Title of Invention

A DEVICE FOR THE QUANTITATIVE ESTIMATION OF CREATININE

Abstract

A device for the quantitative estimation of creatinine, characterised by combining an inert metal plate/foil bottomed (2) top open non-reactive body (1) bienzyme (3) creatinine reactor, the said reactor comprising of immobilized creatinase/creatine amidinohydrolase enzyme and creatininase/ creatinine amidohydrolase enzyme with a protective organically modified sol-gel layer (12) and a solid-state urea bio- sensor (4, 5, 7, 9, 10, 11, 12) comprising of a non-reactive sensor body (4) having a noble metal plate / foil (9) in contact with modified polyaniline electroactive pH sensitive polymer film (10) coated electrode having immobilized urease enzyme (11) with a protective organically modified sol-gel layer (12), the said noble metal plate/foil (9) being provided with an electrical output connection (7) passing through a stand (6) mounted hollow pipe (5) fixed on top of the said sensor body (4), the said combination of bienzyme reactor and biosensor being provided with a reference electrode (13), the said biosensor electrical output (7) and reference electrode (13) being connected to a potential measuring device(8).

Full Text

The present invention relates to a device for the quantitative estimation of creatinine. The present invention particularly relates to a device essentially consisting in combination a novel creatinine reactor and urea bio-sensor, for the quantitative estimation of creatinine. The present invention more particularly relates to a device for the quantitative estimation of creatinine having bienzyme reactor with two immobilized enzymes and a urea bio-sensor for potentiometric estimation of urea. Creatinine has become an important clinical analyte that is used for the determination of renal glomerular filtration rate and kidney dys-functioning and muscle disorder. Reference may be made to (1) K. Spencer, Am. Clin. Biochem., 23 (1986) 1; (2) S. Narayana and H.D. Appleton, Clin. Chem., 26 (1986) 1119. Creatinine is a byproduct of amino-acid metabolism and is the energy source for muscle tissue. The reference ranges for serum/plasma crsatinine and urine creatinine are 35 - 140 uM and 71-265 umokT'Kg"1, respectively, bul during disorder or disease condition the creatinine level may rise to > 1000 pM. Reference may be made to (1) N.W. Tietz, Text Book of Clinical Chemistry, Saunders, Philadelphia, 1st Ed., 1986, 1810; and (2) F. S. Sena, D. Syed, R. B. McComb, Gin. Chem., 34 (1958) 994. Therefore, the assessment of creatinine levels in human blood or urine becomes cynically very important, and it is now the most requested analyte in the clinical laboratory. Spectrophotometric, enzymatic and electrochemical methods are the common methods for the estimation of creatinine. Reference may be made to (1) M. Jaffe, Z. Physiol. Chem., 10 (1886) 391; (2) J. A. Weber and A. P. Van. Zanten, Clin. Chem., 37 (1991) 695; (3) M.T. Joppesen and E. H. Hansen, Anal. Chim. Acta, 214 (1988) 147 and (4) J.K. ;uithony and R. S. Malcolm, Trends in Biotechnology, 18 (2000) 433. 1. Spectrophotometric Method: In most of the clinical laboratories and almost all the commercially available analyzers use Spectrophotometric method based on the Jaffe reaction, in which the active methylene group reacts with alkaline sodium picrate to give red yellow complex. However, this reaction is not specific for creatinine because many substances present in bio-fluids could interfere in this assay e.g. cc-ketoacids, amines or NH4 +, Reference may be made to J. A. Weber and A. P. Van. Zanten, Clin. Chem., 37 (1991) 695 and also the detection level is very poor. 2. Enzymatic Method: Several enzymatic methods (detected Spectrophotometrically) have been reported to increase specificity/selectivity. However, they are not yet widely used in clinical laboratories as they are laborious, costly and time consuming. Moreover, :he low detection limit is another major problem with this method, the reference may be made to (1) T. Konoshita, Y. Hiraga, Chem. Pharm. Bull., 28 (1980) 3501; and (2) N. Per kis and C. M. Wolff, Clin. Chem. 30 (1984) 1792. 3. Electrochemical Methods: Recently bio-sensor systems based on electrochemical methods (amperometric) have been reported which have many advantagi techniques used to analyse creatinine in the clinical laboratories. They reduce t: complexity of routine clinical analysis. These sensors are based on either potent! s over e time, Dmetric or amperometric detection. Each system is characterised by certain advanta disadvantages. Trends in the literature indicate that the amperometric option is bepoming more popular and the first system to be successfully commercialised amperometric device. Reference may be made to, Nova Biomediacl, Rod Germany. Additionally Abbott Inc. has incorporated creatinine amperometric into their i-STAT point of care system. Reference may be made to, T. Mock et.a Biochem., 28 (1995) 187. However, the complicated instrumentation and high co amperometric detectors are one of the major drawback, over potentiometric detect The potentiometric detection is mainly based on detection of ammonia, the product of the enzymatic reaction of creatinine, the reference may be made to W. Matuszewjki, M. Troganowicz, M.E. Meyerhoff et.al., Electroanalysis, 5 (1993) 113. These potentiometric sensors are based on NH/ sensing electrodes, which have the major drawback of inter due to ammonia and several cationic substances. The drawback of these potent sensors is the low detection limit of creatinine in bio-fluids. The main objective of the present invention is to provide a device for the quantitative estimation of creatinine which obviates the drawbacks associated with the other sensors or detectors for creatinine. Another objective of the present invention is to provide a device having in combination a bienzyme reactor and a urea bio-sensor for potentiometric estimation of creatinine converted urea. es and vas an :rmark, etector Clin. tofthe )rs. erence ometnc known Yet another objective of the present invention is to provide a device having a sol stable and reusable bienzyme reactor. earlier nzyme Still another objective of the present invention is to provide a device having a solid-state urea bio-sensor for the indirect detection of creatnine of the order of O.lm M and direct detection of urea of the order of 10 Further objective of the present invention is to provide a cost effective, sensitive, selective and reusable device for the quantitative estimation of creatinine. In the present invention the major drawbacks associated with the methods/sensors/devices for the estimation of creatinine are minimized by using, two specific enzymes immobilized creatinine reactor and urea bio-sensor, thus providing the novel device of the present invention which essentially consists in combination a bi creatinine reactor and an urea bio-sensor. The output of the bio-sensor and a reference electrode being connected to a conventional measuring device such as a pH meter. The present invention describes a device for the quantitative estimation of creatinine based on bienzyme creatinine reactor and a urea bio-sensor. The estimation of the creatinine is based on the three enzymes viz. Creatininase, Creatinase and Urease. The enzymes creatininase and creatinase are immobilised in organically modified sol-gel film of precursor 3-aminopropyltriethoxysilane and 2-(3, 4-epoxycyclohexyl)-ethyltrimethoxysilane at the bottom of a reactor. The reactor is used for the estimation of the creatinine in the biological samples by converting creatinine in urea. Further the urea is estimated with a urea sensor based on urease enzyme, immobilised over a novel solid-state pH electrode. The estimation of the urea is back calculated for the quantification of creatinine in the sample. The sensor is sensitive to detect the urea as low as 10 j^M and creatinine up to 0. 1 mM. In figure 1 of the drawings accompanying this specification is shown a schemati device of the present invention. In the said figure 1 the various numbered parts are: 1 . Non-reactive top open reactor. 2. Inert metal plate / foil (mirror polished). 3. Bienzyme with protective layer of sol-gel film. 4. Non-reactive body of urea bio-sensor. 5. Hollow pipe fitted on top of sensor body. 6. Stand connected to hollow pipe (5) for enabling vertical movement. d-state, of the 7. Electrical connection / output of bio-sensor. 8. Conventional measuring device, such as pH meter. 9. Noble metal plate / foil having electrical connection (7). 10. Polyaniline electroactive pH sensitive polymer film. 11. Urease enzyme. 12. Sol-gel protective layer 13. Reference electrode (Ag/AgCl). The non-reactive top open reactor (1) with noble metal plate/foil (2) at bottom is used to caste a uniform bienzyme film (3) and sol-gel layer (12) is used to protect the enzymes from air oxidation and deactivation. A solid -state potentiometric urea bio-sensor (4,5,7,9,10,11,12) based on novel solid-state pH electrode and urease enzyme (11) is used to detect the urea formed on enzymatic reaction of creatinine with bienzymes cast at the bottom of the reactor. The urea bio-sensor is a solid-state pH electrode having urease enzyme (II) and sol-gel (12) layer over polyaniline electroactive pH sensitive film (10). The potential response of the urea bio-sensor is measured with respect to a standard reference el ectrode (13) such as Ag/AgCl electrode, calomel electrode, using a potential measuring device (8) such as a conventional pH meter. The creatinine reactor of the present invention is developed using two cylindrical p Teflon (diameter 1.25 inch, length 1.5 inch and 1.0 inch) having a well (internal diameter J/2 inch and depth Vi inch) in one top Teflon cylinder. Both the pieces of Teflon cylinders are connectable together with a screw arrangement. Before connecting both the Teflon cylinders a noble metal plate/foil disk (mirror polished) of diameter greater than Vi inch is placed on the screwed rod of the second piece of Teflon cylinder and screwed in a way to get the metal plate at the bottom of the top cylinder. The well of the reactor having metallic bottom is used to cast the mixture of two ejnzymes of high activity i.e. creatinase/creatine amidinohydrolase 1- 50 mg and creatininase/cr amidohydrolase 1-50 mg with a protective layer of organically modified sol-gel precursor 3-aminopropyle triethoxy silane (70 jxl) and 2-(3,4-epoxy cyclo hexyl)-ethyl trimethoxy silane (20 (il) in 600 \a\ of polyethylene glycol and 5 \i\ of 0.1 M H enzymes in sol-gel layer are allowed to dry to get a solid layer and the well is perform the reaction with samples containing creatinine. The two enzymes presen bottom layer convert the creatinine present in the sample to urea. Further the ure eces of atmme ayer of 1 The used to in the sample is estimated by a novel urea bio-sensor and creatinine is calculated in terms of urea detected. The urea bio-sensor of the present invention is developed based on solid-state pH electrode and urease enzyme. The pH electrode is developed based on a neutral (dedoped) polyaniline polymer coated electrochemically using non-aqueous electrolyte of acetonitrile containing aniline monomer and tetraphenyl borate over the noble metal electrode. Further the aqueous solution of urease enzyme (1-50 mg/ml) is immobilized over the polyaniline layer and protected with the sol-gel layer of precursor 3-aminopropyle triethoxy silane (70 JU.1) and 2-(3,4-epoxy cyclo hexyl)-ethyl trimethoxy silane (20 jul) in 600 ul of polyethylene glycol and 5ulof0.1 MHC1. Accordingly, the present invention provides a device for the quantitative estimation of creatinine, characterised by combining an inert metal plate/foil bottomed (2) top open non-reactive body (1) bienzyme (3) creatinine reactor, the said reactor comprising of immobilized creatinase/creatine amidinohydrolase enzyme and creatininase/ creatinine amidohydrolase enzyme with a protective organically modified sol-gel layer (12) and a solid-state urea bio¬sensor (4, 5, 7, 9, 10, 11, 12) comprising of a non-reactive sensor body (4) having a noble metal plate / foil (9) in contact with modified polyaniline electroactive pH sensitive polymer film (10) coated electrode having immobilized urease enzyme (11) with a protective organically modified sol-gel layer (12), the said noble metal plate/foil (9) being provided with an electrical output connection (7) passing through a stand (6) mounted hollow pipe (5) fixed on top of the said sensor body (4), the said combination of bienzyme reactor and biosensor being provided with a reference electrode (13), the said biosensor electrical output (7) and reference electrode (13) being connected to a potential measuring device(8). In an embodiment of the present invention the creatinine reactor is constructed using 1-50 mg creatinase/creatine amidinohydrolase enzyme and 1-50 mg creatininase/creatinine amidohydrolase enzyme over the inert metal plate /foil bottom of the reactor. In another embodiment of the present invention the inert metal bottom of the reactor is a plate /foil of platinum, gold, stainless steel or any inert metal. In yet another embodiment of the present invention the non-reactive reactor body is of material such as Teflon, polyvinyl chloride, any plastic material on which enzymes can be immobilized. 'drolase a noble satmme In still another embodiment of the present invention the organically modified sol-gel layer consists of precursors such as 3-aminopropyltriethoxysilane and 2-(3,4-epoxycyciohexyl)- ethyltrimethoxy silane in polyethylene glycol or any other sol-gel precursor or p alcohol or polyvinyl chloride or any other protective polymer capable of forming solid and stable permeable film. In still yet another embodiment of the piesent invention the urea bio-sensor is constructed using urease enzyme 1-50 mg/ ml over a modified polyaniline electroactive pH sensitive polyrner film coated electrode. In yet another embodiment of the present invention the pH sensitive polymer layer electrochemically or chemically formed polyaniline electroactive polymer doped film through organic or aqueous solvent. In still another embodiment of the present invention the polyaniline electroactive polyrner film is doped with tetraphenylborate, perchlorate or any other soluble organic or inorganic salts In another embodiment of the present invention the reference electrode is such as Ag/AgCl electrode, calomel electrode or any other commercially available reference electrode. In a further embodiment of the present invention the electrical output (7) with respect to the reference electrode (13) being connected to a potential measuring device (8), such as a conventional pH meter. In the present invention the estimation of the creatinine is based on the three enzyijnes viz. Creatininase, Creatinase and Urease as shown below : Creatininase or Creatinine [ O=j NH ] + H2O ^ Creatine -1 = NH creatinine amidohydrolase N CH3 Creatinase Creatine [H2NC(==NH)N(CH3)CH2COOH] + H2O « Sarcosine + Uria - 2 creatine amidinohydrolase lyvinyl used is Urease Urea [NH2CONH2] + H2O + -3 In the reactor the two enzymes creatinase and creatininase immobilized in sol-gel film are cast at the metallic bottom of the reactor. When the sample containing creatinine is placed in the well the first two reactions take place in the reactor and urea formed as a final product as shown above. The urea so formed can be detected by the urea sensor based on polvaniline sensitive pH electrode as shown in the third reaction. The creatinine is nearly conveited into urea after placing the sample in the reactor for a sufficient time (15 minutes). The total volume is made up to 2 ml with tris-HCl buffer (ImM, pH 7.0). The urea is measured by urea bio-sensor along with Ag/AgCl reference electrode. Taking the known amount of creatinine in the reactor for a definite time period and then measuring the urea formed in that time period, a calibration curve can be plotted for creatinine. The pH electrode is tested before construction of the urea bio-sensor by estimat and base. The urea bio-sensor is also calibrated before taking any measurements. The novelty lies in the combination of bienzyme reactor, polvaniline solid-s electrode based urea bio-sensor to function as a device for the estimation of creatn first time a compact arrangement of bienzyme reactor, urea bio-sensor along with reference electrode, in combination, for measurement of creatinine is used as shown in figure-1 of the drawings. The non-obvious inventive steps to achieve the novelty, as stated above, residels in the constructional features of the bienzyme reactor and urea biosensor and combination thereof. The novel pH electrode is first time developed using a polyaniline electroactive conducting polymer synthesised from non-aqueous electrolyte using tetraphenylborate as supporting electrolyte. The urea bio-sensor based on pH electrode is unique, where urease ng acid ate pH ne. The enzyme is immobilised in a sandwich configuration between pH sensitive polyani ine and protective organically sol-gel film. The stability and sensitivity of the bio-sensor enhanced many fold in such type of configuration. The invention is further illustrated with the help of the following examples and tierefore should not be construed to limit the scope of the present invention in any manner whatsoever. Example 1 Development of Urea Bio-Sensor: Polyaniline was potentiostatically coated over Pt-disk electrode (2mm diameter) using non-aqueous electrolyte of acetonitrile containing 0.5 M aniline monomer and 0.5 M tetraphenylborate at 2.0 V vs. Ag/AgCl. Polyaniline coated electrode was washed in acetonitrile and incubated in 0.1 M HC1 to get the pH sensitive electrode. pH sensitivity was tested at pH ranging from 2 to 10 and a linear plot of potential vs. H ion concentration was obtained between 3 to 9 as shown in figure-2 of the drawings accompanying this specification. Electrode was washed thoroughly in tris-HCl buffer (pH 7) and then urease enzyme was immobilized on the polymer film by pouring 50 ^1 of urease aqueous solution (10 rag/ml) in ImM tris-HCl buffer (pH 7) on the polymer film and allowed to dry for 10 hours at 4°C . Finally 50 \jd of sol-gel film was casted from its typical composition as described below: 3-aminopropyltriethoxysilane (70 ul) and 2-(3,4-epoxycyclohexyl)-ethyltrimetho>cysilane (20 ul) in 600 (4.1 of polyethylene glycol and 5 |al of 0.1 M HC1 was cast over th; urease immobilized electrode and dried for 20 hours. Resultant bio-sensor was incubated in ImM trib-HCl buffer for 2 hrs and used for the detection of urea in the solution. Potential response was recorded in potentiometric cell of working volume of 2 ml of ImM tris-HCl buffer (pH 7.0) using bio-sensor electrode with a Ag/AgCl reference electrode (with cell assembly reference electrode/test solution/urea bio-sensor). At the steady-state potential, varying concentrations of the aqueous urea solution were injected into the cell and the new steady-state potential was recorded as shown in figure-3 of the drawings accompanying this specification. A calibration curve was plotted for potential responses vs. urea concentrations. A linear response was observed with the 10 joM detection limit of the urea as siown in figure-4 of the drawings accompanying this specification. Example 2 Estimation of Creatinine using bienzyme reactor and Urea Bio-Sensor: Creatinase/creatine amidinohydrolase 20 mg and creatininase/creatinine amidohydrolase 20 mg, immobilized in organically modified sol-gel film of precursor 3-an,in Dpropyltriethoxysilane (70 jal) and 2-(3,4-epoxycyclohexyl)-ethyltrimethoxysilane (20 |il) in 600 \JLof polyethylene glycol and 5 (4.1 of 0.1 M HC1. 100|j.l above solution was casted in the reactor 9 and allowed to dry for 20 hours at room temperature. A solid layer formed at the bottom of the reactor. A known concentration (0.1 mM) of creatinine sample 600 ^1 was take)n in the sample and allowed to stay for 15 minutes. After 15 minutes 500 ul was taken and injected into the potentiometric cell. Volume was increased to 2 ml with 1 mM tris-HCl (pH=7) and solid-state urea bio-sensor along with Ag/AgCl reference electrode was used to estiijnate the urea as described above in example-1..The potential response was directly plotted against the creatinine concentration. Similarly three higher concentrations of creatinine samples (0.5 mM, 1 mM and 10 mM) were added and the response was plotted as shown in figure-5. A calibration curved for creatinine was plotted against potential response and log concentration, as shown in figure-6. A linear response was obtained with the lowest detection limit of 0. The main advantages of the present invention are: 1. It is easy to make and cost effective device. 2. It is a non-hazardous device for the detection of creatinine. 3. Creatinine can be quantified in biological samples without any interference chemicals or ions. 4. Creatinine can be detected up to 0.1 mM in aqueous samples. mM. f other We claim: 1. A device for the quantitative estimation of creatinine, characterised by combining an inert metal plate/foil bottomed (2) top open non-reactive body (1) bienzyme (3) creatinine reactor, the said reactor comprising of immobilized creatinase/creatine amidinohydrolase enzyme and creatininase/ creatinine amidohydrolase enzyme with a protective organically modified sol-gel layer (12) and a solid-state urea bio- sensor (4, 5, 7, 9, 10, 11, 12) comprising of a non-reactive sensor body (4) having a noble metal plate / foil (9) in contact with modified polyaniline electroactive pH sensitive polymer film (10) coated electrode having immobilized urease enzyme (11) with a protective organically modified sol-gel layer (12), the said noble metal plate/foil (9) being provided with an electrical output connection (7) passing through a stand (6) mounted hollow pipe (5) fixed on top of the said sensor body (4), the said combination of bienzyme reactor and biosensor being provided with a reference electrode (13), the said biosensor electrical output (7) and reference electrode (13) being connected to a potential measuring device(8). 2. A device as claimed in claim 1 , wherein the inert metal bottom of the reactor is a plate / foil of platinum, gold, stainless steel or any inert metal. 3. A device as claimee jn claims 1-2, wherein the non-reactive reactor body is of material, selected from Teflon, polyvinyl chloride, any plastic material. 4. A device as claimed in claims 1-3, wherein the organically modified sol-gel layer comprising of precursors selected from 3-aminopropyltriethoxysilane and 2-(3,4 - epoxycyclohexyl)-ethyltrimethoxy silane in polyethylene glycol or any other sol-gel precursor or polyvinyl alcohol or polyvinyl chloride or any other protective polymer. 5. A device as claimed in claims 1-4, wherein the electrical output (7) with respect to the reference electrode (13) being connected to a potential measuring device (8), selected from pH meter. 6. A device for the estimation of creatinine substantially as herein described with reference to the drawings accompanying this specification.

Documents:

0697-DEL-2002-Abstract-(11-08-2008).pdf

0697-DEL-2002-Correspondence-Others-(11-08-2008).pdf

697-DEL-2002-Abstract-(22-08-2008).pdf

697-del-2002-abstract.pdf

697-del-2002-claims-(12-09-2008).pdf

697-DEL-2002-Claims-(22-08-2008).pdf

697-del-2002-claims.pdf

697-del-2002-complete specification (granded).pdf

697-del-2002-correspondence-others.pdf

697-del-2002-correspondence-po.pdf

697-del-2002-description (complete)-(12-09-2008).pdf

697-DEL-2002-Description (Complete)-(19-08-2008).pdf

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

697-del-2002-drawings.pdf

697-DEL-2002-Form-1-(22-08-2008).pdf

697-del-2002-form-1.pdf

697-del-2002-form-13.pdf

697-del-2002-form-18.pdf

697-del-2002-form-2-(12-09-2008).pdf

697-del-2002-form-2.pdf

697-del-2002-form-3.pdf