Title of Invention

"ELECTROCHEMICAL SENSOR FOR DETERMINING DOPAMINE LEVELS"

Abstract

An electrochemical sensor for determining glucose or dopamine levels in body fluids, said device comprising a sensor strip such as herein described for producing an output signal corresponding to the level of glucose or dopamine present in the body fluid, a converter such as a current voltage converter configured to receive the output signal from the sensor strip and being connected to an amplifier (A), the said amplifier (A) being connected to an analog multiplier (c), which in turn is connected to an analog to digital convener (n) and a timing circuit (Timer) in parallel, the timing circuit and a first output terminal of the analog to digital converter are connected to a decoder (D), which in turn is connected to a first input of a display device and a second output terminal of the analog to digital converter is connected through a switch to a second input of the display device, characterized in that the sensor strip comprises of four screens of micro-band electrode configuration amongst which (1) first one having the unit micro-band electrode dimension of 32 to 38 mm in length, 5 to 7 mm width with a 1 to 1.5 mm gap in center with a total printing area of 80 X 70 mm to provide 9-12 pairs of exposed screen area; the second one (ii) having unit micro-band electrode dimension of 10 to 12 mm length, 2 to 3 mm width superimposing on one of the conductive silver track of screen(l) in alternate fashion to provide 9 to 12 unit of exposed printing area; the dimension of third one (iii) is similar to that of second one however the exposed printed area is shifted to 3 to 4 mm in either direction superimposing the remaining alternate silver conducting track of screen (i); and the fourth one (iv) having unit micro-band electrode dimension of 28 to 32 mm length, 2.2 to 3.2 mm width to with a exposed area of 8 to 9 mm x 1.8 to 3.2 mm at one end to provide 9 to 12 pairs of total printing area.

Full Text

Field of the invention The present invention relates to a biosensor for measuring dopamine or other biochemicals/metabolites such as dopamine glucose. Particularly, the present invention relates to a strip and a process for making disposable micro-band electrode based disposable test strips for dopamine and a Dopamine biosensor made there from. This particular invention is useful for monitoring dopamine concentration present in biological samples. Background and prior art references Mammals, particularly humans, are highly sensitive to external or internal stimuli which alter neurotransmitter impulse activity that indirectly after the steady-state level of messengers RNA encoding based neurotransmission. The peripheral sympathetic nervous system is relatively well defined and is responsible for neural function. Within the blood brain barrier the steady state of neural transmission is maintained based on the following biochemical recycling of neural transmitter : TH DDC DBH PNMT Tyrosine —> L-Dopa —► Dopamine —► Norepinephrine —►Epinephrine TH-tyrosine hydrolase; DDC-aromatic amino acid decarboxylase; DBH=dopamine-ß-hydrolase; PNMT=phenylethanolamine-N- methyltransferase. The status of brain (physiological) is defined by the steady-state autonomous inter-conversion of above neurotransmitters in response to external or internal stimuli. A long term disorder, in response to a particular stimuli in steady-state autonomous inter-conversion of above neurotransmitters, causes disorder (European Patent, 0514490 Al 921125) in brain physiology (US Patent, 4701407, 1987, European Patent, WO 9858076 Al 19881223, European Patent, WO 9807426 Al 19880226) leading to psychotic problem (US Patent, 4772791, 1988) and manifest diseases like Parkinson's, Schizophrenia (European Patent, WO 9637780 Al 961128), mania etc. Additionally Epinephrine and Norepinephrine is also responsible for cardiac physiology, hence these disorder may cause cardiac arrest. One of the important Neurotransmitter i.e. dopamine play a central role during disorder of neuronal autonomous inter-conversion. When such disorder is physiologically adopted by the human, the concentration of dopamine alter in CSF first and subsequently in out side the blood brain barrier e.g. in blood. Both decrease and increase in dopamine concentration severely affects the status of brain. Drugs like cocaine (US Patent 5853696, 1998); morphine etc is likely to cause such disorder (US Patent, 4712561, 1987) and alter the dopamine concentration and in such cases an increased level of dopamine in cerebro-spinal fluids (CSF) and in blood is expected. Accordingly, a biosensor, suitable for detecting dopamine concentration in a drop of blood/CSF is essentially one of the emergent need not only in India but as a whole world. Efforts have been made to detect dopamine following direct or indirect detection systems are available. For examples indirect detection involves : a) the composition and methods for diagnosing schizophrenia based on modification of the dopamine receptor is reported (US Patent, 5686255, 1997); b) allelic diagnosis of susceptibility to compulative disorder (US Patent, 5550021, 1996); c) cDNA encoding a dopamine transporter (US Patent, 5312734, 1994); d) detection reagent and antibody specific for dopamine releasing protein (US Patent, 5330895, 1994); e) allelic association of the human dopamine receptor gene in compulsive disorder such as alcoholism (US Patent, 5210016, 1993); f) method for detecting dopaminergic diseases using fluorine-18 radiolabeled dopamine receptor ligands (US Patent, 4931270, 1990); g) methods land electrodes (individual or composite) for the detection and measurement of hydrogen peroxideformed by electrolytically oxidizing a phenolic compound (especially phenol itself or dopamine (European Patent, 0672167 Al 950920). The direct detection of dopamine is so far conducted by HPLC system. All the above systems for direct or indirect detection of dopamine has following major drawbacks : a) Some of the methods are highly expensive for analysis (> 10 lac casting the system), b) require technical expertise, c) non-portable, d) relatively much longer time of analysis, e) non-suitability for routine analysis, f) Less feasibility of commercialisation. Several other methods of dopamine detection specifically electrochemical one have been developed at laboratory level. Many authors i.e. Hall et al, (Anal. Chim. Acta, 213 (1988) 113-119); Cosnier & innocent (J. Electroanal. Chem., 328 (1992) 361-366), Cosnier and Labbe (in uses of Immobilized biological compounds; Eds. G.G. Guilbault & M. Mascini, Academic Press, 1993. pp 231-244); Wang et al (Labbe (in uses of Immobilized biological compounds, Eds. G.G. Guilbault & M. Mascini, Academic Press, 1993, pp 231-244); Campanella et al (Talanta, 41(1994)1397-1404); Iwuoha et al (Biosensor & Bioelectronics, 10 (1995)661-667) describe the analysis of catacholamine in organic phase which does not suits for clinical purposes. Scheller et al (Biosensor &Bioelectronics, 10 (1995) 717-722) describe a coupled enzymatic system for sensitive detection of catacholamine in aqueous phase however the system is so complicated that test protocol in one step can not be developed for routine analysis of these catacholamine. In all these cases the amperometric detection of catacholamine was conducted. A potentiometric sensor involving poly (crown ether) has also been reported for the detection of catacholamines and other dihydroxybenzene derivatives (Biosensor & Bioelectronics, 10 (1995)705-715). In all these systems, the selectivity for dopamine is not observed and the system could not be put at commercial scale. Accordingly, lack of disposable strip based electronic digital display system for the real time analysis of dopamine and subsequently a commercial biosensor for dopamine is not available so far. Objects: The main object of the present invention is to provide a detection protocol for biochemical and metabolites. Another object of the invention is to provide a biosensor for measuring dopamine and other biochemicals/metabolites. Another object of the invention relates to a process for making electrode based disposable test strips for dopamine and a Dopamine biosensor made there from. Yet another object of the invention relates to monitoring or measuring dopamine concentration present in biological samples. Still another object of the invention relates to a micro-band electrode based disposable test strips screen printed on a PVC substrate for the use of sensing dopamine in real blood/CSF samples in less than 1 min. Another objective of the present invention is to design and fabricate a digital electronic system compatible with dopamine test strips which can display dopamine concentration in ng/ml. Detailed description of the invention The present invention for the first time reports a device for measuring glucose and/or biochemicais/metabolites present in a body fluid. The invention also relates to a process for making a novel micro-band electrode based test strip highly sensitive to dopamine. The test strip is followed by the development of electronic circuit to display to dopamine level in ng/ml which falls in the clinical concentration range. The test strip in combination with electronic unit display dopamine level in Brief description of the accompanying drawings Fig. 1 : shows the diagrammatic representation as to how to use the strip and the time taken to record reading. Fig. 2 : shows the block diagrammatic representation of the present device. Fig.3 : shows the preferred embodiment of the circutary employed in the present invention. Fig.4: shows four stages [4 (a) to (d)] the diagrams for making microband test strip electrodes. The present invention basically provides a sensor strip for measuring glucose or dopamine levels in blood samples, strip comprising a substrate of desired dimension, test strip having two conductive tracks of silver ink, the total printed area of the silver ink has 9 to 12 pairs of conducting tracks, a layers of silver-silver chloride ink located between the conducting tracks of silver ink, layers of graphite ink are placed on the top of silver tracks, layers of enzyme ink located on the graphite tracks and a mask covers both the metal and enzyme printed tracks. Preferably, the substrate is a PVC substrate of 1mm thickness, and the enzyme ink comprising hydroxyethyl cellulose solution (1-2% w/w), polyethylene glycol (Mol.Wt.4000 to 6000), Horseradish peroxidase/other enzymes e.g. glucose oxidose solution in 0.2 M phosphate buffer (25 to 35 % w/w) and graphite power (73 to 63 & w/w) thoroughly mixed together to form a paste. The sensor strip of the present invention preferably comprising four screens of micro-band electrode configuration amongst which the first one (i) having the unit micro-band electrode dimension of 32 to 38 mm in length, 5 to 7 mm width with a 1 to 1.5 mm gap in center with a total printing area of 80 x 70 mm to provide 9-12 pairs of exposed screen area; the second one (ii) having unit micro-band electrode dimension of 10 to 12 mm length, 2 to 3 mm width superimposing on one of the conductive silver track of screen (I) in alternate fashion to provide 9 to 12 unit of exposed printing area; the dimension of third one (iii) is similar to that of second one however the exposed printed area is shifted to 3 to 4 mm in either direction superimposing the remaining alternate silver conducting track of screen (i); and the fourth one (iv) having unit micro-band electrode dimension of 28 to 32 mm length, 2.2 to 3.2 mm width to with a exposed area of 8 to 9 mm x 1.8 to 3.2 mm at one end to provide 9 to 12 pairs of total printing area. The construction of the strip is shown in Fig.4 wherein 4(a) shows the initial step of printing two conductive tracks, 4(b) shows the next step of printing silver/silver chloride ink, 4(c) clearly shows the printing of graphite ink followed by enzyme ink and finally 4(d) clearly shows the diagram of masking ink leaving reactive surface area. For detecting glucose, the enzyme glucoseoxidose alongwith a mediator i.e. 3 amino propyl hydroxy ferrocene is used. As regards dopamine the enzyme used this horseradish peroxidease and dopamine itself access us a mediator. This enzyme is water soluble and has good biocompatibility and not sensitive to environmental factors such as pH, temperature, good redox electro chemistry under the present test condions. The electronic device employed in the present invention basically comprising of a converter such as a current voltage converter which is connected with an amplifier. The amplifier selected is preferably an instrumentation amplifier which is connected to an analog multiplier. The multiplier in turn is connected to an analog to digital converter. A timing control circuit have been digital gates and counter. The said digital gates are connected to a bus-bar linking analog multiplier and analog to digital converter, whereas, the counter is connected to digital gates as well as to the analog to digital converter. The analog to digital converter at one end connected to a switch and also to a decoder which in turn linked to a display. A power supply generating, +5V, -5V and ground (GND) is also required. Preferably, the power supply may be made from IC 7905 along with Oscillator or using IC 7660, 7905 and 7805. The prefer device includes an inlet for providing input signals to a current voltage converter which is connected to an amplifier. The amplifier is connected to an analog multiplexure and also operationally connected to an analog to digital converter. The analog to digital converter and the analog multiplexure are connected to timer device which is also connected to a decoder. The decoder is connected to LCD display. The analog to digital converter and the LCD display are connected to one and another to a switch. The novelty of the present invention lies in the use of highly sensitive test strip for dopamine and required amplification property of recognizing event to a measurable level of the electronic system. The strip of the present invention has a substrate made of PVC, acrylic, polystreyne, polypropylene, the strip comprising two conductive tracks made of metals, alloys, or graphite which are good conductors of electricity, the two conductive tracks have reaction sites at one end, one of the reaction site is made of enzyme and/or a mediator and the other side is made of silver-silver chloride coating or any other reference electrode, the working site of the reaction site will be coated with graphite, since the enzyme will be deactivated by the silver-silver chloride. Generally, the first coating is only silver on both tracks or any suitable conductive material such as copper, platinum, gold, graphite, etc. The second coating on one conducting track at a reactive site is graphite and the other track is silver-silver chloride. Coated on the graphite part is the enzyme. Further, the two tracks are coated with vinyl ink i.e. PVC ink merely exposing the reaction sites. Finally, the reaction sites of both tracks are covered with Nafion solution if necessary. Accordingly, the present invention provides a process for making micro-band electrode based disposable test strips for dopamine comprising: a) PVC substrate (0.5-1 mm Thick) of desired dimension preferably (100 mm x 100 mm), b) Silver ink to be cured at room temperature, c) Silver and silver chloride inks to be cured at room temperature, d) graphite ink to be cured at room temperature. e) Enzyme ink which comprises of (hydroxyethyl cellulose solution (1-2% w/w) containing 0.5% polyethylene glycol (Mol.Wt.4000 to 6000), Horseradish peroxidase solution in 0.2 M phosphate buffer (25 to 35% w/w) and graphite power (73 to 63 & w/w) thoroughly mixed together to form a paste. f) Four screens of desired micro-band electrode configuration amongst which (i) first one having the unit micro-band electrode dimension of 32 to 38 mm in length, 5 to 7 mm width with a 1 to 1.5 mm gap in center with a total printing area of 80 x 70 mm to provide 9-12 pairs of exposed screen area; the second one (ii) having unit micro-band electrode dimension of 10 to 12 mm length, 2 to 3 mm width superimposing on one of the conductive silver track of screen (1) in alternate fashion to provide 9 to 12 unit of exposed printing area; the dimension of third one (iii) is similar to that of second one however the exposed printed area is shifted to 3 to 4 mm in either direction superimposing the remaining alternate silver conducting track of screen (i); the fourth one (iv) having unit micro-band electrode dimension of 28 to 32 mm at one end to provide 9 to 12 pairs of total printing area, The process for making micro-band electrode based disposable test strips screen printed on a PVC substrate for sensing dopamine in real blood/CSF samples in less than 1 min, said process comprising the steps of: a) screen printing of silver ink on PVC substrate using screen (1) to provide two conductive tracks and total printed area has 9-12 pairs of conducting tracks; bj screen printing of silver-silver chloride ink into alternating one of the conducting track obtained from screen (1) using screen (ii) at one and to provide 9-12 units of silver-silver chloride tracks; c) screen printing of graphite ink into alternating conducting track obtained from screen (1) using screen (iii) at one and to provide 9-12 units of graphite tracks over silver tracks. d) Screen printing of enzyme ink into graphite paste ink printed track using screen (iii) and to provide 9-12 units of enzyme sensitive tracks over graphite tracks; e) Screen printing of white vinyl ink over the above metal/enzyme inks printed tracks to provide masking of undesired portion of the conducting tracks and leaving 6-10 mm unexposed conducting tracks for electrical connections at one end and a oval shaped active exposed area on the other Thich is sensitive to reacting analyte. The first three printing steps are followed by curing the metal inks under UV drier at 40°C to 20 min. while after enzyme printing, the same is cured at 25°C for 12 h in vacuum dessicator. Finally the un-printed area of PVC is removed by cutting and the strip in pair comprising of a silver-silver chloride and enzyme printed active area. In an embodiment of the present invention, the screen printer may be a DEK screen printer model J 1202. Further other important part of the invention is to develop electronic meter for holding the test trip having a desired constant dc voltage source and arrangement for digital signal processing in calibrate/test mode, which comprises of following means : a) A printed circuit board having means for holding 3, 8, 14, 16 and 40 pins ICs, zener diode 3 and half digital LCD display; b) The block diagram of the electronic circuit shown in Fig. 1 wherein the various numerals and their corresponding parts are shown as under : In the block diagram of Fig. 1 in which l=strip, 2=varaible voltage supply, 3=current to voltage converter; 4=amplifier; 5=analog multiplexure; 6=analog to digital converter; 7=timer; 8=decoder; 9=L.C.D. display (3.5 digits). c) The circuit diagram of the electronic display unit is shown in Fig. 2. The electronic circuit has a strip holder, arrangement for variable voltage supply across the strip, a amplifier, a multiplexure, timer, analog to digital converter, decoder and a 3.5 digit L.C.D. display which incorporates Ics 741/OP07/OP27, 7660, 7805, 7905, 8069, LM 385, 4053, 4541, 4030, 7116/ 7106/7136. A 3.75 digital LCD display may also be used for autoranging of the data. In the present circuitry, the input i.e. the strip is connected to a current to voltage converter (CVC) and a variable voltage source, the said CVC is connected to and Analogue to digital converter which in turn is connected to Analogue multiplexure. The Analogue multiplexure is connected to the Analogue digital converter wherein, one connection bypassing the strip and the other one through the strip. The bypassed circuitry carries the multiplexure. The Analogue multi plexure is connected to a timer, the timer in turn is connected to a decoder. The decoder is connected to a Analogue to digital converter, an LCD and the timer. Preferably, the strip is connected to OP 27 amplifier as well as to another amplifier to supply a decide voltage or constant operating potential to the strip. The amplifier OP 27 is connected to a multi pluxere-cum-analogue to digital converter. A timer IC 45-41 is connected to the IC 4053 and IC 7116 which is a decoder or display driver. The display driver is in turn connected to an LCD for supply. The power supply comprising a battery and an IC 7660 (power controller). IC 7660 is connected to two capacitors and then to the ground which are recovered for operating all the IC's, operational amplifiers and LCD. The said IC 7660 in turn is also connected to power regulatory IC's 7905 and 7805. The following examples are given by way of illustration of the present invention and therefore should not be constructed to limit the scope of the present invention. EXAMPLE -1 1) Micro-band electrode based test strip for dopamine : The micro band electrode which also referred as test strip is made using a DEK screen printer (DEK J 1202). For the printing of micro-band electrode on PVC substrate initially four different screen on which nylon cloth having mesh size 170 µl exposed by photography using a positive of desired dimension of the test strips with full printing covered area. The four different screens having the exposed area of; (i) having the unit micro-band dimension of 32 to 38 mm in length, 5 to 7 mm width with a 1 to 1.5 mm gap in center with a total printing area of 80 x 70 mm to provide 9-12 pairs of exposed screen area; the second one (ii) having unit microband dimension of 10 to 12 mm length, 2 to 3 mm width to provide 9 to 12 unit of exposed printing area; the dimension of third one (iii) is similar to that of second one however the exposed printed area is shifted to 3 to 4 mm in either direction of total printing area; the fourth one (iv) having unit micro-band dimension of 28 to 32 mm length, 2.2 to 3.2 mm width to with a exposed area of 8 to 9 mm x 1.8 to 3.2 mm at one end to provide 9 to 12 pairs micro-band electrodes. A PVC substrate of 100 mm x 100 mm is used for printing the different inks. First a silver conductive tracks are printed using screen (I) and silver paste followed by printing of silver-silver chloride track on one of end of conducting tracks (single conducting track of one pair micro-band electrode) which again followed by printing a graphite paste on the other conducting tracks which is not covered by silver-silver chloride printing using screen (ii) On the graphite printed trach enzyme ink is printed using screen (iii) The first three inks (silver, silver chloride and graphite paste are purchased from ELTECKS corporation, Bangalore. The enzyme ink was made with (hydroxyethyl solution (1-2% w/w) containing 0.5% polyethylene glycol (Mol. Wt. 4000 to 6000), Horseradish peroxidase solution in 0.2 M phosphate buffer (25 to 35 % w/w) and graphite power (73 to 63 & w/w) thoroughly mixed together to form a paste. The consecutive three printing silver tracks, silver-silver chloride tracks and graphite paste tracks are followed by curing each printing under UV drier at 40°C for 20 min. the enzyme ink printing is followed by its curing at 25°C in vacuum dessicator. Finally the un-desired printed *=— area is masked by printing a vinyl ink which is commercially available and cured at 25°C for 5h. After drying the printed area, each pair of microband electrode comprising of a silver-silver chloride and enzyme tracks over silver conducting tracks are cut out and stored at 25°C. Electronic unit for holding micro-band electrode and subsequent arrangement of variable voltage source followed by signal processing through IC based LCD display: A printed circuit board (general purpose PCB) which commercially available is used for the fabrication of electronic circuit. All the Ics, resistors, capacitors and LCD as given in block diagram (Fig. 1} and circuit diagram (Fig.2) are fixed on PCB/PTH followed by single printing/double printing of desired components as shown in Fig.2. A strip holder is attached to the PCB to hold the strip with the arrangement for polarizing the test strip at desired voltage (200 mV vs Ag/AgCl). The strip function on the following reaction scheme : HRP(I) + peroxide —► HRP (II) + H2O (1) HRP(II) + dopamine —► HRP (I) + oxidized dopamine ...(2) Oxidized dopamine + e- —► dopamine (3) A small current change as a function of dopamine concentration is generated across the circuit. The HRP and peroxide are in excess so that it does not act as a rate limiting step. The activity of HRP on the strip is monitored to be 50 unit and the peroxide concentration choosed its 5 mM. Under this condition the presence of dopamine at ng/ml level could be monitored. The sensing of dopamine is conducted following the adjustment of gain (multiplexure) on calibrate mode. The micro-band electrode is then placed into the strip holder of the electronic unit. A standard solution of dopamine in 0.1 M phosphate buffer pH 7.0 containing 5 mM hydrogen peroxide is made. 5- 10 µl of the standard solution is placed over the active exposed portion of the test strip and the concentration of dopamine is read after 30 s delay. Advantages 1. The test strip is highly sensitive to dopamine. 2. Detect dopamine level is 30 s. 3. Ng/ml sensitivity of the test strip in combination of digital electronic unit. 4. Inexpensive as compared to any other reported method for dopamine detection. 5. Portable unit for dopamine detection. 6. Real time analysis of dopamine. We Claim: 1. An electrochemical sensor for determining glucose or dopamine levels in body fluids, said device comprising a sensor strip such as herein described for producing an output signal corresponding to the level of glucose or dopamine present in the body fluid, a converter such as a current voltage converter configured to receive the output signal from the sensor strip and being connected to an amplifier (A), the said amplifier (A) being connected to an analog multiplier (c), which in turn is connected to an analog to digital converter (n) and a timing circuit (Timer) in parallel, the timing circuit and a first output terminal of the analog to digital converter are connected to a decoder (D), which in turn is connected to a first input of a display device and a second output terminal of the analog to digital converter is connected through a switch to a second input of the display device, characterized in that the sensor strip comprises of four screens of micro-band electrode configuration amongst which (1) first one having the unit micro-band electrode dimension of 32 to 38 mm in length, 5 to 7 mm width with a 1 to 1.5 mm gap in center with a total printing area of 80 x 70 mm to provide 9-12 pairs of exposed screen area; the second one (ii) having unit micro-band electrode dimension of 10 to 12 mm length, 2 to 3 mm width superimposing on one of the conductive silver track of screen (I) in alternate fashion to provide 9 to 12 unit of exposed printing area; the dimension of third one (iii) is similar to that of second one however the exposed printed area is shifted to 3 to 4 mm in either direction superimposing the remaining alternate silver conducting track of screen (i); and the fourth one (iv) having unit micro-band electrode dimension of 28 to 32 mm length, 2.2 to 3.2 mm width to with a exposed area of 8 to 9 mm x 1.8 to 3.2 mm at one end to provide 9 to 12 pairs of total printing area. 2. An electrochemical sensor as claimed in claim 1, wherein the said current voltage converter is provided a bias voltage of about 200 mV using a variable voltage source (s). 3. An electrochemical sensor as claimed in claim 1, wherein the display device comprises of IC 7116 and a 3.5 digit liquid crystal display (LCD). 4. An electrochemical sensor as claimed in claim 1, wherein the dopamine acts as mediator for Horseradish peroxidase. 5. An electrochemical sensor for sensing glucose or dopamine levels in body fluids such as herein described with reference to the accompanying drawings.

Documents:

923-del-2003-abstract.pdf

923-del-2003-claims.pdf

923-del-2003-complete specification (granted).pdf

923-del-2003-correspondence-others.pdf

923-del-2003-correspondence-po.pdf

923-del-2003-description (complete).pdf

923-del-2003-drawings.pdf

923-del-2003-form-1.pdf

923-del-2003-form-19.pdf

923-del-2003-form-2.pdf

923-del-2003-form-26.pdf

923-del-2003-form-3.pdf

923-del-2003-form-5.pdf