Title of Invention

"A PROCESS FOR THE PREPARATION OF A SOLID-STATE BIO-SENSOR FOR UREA"

Abstract

In the present invention a new type of solid-state pH electrode based on a neutral conducting polymer such as poly(3-cyclohexyl thiophene) is used and further this electrode was modified under a new configuration. For the first time a solid-state urea bio-sensor is reported based on a solid-state conducting polymer pH electrode with a sol-gel film using urease enzyme. In the present invention conducting polymer such as poly(3-cyclohexyl thiophene) [PSCHTh] based pH electrode was made urea sensitive by immobilizing urease enzyme over neutral/undoped conducting polymers layer coated metal electrode and then coated with a sol-gel film to get a configuration of type: P3CHTh/Urease/Sol-Gel membrane.

Full Text

The present invention relates to a process for the preparation of a solid-state biosensor for urea, a solid-state bio-sensor for urea prepared thereby and a urea meter made therefrom. Need of detection of urea is important in clinical areas as well as environmental areas such as blood, water, agriculture and also as toxins for synthetic milk (adulteration of milk with urea) and other products. A number of urea sensors based on urease enzyme have been developed. These sensors are mainly based on the detection of C02, NH3 gas or NH4+ / H+ ions. Gas sensors have its own disadvantages while many sensors have been developed based on change in pH (H+) using glass membrane pH electrodes [(1) C. Tran-Minh, G. Brown; Anal. Chem., 47, 1359, 1975 (2) R. Tor and A. Freeman, Anal. Chem., 58, 1042, 1986 (3) P.O. Pandey et al, Indian Journal of Technology, 30, 404, 1992]. Slow response, low sensitivity and stability of the sensors are the major drawbacks associated with urease based electrodes made so far. These drawbacks are mainly due to poor immobilization of the enzyme over glass membrane pH electrode. Apart this glass membrane pH electrodes are also not suitable for such modifications. The main object of the present invention is to provide a process for the preparation of a solid-state urea bio-sensor, which obviates the drawbacks associated with urease based bio-sensors. Another objective of the present invention is to provide a solid-state urea bio-sensor. Yet another objective of the present invention is to provide a urea meter. In the present invention above drawbacks associated with urease based biosensors were minimized by using a new type of solid-state pH electrode based on a neutral/undoped conducting polymer such as poly(3-cyclohexyl thiophene) and further this electrode was modified under a new configuration. For the first time we have reported a solid-state urea bio-sensor based on a solid-state conducting polymer pH electrode with a sol-gel film using urease enzyme. In the present invention conducting polymer such as poly(3-cyclohexyl thiophene) [PSCHTh] based pH electrode was made urea sensitive by immobilising urease enzyme over neutral / undoped conducting polymers layer coated metal electrode and then coated with a sol-gel film to get a configuration of type: P3CHTh/Urease/Sol-Gel membrane. Accordingly, the present invention provides a process for the preparation of a solid-state biosensor for urea which comprises preparing a saturated solution of conducting polymer poly(3-cyclohexyle thiophene) or its copolymers or its derivatives in organic solvent such as herein described, coating the said saturated solution on to a metallic electrode by conventional methods to obtain a solid-state polymer electrode, incubating this electrode in dilute acid selected from HCI, H2SO4, HCIO4, HCIO3 to make it pH sensitive, further impregnating the said pH sensitive polymer electrode with urease enzyme, casting a sol-gel film such as herein described over the urease enzyme impregnated pH sensitive polymer electrode , providing an insulating protective outer cover such as herein described to the resultant electrode , getting the desired solid-state bio-sensor for urea , the said process is characterized in that use of conducting polymer poly(3-cyclohexyle thiophene) or its copolymers or its derivatives for preparation of polymer electrode. In an embodiment of the present invention, the saturated solution may be prepared using organic solvents such as chloroform, tetrahydro furan (THF), dichloromethane (CH2CI2). In another embodiment of the present invention, the metal electrode used may be such as gold (Au), platinum (Pt), titanium (Ti), stainless steel. In yet another embodiment of the present invention, the coating of the metal electrode with saturated solution of poly(3-cyclohexyle thiophene) may be effected using known methods such as brushing, spin coating or simple solvent evaporation method. In still another embodiment of the present invention, the sol-gel film used may be such as consisting of precursors 3-aminopropyle triethoxy silane and 2-(3,4 - epoxy cyclo hexyl) - ethyl trimethoxy silane in polyethylene glycol. In still another embodiment of the present invention, the insulating protective outer covering, may be of polyvinyl chloride, acrylates, Teflon or any commercially available plastic or metals/allows having perforation. Accordingly, the present invention provides a solid-state bio-sensor for urea prepared by the process described above. Accordingly, the present invention provides a urea meter incorporating the solid-state urea bio-sensor as described above, which comprises placing the solid-state urea bio-sensor and a reference electrode in a container capable of holding the fluid, in which urea is to be measured, connecting the out put leads of the said electrode pair through a potential measuring means. In an embodiment of the present invention, the reference electrode used may be such as calomel electrode, Ag/AgCI electrode or any commercially available standard electrode. In another embodiment of the present invention, the means used for measuring the potential difference may be such as multimeter, voltmeter, digital display meter, computer. In the process of the present invention we have used undoped conducting polymer, more specifically poly(3-cyclohexyle thiophene), which we have found to be pH sensitive after incubation in dilute acid for few hours. We have also found that the said conducting polymer may or may not be pH sensitive in its normal form due to its doped sate (having positive charges on the chain and incorporated anions in the matrix) mainly due to repulsion of positively charged polymer chain to H+ ions and also non-availability of sites because of occupied by doped anions. So to make the polymer sensitive for the H+ ions (for pH) it is necessary that it should be in its neutralised/undoped sate and further incubated in dilute acid. To make the pH electrode to sense urea we immobilised the urease enzyme on the pH sensitive polymer after few washings with tris-HCI buffer (pH 7), by pouring a saturated urease solution in tris-HCI buffer (pH 7). In order to protect the urease enzyme a sol-gel film of a typical composition of the precursors 3-aminopropyle triethoxy silane and 2-(3,4-epoxy cyclo hexyl)-ethyl trimethoxy silane in polyethylene glycol was casted over the urease enzyme immobilized pH sensitive polymer and allowed to dry for 20 hours. Further, above electrode was dipped in the tris-HCI buffer (pH 7) for 10 hours to open the channels in the sol-gel film so that urea can move inside the sol-gel film and urease catalysed reaction can be performed at the interface of the urease enzyme immobilized pH sensitive polymer and sol-gel film. Urease acts as a bio-catalyst and decomposes urea in ammonium, carbondioxide gas, water and remains as it is after the reaction. The ammonium ion as a result of interaction with water molecules form NH4OH which causes change in the pH at the interface of the urease enzyme immobilized pH sensitive polymer and sol-gel film, which is detected by the pH sensitive electrode. The change of pH can be related to the amount of urea reacting and hence the total concentration of the urea in the sample. The typical sol-gel film provides the stability to the urease enzyme at the room temperature and hence provides long life of the urea bio-sensor. The novelty of the present invention lies in the use of the conducting polymer such as poly(3-cyclohexyle thiophene) based solid state pH electrode and further modification of this electrode as urea sensor in a new configuration with sol-gel. Urease enzyme was immobilised on the solid-state pH electrode and a sol-gel film [precursors A) 3-aminopropyle triethoxy Silane and B) 2-(3,4-epoxy cycle hexyl)-ethyl trimethoxy silane] was casted over the urease immobilized polymer film. The invention is further illustrated with the help of the following example and therefore should not be construed to limit the scope of the present invention in any manner. Example 1: Neutral poly (3-cyclohexyle thiophene) (PSCHTh) was coated over Pt-disc electrode (2mm diameter) from its saturated solution in chloroform, and dried completely in deccicator. PSCHTh coated Pt-disc electrode was incubated in 0.1 M HCI for 10 hours to get the pH sensitive electrode. pH sensitive polymer electrode was washed properly in tris-HCI buffer (pH 7) and then urease enzyme was immobilized on the polymer film by pouring 50 µlof saturated solution of urease enzyme in 1mM tris-HCI buffer (pH 7) on the polymer film and allowed to dry for 15 hours at 4°C . Finally 50 µl of sol-gel film was casted from its typical composition as described below: 3-aminopropyle triethoxy silane (70 µl) and 2-(3,4-epoxy cyclo hexyl)-ethyl trimethoxy silane (20 µl) in 600 µlof polyethylene glycol and 5 nl of 0.1 M HCI was cast over the urease immobilized electrode and dried for 20 hours. Resultant biosensor was incubated in 1mM tris-HCI buffer for 10 hrs and used for the detection of urea in the solution. Potential response was recorded in stirred cell of working volume of 20 ml of 1mM tris-HCI buffer (pH 7.0) using bio-sensor electrode with a double junction Ag/AgCI reference electrode (with cell assembly Reference electrode/test solution/urea bio-sensor). The electrode potential was monitored with a Keithley 2000 multimeter interface with computer and recorded on computer using Keithley software. At the steady-state potential, three varying concentrations (1mM, 2.5mM and 5mM) of the aqueous urea solution were injected into the cell and the new steady-state potential was recorded. A calibration curve was made by plotting potential responses vs. corresponding urea concentrations. A linear response was observed with the 1 mM detection limit of the urea. Example 2: Detection of the urea in the milk sample was also made in the similar ways as described in the example-1. Urea bio-sensor was made as described in example-1. Potential response was recorded in stirred cell of working volume of 20 ml of 1mM tris-HCI buffer (pH 7.0) using bio-sensor electrode with a double junction Ag/AgCI reference electrode (with cell assembly Reference electrode/test solution/urea bio-sensor). The electrode potential was monitored with a Keithley 2000 multimeter interface with computer and recorded on computer using Keithley software. At the steady-state potential, three varying concentrations (1mM, 2.5mM and 5mM) of the milk sample having urea were injected into the cell and the new steady-state potential was recorded. A calibration curve was made by plotting potential responses vs. corresponding urea concentrations in the milk. A linear response was observed with the 1mM detection limit of the urea. The responses were similar to that of the urea response in the water. The main advantages of the present invention are: 1. Solid-State urea bio-sensor. 2. Simple configuration of the urea sensor electrode. 3. Ease in construction of electrode. 4. Cost effective. 5. Occurrence of constant steady-state potential. 6. 1 mM detection limit for urea. 7. Electrode is stable for a very long time (> 3 months). We Claim: 1. A process for the preparation of a solid-state bio-sensor for urea which comprises preparing a saturated solution of conducting polymer poly(3- cyclohexyle thiophene) or its copolymers or its derivatives in an organic solvent such as herein described, coating the said saturated solution on to a metallic electrode by conventional methods to obtain a solid-state polymer electrode, incubating this electrode in dilute acid selected from HCI, H2SO4, HCIO4, HCIO3 to make it pH sensitive, further impregnating the said pH sensitive polymer electrode with urease enzyme, casting a sol-gel film such as herein described over the urease enzyme impregnated pH sensitive polymer electrode , providing an insulating protective outer cover such as herein described to the resultant electrode , getting the desired solid-state bio-sensor for urea , the said process is characterized in that use of conducting polymer poly(3-cyclohexyle thiophene) or its copolymers or its derivatives for preparation of polymer electrode. 2. A process as claimed in claim 1, wherein the organic solvents used is selected from tetra hydro furan (THF), chloroform, dichloromethane. 3. A process as claimed in claims 1 and 2, wherein the metal electrode used is selected from gold(Au), platinum (Pt), titanium (Ti) and stainless steel. 4. A process as claimed in claims 1 - 3, wherein the coating of the metal electrode with saturated solution of poly(3-cyclohexyle thiophene) is effected by conventional methods selected from brushing, spin coating or simple solvent evaporation method. 5. A process as claimed in claims 1 - 3, wherein the sol-gel film used is selected from a group consisting of precursors 3-aminopropyle triethoxy silane and 2-(3,4- epoxy cyclo hexyl)-ethyl trimethoxy silane in polyethylene glycol. 6. A process as claimed in claims 1 - 5, wherein the insulating protective outer covering is made of polyvinyl chloride, acrylates, Teflon having perforation 8. A process for the preparation of a solid-state bio-sensor for urea substantially as herein described with reference to the examples.

Documents:

1282-del-1999-abstract.pdf

1282-del-1999-claims.pdf

1282-DEL-1999-Correspondence-Others.pdf

1282-del-1999-correspondence-po.pdf

1282-del-1999-description (complete).pdf

1282-del-1999-form-1.pdf

1282-del-1999-form-19.pdf

1282-del-1999-form-2.pdf

1282-del-1999-form-3.pdf