Title of Invention

A BIOSENSOR DEVICE USEFUL FOR THE MEASUREMENT OF ORGANIC ACIDS AND THEIR DERIVATIVES

Abstract

The present invention provides a biosensor device useful for the measurement of organic acids and their derivatives which comprises of a conventional dissolved oxygen electrode, a sensor element consisting of conventional bio-compatible membrane, immobilized with corresponding enzymes on membrane and the said enzyme being capable of utilizing organic acids and their derivatives having carbon number C3 to C6, firmly held on the sensing element of the said dissolved oxygen electrode using o ring conventional signal conditioning circuitry, detector and display system.

Full Text

This invention relates to a device useful for the measurement of organic acids and their derivatives. This invention particularly relates to an amperonietric device for the measurement of lactate, ascorbate and oxalate and more particularly L-lactate. Various organic and inorganic acids are present in the food and influence quality, flavour and the possibility of microbial growth. These compounds are determined by various chemical, chromatographic or biochemical methods. L- glutamate is a well known taste component that is widespread in food and is also used as a seasoning or flavour enhancer in food products. It is a potent neuroexcitatory amino acid associated with certain behaviour pattern such as aggressive behavior, retarded learning, and retrograde amnesia. Determination of this is very essential for appropriate quality and flavour of the food product as well as from health considerations. Ascorbic acid is also a very important component of food flavours and additives and its analysis is. very essential for quality of foods. Also ascorbic acid is an essential vitamin and commonly known as vitamin "C" and is widely used for therapeutic applications. Lactic acid (2-hydroxy propionic acid) is an organic hydroxy acid whose occurrence in nature is widespread. It has the molecular formula (Formula Removed) and occurs as L- and D - optical isomers. Although both these forms are known to exist in biological systems, mostly it is the L-form that is more important. The presence of L-lactic acid in food is taken as an index of spoilage, contamination by microorganisms and also the quality of food in general. Similarly in clinical medicine, the presence of lactic acid is associated with diseases. Therefore efficient and rapid analysis of L-lactate in food, fermentation and clinical samples is of great importance. Conventional methods of L- lactate determination including spectrophotometric (Lowrence, A (1975), Aust.J.Dairy Technol. 30 14-15; Barber S.B. and Summerson, W.H(1941), J.Biol . Chem. 38 535-554 ) , thin layer chromatography (Tanner, H., and Sandoze, M. (1972) Scweinzerische- zeitschrift- ferer-obst-und weinban, 108.182-186) , gas liquid chromatography ( Nassos, P., Schade, J.E., King, A.D. and Stanford, AE, (1984). J. Food Sci. 49: 671-674) and High Performance Liquid Chromatography (Change, P., Mueller, RE, Jaguar, S., Bajpai, R. and Donatti, E.L. 1991, J. Ind. Microbial. 7 27-34) are nonspecific, time consuming, laborious, expansive and require skilled technical personnel. Spectrophotometric methods, which give total lactic acid, are based on converting lactic acid chemically into acetaldehyde and then measuring the optical density. A serious disadvantage in this is that biological samples contains sugars and carbohydrates which get caramelized on contact with the reagents and thus interfere in the estimation of lactic acid. Enzyme based biosensors for determination of lactic acid in milk and other fermented milk product have been reported ( Macini M., Moscone, D and Pallesche, G.(1984) Anal.Chim, Acta 157 45-51; Moolchandani A., Bassi A., and Nguyen ,A (1995), J. Food Sci 60 (1), 74-77). However, these are based on Lactate Oxidase (LOD) where the possibility of interference from other oxidizing and reducing substances prominently exist. Enzyme electrodes for lactic acid based on Lactic Dehydrogenase (LDH) have also been reported but have reduced stability due to fouling of electrodes because of oxidation of products of reduced Nicotinamide Adenine Dinucleotide (NADH) or mediators (Schaller , F.W., Pfeiffer,D., Schubert, F., Renneberg, R. And Kirsten, D.(1987) in: Biosensors, Fundamentals and Applications, APF Turner, I. Karube, and G, Wilson Eds., Oxford University Press, pp 315-346. Further, they have a high cost due to the requirement of NAD (Nicotinamide Adenine Dinucleotitde), an expensive and unstable cosubstrate (Kulys J., Wang L. and Macsimoviens A (1993), Anal.Chim Acta 274, 53 - 58). LOD based biosensors have the disadvantages of enzyme inhibition of Hydrogen Peroxide (H2O2)and also nonspecific electro-oxidation of compounds such as ascorbic acid, uric acid, glutathione etc., at the relatively high potential required for electrochemical oxidation of H2O2. The presence of reducing substances such as ascorbic acid pose a problem in the detection for H2O2 and these interfere in the analysis. In the present device, the enzyme Lactate Mono Oxygenase (LMO) (EC 1,13,12,4) which converts lactate to acetate, carbon dioxide and water without the production of hydrogen peroxide has been used, thus overcoming the problems with LDH and LOD based biosensors outlined above. Since there is no production or detection of H2O2 involved , there is neither the inactivation of the enzyme nor interference from compounds like ascorbic acid. LMO neither requires the cosubstrate NADH nor produces H2O2. There are no reports so far on LMO based biosensor for L-lactate determination and this is a new development. The principle in which this device is developed is based on the electrochemical changes brought about by the biochemical reactions catalysed by the enzyme LMO in the vicinity of the sensing elements of a Clark electrode, resulting in a changing currents, which, when suitably conditioned and amplified and detected through a signal handling system, gives a voltage which is proportional to the L-lactate contents in the sample in which the biosensor element is placed. The biochemical reaction taking place is (Formula Removed) The L-lactate in the sample is oxidized to acetate catalysed by LMO, resulting in the consumption of oxygen. This oxygen consumption, or an applied potential of (-)650mV is accompanied by acceptance of electrons resulting in the following cathodic amperometric reaction. (Formula Removed) The consumption of electrons decreases the current and this is related to the concentration of L-lactate in the sample in a quantitative manner. The main objective of the present invention is to provide a device for the measurement of organic acids and their derivatives which obviates the drawbacks described above. Another objective of the present invention is to obtain a device for the quantitative measurement of organic acids and their derivatives based on immobilized enzymes. Still another objective of the invention is to have a device as described above wherein accurate measurement of L-lactate can be carried out in the presence of sugar and carbohydrates. Yet another objective of the invention is to have a device as described above wherein the analysis is rapid, the average time taken for analysis being less than 5 minutes. Yet another objective of the invention is to have a device as described above wherein the sample for analysis can be used directly or with minimal pretreatment. Yet another objective of the invention is to have a device as described above wherein the stable operational life of the enzyme sensing element of the device is over 2 months. Figure 1 of the drawing accompanying the specification illustrates the configuration of the device. Figure 2 of the drawing accompanying the specification illustrates a schematic diagram of the device of the present invention. Accordingly, the present invention provides a biosensor device useful for the measurement of organic acids and their derivatives which comprises of a conventional dissolved oxygen electrode, a sensor element consisting of conventional bio-compatible membrane, immobilized with corresponding enzymes such as herein described on membrane and the said enzyme being capable of utilizing organic acids and their derivatives having carbon number C3 to C6, firmly held on the sensing element of the said dissolved oxygen electrode using o ring conventional signal conditioning circuitry, detector and display system. In an embodiments of the present invention, the organic acids and their derivatives to be measured may be such as lactate, ascorbate, glutamate, oxalate and their respective acids. In another embodiment of the present invention, corresponding enzymes capable of utilizing organic acids and their derivatives may be such as lactate monooxygenase, ascorbic acid (ascorbate) oxidase, L-amino acid oxidase, glutamate oxidase. In still another embodiment of the present invention, the biocompatible membranes used for immobilization of enzymes may be such as cellophane, nylon, teflon, cellulose acetate, polyethylene and more particularly such as cellophane and teflon. Details of the device are given below: The biosensor device consists of a sensor element (Figure 1, 1 and 2) made up a set of two membranes (No. 3 and 5) immobilized with corresponding enzymes capable of utilizing organic acids and their derivatives to be measured and a Clark dissolved oxygen electrode, conventional signal conditioning, amplification, detector and display system (Fig 2, 6,7,8,9,10). The sensor element is the heart of the device consisting of a cellophane membrane (5) on which corresponding enzyme is immobilized (4) using gelatin and BSA (Bovin Serum Albumin) and glutaraldehyde crosslinking and a Teflon membrane (3). The teflon membrane used is commercially available, for example, manufactured by M/S Wissenschaftlich- Technische Werkstatten (WTW) GmbH - 812 Weilheim I. OB Germany. The immobilized enzyme membrane system is constructed in the following manner. Fifty to hundred microlitres of melted gelatin solution was placed on a cellophane membrane ( Spectra/Por Membrane MWCO 6,000-8,000, M/S Spectrum Industries, Inc., California, USA) followed by the addition of lmg-2 each of BSA and the enzyme (M/S Sigma, Chemicals, USA). The materials were mixed thoroughly with the help of a glass rod and spread over an area. 50 -60 microlitres of glutaraldehyde solution were added to this mixture, kept for a period upto 30-40 sec and then washed several times with distilled water. The membrane with immobilized enzyme is now ready and stored in a refrigerator at4°Ctill use. The cellophane membrane prepared as above was placed over a teflon membrane and then firmly held on the sensing element of a commercial Clark electrode ( M/S Century Instruments Ltd., Chandigarh ) using an 'O' ring. This dissolved oxygen electrode contains a gold anode and Ag/AgCl cathode (1 and 2). The construction of the immobilized enzyme sensing element is shown in Figure 1. A polarizing potential of -650mV was applied to the Ag/AgCl cathode and kept for polarization in the buffer for 12 hrs. Sample cell; A 25 ml glass beaker was used as a sample cell ( Figure 2,13) with a working volume of 5ml buffer. The liquid was continuously supplied air with a portable air pump ( super 666 aquarium pump, 11 ) to keep the contents thoroughly mixed and also to see that oxygen is not rate limiting. The volume of sample injected (14) was fixed at 100 -200 µl. Signal conditioning, amplification and detector system: Signals obtained from the sensor element, (12) were amplified using OP amplifiers and signal conditioning circuit (6) . The data acquisition and analysis system has an interfacing unit consisting of an analog to digital converter (7) (AD574AJ, Analog Devices,USA) a control system (8), a menu driven software for calibration of the electrode, data acquisition, analysis, display (10) and storage (9). The A/D converter (7) used is a successive approximation type which has an advantage of both high speed and high resolution. The interface system developed is of RS-232 standard (specifications given by the Electronic Industry Association) with the aid of the interfacing system, a microcomputer (9) was used for data acquisition from the enzyme electrode. The total system configuration consisting of sensing element, signal oning, A/D converter, interfacing and display is shown schematically in Figure 2. OPERATION OF THE DEVICE: Measurement; The enzyme electrode kept in the sample cell containing buffer was polarized by applying -650mV at cathode for 30 min. The buffer was now changed and the initial out put voltage was measured. 100 µl of sample was injected and voltage output was monitored till steady state which take atleast 2-3 min. After each measurement, the electrode was washed with distilled water and kept in fresh buffer till next analysis. Calibration of the device was carried out by injecting through a syringe 100 µl samples of known concentrations of respective organic acids and their derivatives. For each analysis , the drop in voltage was measured at 3 minutes by which time steady state is reached. Operational stability The enzyme electrode being always kept in buffer was tested for its response daily, and was found stable for more than 60 days. Effect of interfering substances The response of the device was tested in the presence of several possible interfering substances such as carbohydrates , organic acids, amino acids, heavy metals and enzymes. It was found that these substances did not affect the results of the analysis. Details are given in example No.2. The following examples are given by way of illustration only and therefore should not be construed to limit the scope the invention. Example 1 Calibration with pure L- lactate Calibration of the biosensor device was done by injecting different concentrations of L-lactate ( 50,100,200, 300, 400,500, 600, 700 & 800 mg/dl) as described earlier. The citrate buffer (pH 6.0) volume was kept 5-10 ml, and the initial out put voltage was measured. 100 µ1 of sample was injected and voltage output was monitored till steady state which take atleast 2-3 min. After each measurement, the electrode was washed with distilled water and kept in fresh buffer till next analysis. Calibration of the device was carried out by injecting through a syringe 100µ1 samples of known concentrations of L-lactate in the range of 50,100,200, 300, 400,500, 600, 700 & 800 mg/dl. For each analysis , the drop in voltage was measured at 3 minutes by which time steady state is reached. Drop in voltage at 3 min plotted against L- lactate concentration gave an excellent linearity with a regression value of 0.9973 for 50 - 800 mg/dl. The excellent linear response in terms of voltage drop vs L-lactate concentrations is illustrated in Figure 3. Example 2: Effect of interfering substances on the response of the electrode To 5 ml of the citrate buffer pH 6 taken in the sample cell, potential interfering substances ( carbohydrates, organic acids, amino acids, heavy metals and enzymes) at various concentrations indicated (see Table 1) are injected with and without L-lactate (100 µ 1 of 500 mg/dl cone.) and the voltage response indicated are shown in Table 1. Table 1. Effect of various possible interfering substances on the biosensor performance. (Table Removed) * Drop in voltage V3 is defined as V3 = V0 (initial voltage) - v3 (Voltage at 3 minutes) Example 3 Lactic acid concentration in milk 100 ml of commercially available pasteurized milk was inoculated with 1ml curd containing (Lactic acid bacteria) LAB ( 10110) at room temperature. Samples were withdrawn every 4hr and analysed for L-lactic acid by the biosensor device as well as the conventional spectrophotometric method (Lawrence, 1975). The results compared in Table 2 show excellent agreement. Table 2 : Comparative analysis of L-lactate by using biosensor and spectrophotometric methods during milk fermentation (Table Removed) Example 4; L- lactate concentration in Idli fermentation batter The idli batter was prepared in the traditional way in which rice and urad (Phaceolus mungus) was taken in the ratio of 4:1. After 5-6 hrs. soaking in water both grains were mixed together and thick paste was made using grinder. The mixture was allowed for ferment naturally for 18 hrs. During the natural fermentation there is an increase in acidity and change in flavour. The acidity is mainly due to lactic acid which may affect the quality of the batter. The lactate concentration was with samples drawn regular intervals of time as shown in table 3 by the biosensor device. The lactic acid concentration was simultaneously measured by the spectrophotometric method. The results compared in Table 3 shows excellent agreement. TABLE 3 : Comparative analysis of L-lactate by the Lactate biosensor and spectrophotometric method during Idli batter fermentation . (Table Removed) The main advantages of the present invention are 1. The biosensor device developed for analysis of L-lactate over comes a major disadvantage of chemical conversion to acetaldehyde and spectrophotometric measurement which fails in case of biological samples containing sugars and carbohydrates. 2. The method is rapid, requiring an average less than 5 minutes per analysis. 3. The method requires practically no pretreatment such as centrifugation, colour removal, precipitation of the sample. At most filtration may be required to remove suspended solids. 4. The volume of sample requires for analysis is small at 100 (4,1. 5. The method is faster and less expensive than the chiral stationary phase gas chromatography and chiral ligand exchange High performance Liquid Chromatography (HPLC). 6. The biosensor device developed here has a stable operating life of over 2 months at room temperature (28± 2°c). 7. The device is capable of analysing L-lactate and similar organic acids and their derivatives, in food and fermentation samples. It will be usable for clinical applications also. We Claim: 1. A biosensor device useful for the measurement of organic acids and their derivatives which comprises of a conventional dissolved oxygen electrode, a sensor element consisting of conventional bio-compatible membrane, immobilized with corresponding enzymes such as herein described on membrane and the said enzyme being capable of utilizing organic acids and their derivatives having carbon number C3 to C6, firmly held on the sensing element of the said dissolved oxygen electrode using O ring conventional signal conditioning circuitry, detector and display system. 2. A biosensor device as claimed in claim 1, wherein the corresponding enzymes used for immobilization are such as lactate monooxygenase, ascorbic acid oxidase, L-amino acid oxidase and glutamate oxidase. 3. A biosensor device as claimed in claim 1, wherein the biocompatible membranes used for immobilization of enzyme are such as cellophane, nylon, teflon, cellulose acetate, polyethylene and more particularly cellophane and teflon. 4. A biosensor device for the measurement of organic acids and their derivatives, substantially herein described with reference to the figures accompanying this specification.

Documents:

2159-del-1998-abstract.pdf

2159-del-1998-claims.pdf

2159-del-1998-correspondence-others.pdf

2159-del-1998-correspondence-po.pdf

2159-del-1998-description (complete).pdf

2159-del-1998-drawings.pdf

2159-del-1998-form-1.pdf

2159-del-1998-form-19.pdf

2159-del-1998-form-2.pdf