Title of Invention

"BIOSENSOR DEVICE FOR THE DETERMINATION OF CAFFEINE IN FOOD SAMPLES"

Abstract

The development and application of new caffeine detection methods remains an active area of investigation, particularly in food and clinical chemistry. Significant research and development activity has been devoted to preparing compact analytical devices comprising a bioactive sensing element integrated with a suitable transuding system, also known as biosensors, for determination of various inorganic, organic and biological substances. The main advantages of these devices are their specificity, sensitivity and simple preparation, and the fact that no other reagents besides a buffer and a standard are usually required. Keeping all the above factors in view work was done to develop a biosensor for the estimation of caffeine in food and beverage samples.

Full Text

The present invention relates to a biosensor device for the determination of caffeine in food samples. Caffeine (C8H10N4O2) is an alkaloid naturally occurring in coffee and coca beans cola nuts and tea leaves. Caffeine is described as a methylated xanthine alkaloid derivative (1,3-7 Trimethyl xanthine). Coffee and tea are among the most popular drinks across the world and their commercial and social importance so obvious. In mammals ingested caffeine is rapidly absorbed, metabolized and excreted in the urine as methyl Xanthine derivatives. Caffeine has become a ubiquitous drug and is widely distributed in pharmaceutical preparations and beverages. It has a variety of biological effects. It stimulates the central nervous system, shows toxicity when fed excessively and is even mutagenic in-vitro. Because of the relative important role of the caffeine in determining the quality of coffee beverages and in account of its harmful effects, the development of a sensitive, fast and cost effective method for monitoring caffeine is greatly needed. The development and application of new caffeine detection methods remains an active area of investigation, particularly in food and clinical chemistry. Significant research and development activity has been devoted to preparing compact analytical devices comprising a bioactive sensing element integrated with a suitable transuding system, also known as biosensors, for determination of various inorganic, organic and biological substances. The main advantages of these devices are their specificity, sensitivity and simple preparation, and the fact that no other reagents besides a buffer and a standard are usually required. Keeping all the above factors in view work was done to develop a biosensor for the estimation of caffeine in food and beverage samples. Principle of caffeine biosensor: This is an amperometric microbial-based biosensor. As it is well known that oxidoreductase enzymes oxidize the substrate, which involves oxygen uptake, or release, which can be monitored when these enzymatic reactions are brought about in the vicinity of a Clarke electrode as it monitors the increase or depletion of dissolved oxygen. The possible mechanism involved in the degradation of caffeine is supposed to be the oxidation of the substrate by an Oxidase, which by the utilization of oxygen degrades it into theobromine or paraxanthine. Caffeine+O2 Oxidoreductase-» Dimethyl xanthine + H20 Reference may be made to Moriyasu, Saito, K., Nakazato.M., Ishikawa.F., Fujinuma.K., Nishima.T and Tamura,Y.,(1996).Simultaneous determination of caffeine, theobromine and theophylline in food by HPLC. J. Food Hyg. Soc. Japan 37: 14-19, wherein they have reported the high-performance liquid chromatography analysis of caffeine. However this method suffers from the disadvantage of being highly labour intensive, time consuming, requires highly skilled personnel and high cost of analysis involving solvents and costly instrumentation. Reference may be made to Turk, J. C, and Guzin (2002), Derivative Spectrophotometric determination of caffeine in some Beverages, ALPDOGAN, 62: 295 - 302, wherein they have reported the analysis of caffeine by UV-spectrophotometric method. This method suffers from the disadvantage of being non-specific, interference due to other methyl xanthines and aromatic compounds and extensive sample preparation. Reference may be made to Zhao, Y.P. and Lunte, C.E., (1997), Determination of caffeine and its metabolites by micellar electrokinetic capillary electrophoresis, Journal of Chromatography B, 628: 265-274; wherein they had reported the analysis of caffeine by Capillary electrophoresis. This method suffers from the disadvantage of the use of hazardous chemicals, time consuming, labour intensive, requirement of skilled man power and increased cost per analysis. Reference may be made to Statheropoulos, M., Samargadi, E., Tzamtzis, N and Geogakopoulous, C, (1996), Principle component analysis for resolving coeluting substances in gas chromatography, mass spectroscopy doping control analysis. Analytica Chimica Acta, 113: 292-301; wherein they have reported the analysis of caffeine by gas chromatography. This method suffers from the disadvantage of the requirement of extensive sample preparation and derivatization, matrix interferences and the use of highly expensive instrumentation. Reference may be made to Statheropoulos, M., Samargadi, E., Tzamtzis, N and Geogakopoulous, C, (1996), Principle component analysis for resolving coeluting substances in gas chromatography, mass spectroscopy doping control analysis. Analytica Chimica Acta, 113: 292-301; wherein they have reported the analysis of caffeine by mass spectroscopy. This method suffers from the disadvantage of the requirement of extensive sample preparation and derivatization, matrix interferences and the use of highly expensive instrumentation. Reference may be made to Norton, K.L. and Griffith, P.R., (1995), Performance characteristics of real direct deposition superficial fluid chromatography, Fourier Trasnform Infrared Spectrophotometry system, Journal of Chromatography A, 703: 503-522; wherein they had reported the analysis of caffeine by FTIR Spectrophotometry measurement. However, this method suffers from the disadvantage of the requirement of very costly instrumentation, highly skilled technicians and complicated and time-consuming procedures. Reference may be made to Rico, CM., Fenandez, M.D., Gutirrez, A.M., Golovela, L.A., Stein, H.J and Scheller, F (1991), Development of a flow fluoroimmunosensor for the determination of theophylline, Analyst 120: 2589-2591; wherein they had reported the analysis of caffeine by a flow injection immunoassay using a solid phase reactor. However this method also suffers from the disadvantage of the time and cost for monoclonal antibody production and purification, and the need for their manipulation with extreme care. The novelty of the present invention is the estimation of Caffeine in food, fermentation and pharmaceutical samples using simple amperometry based immobilized microbial cells biosensor. The main objective of the present invention is to provide "a biosensor device for the analysis of caffeine in foods, beverages and pharmaceutical samples", which obviates the drawbacks detailed above. In the drawings accompanying the specification, Figure 1 represents the Schematic diagram of the Biosensor for caffeine, and Figure 2 represents the immobilized whole cell based bioelectrode used for construction of Biosensor for caffeine and Figure 3 represents the calibration curve for caffeine using immobilized whole cell based amperometric Biosensor. Another objective of the present invention is to induce microbial cells to utilize caffeine. Still another object of the present invention is to immobilize microbial cells for effective determination of caffeine. Yet another object of the present invention is to prepare an amperometric electrode for integration of the immobilized cell based biological sensing element to the electronic transducer system. Another object of the present invention is to optimize conditions for effective determination of caffeine in solutions. Another object of the present invention is to prepare a calibration curve for caffeine using the biosensor. Yet another objective of the present invention is to analyze caffeine present in solutions by using the biosensor. The novelty of the present invention is to analyze caffeine rapidly without the use of any chemical reagents. Description of the Invention: In order to construct the biosensor {Fig1. (1)} for caffeine, immobilized cells were sandwiched between two membranes {Fig.2: (5,6&7)} and fixed on to the electrode surface {Fig. 2: (4)} by using 'O' ring. Gas permeable membrane ({Fig.2: (5) is used as internal membrane in the sensor element. Since it is permeable only to the gases, electrode poisoning due to electrochemically interfering compounds like metal ions and ascorbic acid and metal chelating agents like citric acid, is avoided during real sample analysis. A 10 ml glass container with 5ml phosphate buffer {Fig. 1:(7)} was used as the sample cell {Fig. 1: (6)}. Air was continuously bubbled through a simple air pump {Fig.1: (2)} to keep the contents mixed as well as oxygen supplied continuously. Initially for the new electrode, the electrode {Fig.2: (2&3)} kept in the sample cell-containing buffer was polarized for 1 hour. Construction of the transducer-amplifier-detector system {Fig.1: (1)} In order to apply the potential to the electrode and to process the signal, which is obtained from the electrode, an amperometric electrode {Fig.1 :(4)} was used. The reduction of oxygen at the cathode gives an output voltage/current, which is proportional to the oxygen concentration in solution, which can be correlated, with analyte concentration. Immobilization of microbial cells was done with several immobilizing agents such as Gelatin, Polyvinyl alcohol, and polyvinyl pyrrollidone. Following steps were carried out for the immobilization of the microbial cell: Microbial cells were harvested and washed several times. Appropriate volume of pellet and immobilizing agent were added to membrane and were mixed with a glass rod to form a fine layer of the cells. The membrane was kept at room temperature for one hour. These membranes were preserved in buffer at 4 ° C for further use. Immobilized cell membrane was secured tightly on the tip of the amperometric probe and bubbled continuously. Then sample was injected {Fig.1: (3)} and the decrease in dissolved oxygen was recorded which was proportional to the concentration of the caffeine in the sample. A linear calibration curve for caffeine using the biosensor was prepared by plotting the concentration of caffeine on the X-axis and the biosensor response in terms of drop in the percentage dissolved oxygen in the reaction on the Y-axis with a good linearity and a regression value of 0.9602. In the drawings accompanying this specification, figure 1 represents the schematic diagram of the biosensor device for caffeine, figure 2 represents the construction of the biosensing element and figure 3 represents the calibration graph for caffeine using the constructed biosensor device. Accordingly the present invention provides a biosensor device for determination of caffeine in food samples comprising: an electrode (4), microbial sensing element (5), sample injection device (3), sample cell(6), air bubbler (2), display unit (1); characterized in that the said electrode (4) is attached with a membrane immobilized with acclimatized microbe Pseudomonas alcaligenes whole cells capable of detecting the caffeine in the range of 0.01 g/L to 10 g/L, wherein the stabilizer used in the said biosensor is in the range of 10 g/L to 100 g/L. In an embodiment of the process, the buffer employed for analysis contains 20-100 gram per litre (g/L) of disodium hydrogen orthophosphate septahydrate and 10-15 g/L of sodium dihydrogen orthophosphate, and the pH of the buffer is in the range of 5.0-9.0. In yet an another embodiment of the process the operational temperature of the biosensor is in the range of 25-40 degree C. The following examples are given by way of illustration of the present invention only and should not be construed to limit the scope of the invention. Example-1 A loop full of actively growing culture of Pseudomonas alcaligenes CFR 1708 is transferred to 100 ml of nutrient broth containing 0.3-g/L caffeine and incubated at 30°C in a rotary shaker at 120 rpm for 24 hrs. 5 ml of the 24 hrs grown pre inoculum is transferred to 100 ml of nutrient broth containing 1g/L caffeine and incubated at 30°C for 48 hours on the rotary shaker. Biomass accumulated for 24 hours is harvested by centrifugation for 20 min in a bench top centrifuge at 16,000-x g at 4° C. This biomass is aseptically transferred into a 500 ml flask containing 100 ml of Caffeine liquid medium containing 0.5-g/L g of caffeine and incubated at 30°C on the rotary shaker for 48 hrs. Example- 2 1gm wet weight of the pellet of above induced biomass is suspended in 10 ml of 0.1 M phosphate buffer. 25 ^l of the above suspension is then pipetted out and carefully layered on a cellophane membrane (molecular weight cut-off = 3000-6000). 10Ojal of the stabilizer i.e. Poly vinyl chloride at a concentration of 10%w/v) was then added and mixed carefully avoiding any air bubbles. The cell containing membrane was then incubated at 4-8°C in a refrigerator for 3 hours. After the incubation, the membrane was gently washed with distilled water and cold phosphate buffer saline for three times. The enzyme membrane was tightly secured to the electrode with a "O" ring. 100 [i\ of standard caffeine solution was then injected and the response was recorded as a drop in the dissolved content of the reaction mixture. Example-3 The preparation of biosensor membrane is carried out as in Example 2. Standard caffeine at a concentration of 0.05% is injected and the response of the biosensor to caffeine is recorded as 9.1% drop in the dissolved oxygen. Example -4 The preparation of biosensor membrane is carried out as in Example 2 and the analysis carried out as in example 3, except that standard caffeine at a concentration of 0.1% is injected and the response of the biosensor to caffeine is recorded as 15.5% drop in the dissolved oxygen. Example -5 The preparation of biosensor membrane is carried out as in Example 1&4, except that standard caffeine at a concentration of 0.2% is injected and the response of the biosensor to caffeine is recorded as 18% drop in the dissolved oxygen. Example -6 The preparation of biosensor membrane is carried out as in Examples 1-5 and 7 except that standard caffeine at a concentration of 0.4% is injected and the response of the biosensor to caffeine is recorded as 24% drop in the dissolved oxygen. Example -7 The preparation of biosensor membrane is carried out as in Examples 1-6 except that standard caffeine at a concentration of 0.6% is injected and the response of the biosensor to caffeine is recorded as 26.2% drop in the dissolved oxygen. Example -8 The preparation of biosensor membrane is carried out as in Examples 1-7 except that standard caffeine at a concentration of 0.8% is injected and the response of the biosensor to caffeine is recorded as 29% drop in the dissolved oxygen. Example-9 The preparation of biosensor membrane is carried out as in Examples 1-8 except that standard caffeine at a concentration of 1.0% is injected and the response of the biosensor to caffeine is recorded as 33% drop in the dissolved oxygen. Example 10 The preparation of biosensor membrane is carried out as in Examples 1-9 except that standard caffeine at a concentration of 2.0% is injected and the response of the biosensor to caffeine is recorded as 49.6% drop in the dissolved oxygen. Example 11 The analysis of standard caffeine samples by the biosensor membrane is carried out as in Examples 4-10 and the response of the biosensor to caffeine is recorded as drop in the dissolved oxygen. A calibration graph for caffeine is plotted with Concentration on the X-Axis and Response (% Dissolved oxygen drop) on the Y-axis with good linearity with a regression value of 0.9602. The main advantages of the present invention are: 1. It employs a simple amperometric probe integrated with membrane with immobilized cells of Pseudomonas alcaligenes CFR 1708. 2. The biosensor device is easy to operate and does not require reagents 3. The device has a fast response time with the analysis completed within 3-5 minutes. 4. It is an economical alternative for the conventional methods of caffeine analysis. 5. The analysis does not require sample preparation. We claim: 1. A biosensor device for determination of caffeine in food samples comprising: an electrode (4), microbial sensing element (5), sample injection device (3), sample cell(6), air bubbler (2), display unit (1); characterized in that the said electrode (4) is attached with a membrane immobilized with acclimatized microbe Pseudomonas alcalipenes whole cells capable of detecting the caffeine in the range of 0.01 g/L to 10 g/L, wherein the stabilizer used in the said biosensor is in the range of 10 g/L to 100 g/L. 2. A process for the detection of caffeine in food samples in combination with the biosensor device as claimed in claim 1, wherein the process steps comprises; i) securing the said immobilized cell membrane to the said electrode, ii) incubating the assembly of step (i) in air saturated 0.1 M to 9M phosphate buffer, iii) dispensing of 0.1-10% w/v of caffeine in the incubating assembly of step (ii), iv) recording the response of the biosensor in terms of decrease in the dissolved O2 in the sample cell for a period ranging from 1-10 minutes. 3. A process as claimed in claim 2, wherein the buffer employed for analysis contains 20-100 gram per litre (g/L) of disodium hydrogen orthophosphate septahydrate and 10-15 g/L of sodium dihydrogen orthophosphate, and the pH of the buffer is in the range of 5.0-9.0. 4. A process as claimed in claim 2, wherein the operational temperature of the biosensor is in the range of 25-40 degree C. 5. A biosensor device for the determination of caffeine in food samples, substantially as herein described with reference to the examples and drawings accompanying this specification.

Documents:

760-del-2005-Abstract-(16-01-201).pdf

760-del-2005-abstract.pdf

760-del-2005-Claims-(12-06-2014).pdf

760-del-2005-Claims-(16-01-201).pdf

760-del-2005-Claims-(27-08-2014).pdf

760-del-2005-claims.pdf

760-del-2005-Correspondence Others-(12-06-2014).pdf

760-del-2005-Correspondence Others-(16-01-201).pdf

760-del-2005-Correspondence Others-(27-08-2014).pdf

760-del-2005-correspondence-others.pdf

760-del-2005-description (complete).pdf

760-del-2005-Drawings-(16-01-201).pdf

760-del-2005-drawings.pdf

760-del-2005-form-1.pdf

760-del-2005-form-18.pdf

760-del-2005-form-2.pdf

760-del-2005-Form-3-(16-01-201).pdf

760-del-2005-form-3.pdf