without any enzyme activity. This apoenzyme gets reactivated by exposing to the metal ions contained in a sample to be analysed for its metal content, enabling them to be taken up by the active site of enzyme and regain its activity. This regain of enzyme activity by the apoenzyme is proportional to the metal content of the solution. Copper (II) ions have been chemically analysed spectrophotometrically and Atomic Absorption Spectroscopy (AAS) (AOAC 1990) In spectrophotometric analysis sample is treated with acids and digested and color is developed using diethyldithiocarbamate disadvantages of this method are non specificity, color development by contaminants such as Te and Bi and also labor involved, AAS is expensive and require tedious preparations. Reference may be made to report on the construction of biosensors for the analysis of heavy metals like copper (Satoh et al, 1992). I. SATOH, H. ANZAI and H. ITOH 1992, FLOW-amperometricb biosensing of copper (ii) ions using an apoenzyme-membrane.Vol. 8, upplement A (1992) Digest of the 14th Chemical Sensor Symposium April 2-4, 1992 Tokyo Metropolitan University . Ascorbate oxidase covalently immobilized onto a porous polymer membrane was used as the recognition element for copper(ll) ions. The biosensing system was assembled with the enzyme-membrane attached onto an oxygen electrode housed in a flow-through cell for monitoring the enzymic activity. Copper(ll) ions were amperometrically determined by the activation of the immobilized metalfree enzyme (apoenzyme). The activity was assessed by injecting a 0.1 ml of 10 mM L-ascorbate as the substrate and measuring, the change in oxygen uptake due to the enzymic reaction. The membrane was regenerated by pumping N,Ndiethyldithiocarbamate solution (pH 8.0). In this type of biosensor, the enzymatic reaction is inhibited by metal ions causing a decrease in the biosensor response signal due to decreased enzymatic activity. On other hand, in the enzyme activation based biosensors, metalloenzymes have been used for the detection of metal ions by inhanced enzyme activity in presence of metal ions. However, the application of the apoenzyme based biosensors for metal ion detection has been restricted due to the difficulties encountered in the regeneration of the immobilized apoenzyme for repeated analysis. It has been reported ( Sigel, 1981; Sigel, H. (1981). In: Metal Ions in Biological Systems Vol. 12, Marcel Dekker Inc. New York pp73-106.) that the removal of copper ions from soluble (free) Ascorbic acid oxidase (ASCORBIC ACID OXIDASE ) can be achieved by using chelating agents like EDTA in the presence of substrate. However information on the preparation of immobilized Apo-ascorbic acid oxidase is not available .which is very essential for biosensor development for repeated use. Additionally in the case of immobilized enzyme, due to the compactness through multipoint attachment (such as in the crosslinking method) can reduce the efficiency of action of the chelating agents in removing the copper ions can be adversely affected. Moreover, reconstitution of the immobilized apo-enzyme may not be efficient which may lead to poor reproducibility of the constructed biosensor for the copper ion detection. Thus It is difficult to prepare Apoascorbic acid oxidase and information on respect of this is not available. Related work in our laboratory on stabilization of biosensor has indicated that incorporation of lysozyme as a stabilizing agent (CSIR Patent No. 1454/DEL/99, Development of a process for the stabilization of enzymes), during immobilization can help in efficient reversible denaturation of enzymes against guanidine hydrochloride (GdmCI). Taking a clue from this, a novel approach has been used in this invention, involving the preparation of the Apo-ascorbic acid oxidase immobilized with lysozyme through the denaturation by GdmCI followed by removal of copper ions by EDTA and then use this in the construction of a biosensor for the determination of copper ions. Novelty of the present invention is the is the estimation of copper ions by the biosensor device which uses guanidine chloride as a denaturation agent and EDTA as a chelating agent to make apo-ascorbic acid oxidase enzyme which can be immobilized together with lysozyme for repeated use. It is known that ascorbic acid oxidase enzyme contains copper ions in its active sites. The metal ions have been built into the structure of the enzyme molecule and cannot be removed without destroying that structure. In this invention, a novel method was used to remove copper ions from immobilized ASCORBIC ACID OXIDASE using glutaraldehyde cross linking method with lysozyme as stabilizing agent, in order to prepare the Apo-ascorbic acid oxidase for the analysis of copper (II) ions, the immobilized ascorbic acid oxidase was treated with GdmCI and 0.1M of EDTA for 30-60 minutes. During these reactions the enzyme gets denatured and metal-ions are leached out from active center. The biosensor constructed could analyze copper (II) ions at a sensitivity of 2 ppm. The enzyme membrane could be used for more than 50 analyses. The advantage of GdmCI and EDTA treatment of the ascorbic acid oxidase immobilized with lysozyme is the highly efficient removal and incorporation of copper (II) ions from the immobilized enzyme through the reversible denaturation behaviour of ASCORBIC ACID OXIDASE enzyme. Accordingly, the present invention provides a process for the preparation of Apo-ascrobic acid oxidase immobilized membrane electrode for the detemnination of copper ions, which comprises : a) charcacterized in that mixing the 10 µI of ascorbic acid oxidase in 20 µl of 10% solution of lysozyme and 5 % glutaraldehyde to obtain the mixture, immobilizing the said mixture on a membrane cellophane, b) allowing the said enzyme immobilized membrane to dry for a period of 1-2 hours, c) washing the above said enzyme membrane with phosphate buffer at a pH of 5.6 to remove the excess of glutaraldehyde, d) sandwiching the above said enzyme membrane between a Teflon towards electrode and cellophane membrane towards analytic solution and placing it on the Clark dissolved oxygen electrode to obtain the electrode assembly, e) immersing the above said electrode assembly in a buffer solution containing guanidine chloride and Ethylene diamine tetra acetic acid to prepare the desired Apo-ascrobic acid oxidase immobilized membrane electrode. In another embodiment of the present invention, the units per enzyme membrane of ascorbic acid oxidase may be effected in the range of 8-12 units per enzyme membrane. Yet in another embodiment of the present invention units per enzyme may be selected from a range of 8 to 12 units. Still in another embodiment of the present invention, the concentration of glutaraldehyde solution may be effected used is 5% by weight. In an yet another embodiment, the thickness of the enzyme membrane may be obtained is 130 microns. In yet another embodiment of the present invention, the response time of the enzyme electrode obtained may be 5 min. An Immobilized enzyme and amperometric biosensor for estimation copper (II) ions which comprises an immobilized ascorbic acid oxidase membrane, gold and silver electrode and an amperometric detection system patented earlier (CSIR ) patent No. (2159/DEL/98) A device useful for the measurement of organic acids and their derivatives ). For copper ion determination, conversion of the holoenzyme to apoenzyme is essential. The apoenzyme can be prepared by removing the metal ions using chelating agents like EDTA. For detection of copper ions, an apoenzyme of ascorbic acid oxidase was made using GdmCI and EDTA. ascorbic acid oxidase immobilized together with lysozyme was treated with phosphate buffer containing GdmCI (0.1- 4M) and EDTA (0.1-3 mM). After 1-2 hours it was observed that 100% of native enzyme activity was lost. The efficient removal of copper ions by GdmCI treatment in presence of EDTA, can be attributed to the conformational changes (denaturation) in the enzyme structure due to the unfolding of the immobilized and the consequent access to remove the copper ions by EDTA. An excellent regain in the original enzyme activity was then observed by treating the immobilized enzyme preparation with known concentration of copper ions (1-20 ppm) soon after the treatment with GdmCI and EDTA. However, immediately after the GdmCI and EDTA treatment, by immersing the apoenzyme electrode in the buffer with out copper ions, there was no regain in the activity of the enzyme. This indicates that the copper ions are essential to regain the activity of enzyme. Another significant observation is that by keeping the enzyme electrode first in the buffer out with copper ions (after GdmCI and EDTA treatment) for relatively longer times (1-5 hours), and then immersing in the buffer containing copper ion, has not shown any regain in the enzyme activity . This can be attributed to the folding of denatured immobilized ascorbic acid oxidase in the absence of copper ions suggests the misfolding of the Ascorbic acid oxidase immobilized with lysozyme after undergoing denaturation with GdmCI. Further, even by addition of copper ions, no regain in the activity of enzyme can be attributed to the process of misfolding of the immobilized Ascorbic acid oxidase was complete by the time of copper ion addition. At this moment, contacting for 30 - 60 minutes with 0.1- 4 M GdmCI leads to unfolding of immobilized Ascorbic acid oxidase and then addition of copper ions leads to reactivation of the enzyme. A sensitivity for copper ion as low as 2 ppm was possible and a maximum regeneration of the native enzyme activity (85-90 %) was possible effectively with 7 ppm of the copper ion concentration. In an embodiment of the present invention the immobilization of ascorbic acid oxidize is done using lysozyme which is an efficient stabilizer of the enzyme and useful in making immobilized apo-Ascorbic acid oxidase efficiently. In another embodiment of present invention GdmCI and EDTA are used in combination for efficient removal of the copper ions and denaturation of the ascorbic acid oxidase for copper ion detection. Treatment by the chemical denaturant GdmCI in conjunction with EDTA overcomes the difficulties associated in the removal of copper ions from the native ascorbic acid oxidase , which is crucial for the construction of biosensor for copper ions. An effective removal of the copper (II) ions from immobilized ascorbic acid oxidase is not possible by using the EDTA solution alone. In yet another embodiment of present investigation the biosensing of the copper (II) ions has been made possible by using immobilized apoAscorbic acid oxidase . Following is the general scheme of copper analysis using biosensor device Immobilization of ascorbic acid oxidase using lysozyme Substrate response checked for enzyme activity Treating immobilized enzyme with GdmCI and EDTA Apo Ascorbic acid oxidase was prepared Substrate response checked for enzyme activity ( Should give No response) Sample containing Cu(ll) added Substrate response checked Finally Copper ion concentration was determined The following examples are given by way of illustration of the present invention only and should not be contrued to limit the scope of invention. EXAMPLE 1: 10 units of Ascorbic acid oxidase from Cucumber sp., (E.C.1.10.3.3) was used for immobilization using glutaraldehyde cross linking method by adding 100 |j,l of the phosphate buffer pH 5.6 (10 jjJ corresponds to 10 units of Ascorbic acid oxidase ). For immobilization , Ascorbic acid oxidase (10^1) and lysozyme (20 jJ of 10% solution ) and 5% of glutaraldehyde solution were placed on a cellophane membrane and mixed thoroughly by using a thin glass rod. The mixing enables a uniform distribution of the enzyme, the stabilizing agent and the glutaraldehyde throughout the enzyme membrane. This membrane was allowed to dry for an hour and then washed repeatedly with phosphate buffer solution pH 5.6 to remove the excess of glutaraldehyde. Thickness of the immobilized ascorbic acid oxidase incorporating lysozyme along with cellophane was 130 ^. The enzyme membrane was sandwiched between a teflon membrane (towards electrode) and cellophane membrane (towards analyte solution) and placed on the Clark dissolved oxygen electrode with the help of an 'O' ring. The details of construction of cathodic amperometry ( Figure 1) based detector system has been reported elsewhere by Gouda, M.D. , Thakur, M.S. and Karanth, N.G., Stability studies on immobilized glucose oxidase using an amperometric biosensor-effect of protein based stabilizing agent. Electroanalysis 2001,13,849-855. Initially immobilized enzyme activity was measured by first allowing 1.5 ml of phosphate buffer (20 mM) at pH 5.6 to be saturated with oxygen using aquarium pump and then 7.41, 4.56, 2.85, 2.28, 1.98, 1.71, 1.14 and 0.57 mM substrate (50}J) was injected. The electrode response was measured in terms of voltage decrease against time from the time of injection. The reading was observed every 30 seconds until steady state was reached in five minutes. The difference between the zero and the fifth minute reading corresponds to the catalytic activity of the enzyme. Voltage response was 2.5, 1.9, 1.8, 1.5, 1.2, 0.6, 0.5 and 0.2 volts respectively. Example 2: (Figure Removed)In order to remove the copper ions from Ascorbic acid oxidase (native enzyme) to make the Apo- Ascorbic acid oxidase the following procedure was followed. The native enzyme activity 'a1 (2.5 volts which is 100% activity) was measured using the freshly immobilized Ascorbic acid oxidase membrane containing electrode, by injecting 3% substrate (50 |al) in 1.5 ml of phosphate buffer (20 mM) pH 5.6, at 25 ± 1°C. The enzyme electrode was then immersed in 1.5 ml of the same phosphate buffer but containing 4 M GdmCI and 3 mM EDTA for 30 minutes ( which is sufficient to remove the copper ions from the ascorbic acid oxidase It was found that this Cu(ll)" less enzyme did not respond for ascorbic acid. The residual enzyme activity 'b' of the immobilized enzyme membrane was measured (0 voltage which is 0% activity of enzyme). Further by again injecting 3% substrate (50 jJ). In order to confirm the Apo- Ascorbic acid oxidase preparation, the enzyme electrode was washed repeatedly with buffer to check the regain in the enzyme activity in the absence of GdmCI and EDTA. After confirming that no regain in the enzyme activity, the apoenzyme membrane based electrode was ready for use. Example 3: While estimating the copper ions in the solution, soon after GdmCI and EDTA treatment the enzyme electrode was washed with phosphate buffer pH 5.6 and immersed in 1.5 ml of buffer (20 mM) containing known concentration of the copper ions for 40 minutes and the enzyme activity 'c (2.0 voltage) ' was measured. The % regeneration of activity was calculated as (c-b/a) x 100, where 'a' is the initial activity, 'b' is the residual activity after GdmCI and EDTA treatment and 'c' is the regained activity. Repeated inactivation and reactivation (by adding higher copper ion concentration 20 ppm) was done to check the efficiency of the method developed in this study for copper ion estimation. Example 4: A calibration curve was prepared by subsequent addition of the 2, 3,4,5,6 and 7 ppm Cu (II) ions concentration of the copper ions to the apoenzyme electrode. The % regain in the activity was 12, 30, 52, 65, 70 and 80 respectively. With this biosensor it was possible estimate 2-7ppm cu(ll) concentration in the solution. We claim : 1. A process for the preparation of Apo-ascrobic acid oxidase immobilized membrane electrode for the determination of copper ions, which comprises: a) charcacterized in that mixing the 10 µI of ascorbic acid oxidase in 20µl of 10% solution of lysozyme and 5 % glutaraldehyde to obtain the mixture, immobilizing the said mixture on a membrane cellophane, b) allowing the said enzyme immobilized membrane to dry for a period of 1-2 hours, c) washing the above said enzyme membrane with phosphate buffer at a pH of 5.6 to remove the excess of glutaraldehyde, d) sandwiching the above said enzyme membrane between a Teflon towards electrode and cellophane membrane towards analytic solution and placing it on the Clark dissolved oxygen electrode to obtain the electrode assembly, e) immersing the above said electrode assembly in a buffer solution containing guanidine chloride and Ethylene diamine tetra acetic acid to prepare the desired Apo-ascrobic acid oxidase immobilized membrane electrode.

Documents:

363-del-2002-abstract-(13-01-2009).pdf

363-DEL-2002-Abstract-(28-11-2008).pdf

363-del-2002-abstract.pdf

363-del-2002-claims-(13-01-2009).pdf

363-DEL-2002-Claims-(28-11-2008).pdf

363-del-2002-claims.pdf

363-del-2002-complete specification (granted).pdf

363-del-2002-correspondence-others-(13-01-2009).pdf

363-DEL-2002-Correspondence-Others-(28-11-2008).pdf

363-del-2002-correspondence-po.pdf

363-DEL-2002-Description (Complete)-(28-11-2008).pdf

363-del-2002-description (complete)-13-01-2009.pdf

363-del-2002-description (complete).pdf

363-DEL-2002-Drawings-(28-11-2008).pdf

363-del-2002-form-1-(13-01-2009).pdf

363-del-2002-form-1.pdf

363-del-2002-form-18.pdf

363-del-2002-form-19.pdf

363-del-2002-form-2-(13-01-2009).pdf

363-DEL-2002-Form-2-(28-11-2008).pdf

363-del-2002-form-2.pdf

363-DEL-2002-Form-3-(28-11-2008).pdf

363-del-2002-form-3.pdf