Process for making biosensor

Abstract

A process for making a biosensor comprising a hollow coil having wires coiled in parallel and an electronic circuit component connected to the coil, the process including: 1) providing a mandrel on which wires including at least a first wire, a second wire and a third wire are wound in parallel, 2a) immersing the mandrel in a first buffer solution comprising a first bioreceptor, a first monomer and optional additives, 2b) arranging the wires such that the first wire may be used as a working electrode, the second wire may be used as a counter electrode and the third wire may be used as a reference electrode of a three electrode electrochemical cell used in an electropolymerization process, 3) passing electric current through the first wire to form a first biocompatible coating of a first polymer polymerized from the first monomer comprising the first bioreceptor on the first wire, 4) removing the coil from the mandrel, 5) connecting the wires to their respective points of the electronic circuit component such that the first wire may be used as a working electrode, the second wire may be used as a counter electrode and the third wire may be used as a reference electrode and wherein the electronic circuit component is configured such that it can generate an input signal for a wireless receiver based upon the activity of the bioreceptor and wirelessly send the input signal to the wireless receiver.

Claims

1. A process for making a biosensor comprising a hollow coil comprising wires coiled in parallel and an electronic circuit component connected to the coil, the process comprising: 1) providing a mandrel on which wires including at least a first wire, a second wire and a third wire are wound in parallel, 2a) immersing the mandrel in a first buffer solution comprising a first bioreceptor and a first monomer, 2b) arranging the wires such that the second wire is used as a working electrode, the first wire is used as a counter electrode and the third wire is used as a reference electrode of a three electrode electrochemical cell used in an electropolymerisation process, 3) passing electric current through the second wire to form a first biocompatible coating of a first polymer polymerized from the first monomer comprising the first bioreceptor on the second wire, 4) removing the coil from the mandrel, 5) connecting the wires to their respective points of the electronic circuit component such that the second wire may be used, as a second working electrode, the first wire is used as a second counter electrode and the third wire is used as a second reference electrode, wherein the electronic circuit component is configured such that the electronic circuit component can generate an input signal for a wireless receiver based upon the activity of the bioreceptor and wirelessly send the input signal to the wireless receiver, wherein the first monomer is a five-membered heterocycle of formula (I) ##STR00003## wherein R.sup.1 stands for a hetero atom, wherein R.sup.2, R.sup.3 are each, independently, selected from the group consisting of: H, an alkyl of 1 to 4 C atoms, an alkyl of 1 to 4 C atoms having a hydroxyl group, an alkyl of 1 to 4 C atoms having an alkyl group, an alkyl of 1 to 4 C atoms having an alkyl ether group, and o-alkyl, and wherein R.sup.2 and R.sup.3 may form a ring together with the carbon atoms to which R.sup.2 and R.sup.3 are connected, and wherein the bioreceptor is an oxidoreductase.

2. The process according to claim 1, wherein the wires include a further wire and the process further comprises the steps of: 2a) immersing the mandrel in a second buffer solution comprising a second bioreceptor and a second monomer, wherein the second bioreceptor is different from the first bioreceptor, 2b) arranging the wires such that the further wire may be used as a third working electrode, the first wire is used as a third counter electrode and the third wire is used as a third reference electrode of a second three electrode electrochemical cell used in an electropolymerisation process, 3) passing electric current through the further wire to form a second biocompatible coating of a second polymer polymerized from the second monomer comprising the second bioreceptor on the further wire and wherein step 5) comprises the step of connecting the further wire to the electronic circuit component such that the further wire may be used as a fourth working electrode.

3. The process according to claim 2, wherein the process further comprises the step of providing a biocompatible resin capping on the electric circuit component after step 5).

4. The process according to claim 3, wherein the wires further include a fourth wire coated with an insulating layer and wherein step 5) comprises the step of connecting the fourth wire to the electronic circuit component such that the fourth wire is used as an antenna.

5. The process according to claim 4, wherein the wires further include a fifth wire coated with a fifth wire insulating layer which is used as a spacer.

6. The process according to claim 5, further comprising the step of cutting the coil into a length of 5-50 mm between steps 4) and 5).

7. The process according to claim 6, further comprising the step of providing a top coating of a biocompatible material on the biosensor after step 5).

8. The process according to claim 7, wherein the sensor has a diameter of 0.1 to 3 mm.

9. The process according to claim 8, wherein the first coiled wire has a Pt surface.

10. The process according to claim 9, wherein the third coiled wire is a silver/silver chloride reference electrode, wherein the bioreceptor is an oxidoreductase of the enzyme commission groups EC 1.X.3 where X=1-17, and wherein the bioreceptor is glucose oxidase, lactate dehydrogenase, pyruvate dehydrogenase or pyruvate oxidase.

11. The process according to claim 1, wherein the process further comprises the step of providing a biocompatible resin capping on the electric circuit component after step 5).

12. The process according to claim 1, wherein the wires further include a fourth wire coated with an insulating layer and wherein step 5) comprises the step of connecting the fourth wire to the electronic circuit component such that the fourth wire is used as an antenna.

13. The process according to claim 12, wherein the wires further include a fifth wire coated with a fifth wire insulating layer which is used as a spacer.

14. The process according to claim 1, further comprising the step of cutting the coil into a length of 5-50 mm between steps 4) and 5).

15. The process according to claim 1, further comprising the step of providing a top coating of a biocompatible material on the biosensor after step 5).

16. The process according to claim 1, wherein the sensor has a diameter of 0.1 to 3 mm.

17. The process according to claim 1, wherein the first coiled wire has a Pt surface.

18. The process according to claim 1, wherein the third coiled wire is a silver/silver chloride reference electrode.

19. The process according to claim 1, wherein the bioreceptor is an oxidoreductase of the enzyme commission groups EC 1.X.3 where X=1-17.

20. The process according to claim 1, wherein the bioreceptor is glucose oxidase, lactate dehydrogenase, pyruvate dehydrogenase or pyruvate oxidase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The invention will be described in detail below referring to the drawings in which:

(2) FIG. 1 schematically illustrates an embodiment of the sensor according to the invention;

(3) FIG. 2 is a schematic diagram of an embodiment of the sensor according to the invention in which the details of the electronic circuit component are shown;

(4) FIG. 3 is a schematic diagram of a further embodiment of the sensor according to the invention in which the details of the electronic circuit component are shown;

(5) FIG. 4 illustrates the working principle of the potentiostat in the electronic circuit component in the sensor according to the present invention.

(6) FIGS. 5-8 illustrate various examples of the configuration of the biosensor according to the present invention.

(7) FIG. 9 illustrates an example of the process according to the invention.

(8) FIGS. 10-13 show various graphs obtained by experiments relating to the invention.

DETAILED DESCRIPTION OF THE INVENTION

(9) FIG. 1 illustrates an embodiment of the sensor 100 according to the present invention. The sensor 100 comprises a hollow coil 10 comprising a first coiled wire 1, a second coiled wire 2, a third coiled wire 3, a fourth coiled wire 4 and a fifth coiled wire 5 coiled in parallel. The hollow coil 10 is connected to an electronic circuit 20. The electronic circuit 20 is attached at the end of the coil 10. The hollow coil 10 in this example has a diameter of 1 mm.

(10) The first coiled wire 1 functions as a counter electrode. The second coiled wire 2 functions as a working electrode. The third coiled wire 3 functions as a reference electrode. The fourth coiled wire 4 functions as an antenna. The fifth coiled wire 5 functions as a spacer.

(11) The hollow coil 10 and the electronic circuit 20 are covered in a continuous top layer 11. The electronic circuit 20 is embedded in a resin layer 21 under the top layer 11.

(12) The first coiled wire 1 is made of a platinum-plated stainless steel and is provided only with the top layer 11.

(13) The second coiled wire 2 is made of a platinum-plated stainless steel and is provided with a polymer layer 2A under the top layer 11. The polymer layer 2A is electron conductive and comprises a bioreceptor 2B, glucose oxidase in this example.

(14) The third coiled wire 3 is a silver plated stainless steel and is coated with a silver chloride layer 3A under the top layer 11.

(15) The fourth coiled wire 4 is made of a stainless steel and is coated with an insulating layer 4A under the top layer 11. The insulating layer 4A is made of e.g. PTFE.

(16) The fifth coiled wire 5 is made of a stainless steel and is coated with an insulating layer 5A under the top layer 11. The insulating layer 5A is made of e.g. PTFE.

(17) A return wire 22 for the antenna 4 extends from the electronic circuit component 2 to a different loop (not shown) of the fourth coiled wire 4, so that a closed loop antenna is formed.

(18) During use, the sensor of this embodiment is placed in the lower eyelid filled with a tear fluid. Glucose in the tear liquid produces H.sub.2O.sub.2 by the catalytic function of glucose oxidase in the polymer layer 2A of the second coiled wire 2.

(19) The sensor operates by an electromagnetic field generated by a transceiver (not shown) placed close to the lower eyelid. The electromagnetic field induces an electric current through the coil. The level of the electric current depends on the level of H.sub.2O.sub.2 which in turn depends on the level of glucose in the tear liquid. The electronic circuit 20 generates a signal indicating the level of glucose and sends it to the external device through coil 4.

(20) FIG. 2 schematically illustrates an embodiment of the sensor according to the invention. The electronic circuit 20 component is illustrated more in detail. In this example, the coil 10 comprises a coiled wire used as an antenna. The electronic circuit component 20 consists of a potentiostat 40, a reference source 80, an A/D converter 50, a microprocessor 60 and a RF transceiver 70. The potentiostat 40 translates the current of the working electrode into a voltage. This voltage is digitized by the A/D converter 50 into counts. The reference source 80 provides necessary bias voltages to the potentiostat 40. The microprocessor 60 controls the processing of the sensor. The counts, i.e. the sensor raw data, are converted into a transmit data packet, for example as described in the Norm ISO 18000-3, by the microprocessor 60. The RF transceiver 70 is wirelessly connected to a reader unit (not shown here) using inductive coupling. The RF transceiver 70 is connected to the antenna coil by a return wire 30. The RF transceiver 70 transmits the data packet containing the sensor raw data to the reader unit using the antenna coil. The sensor is wirelessly powered also using inductive coupling. For data and power transmission the same antenna coil is used.

(21) FIG. 3 schematically illustrates an embodiment of the electronic circuit component of the sensor according to the invention. FIG. 3 is identical to FIG. 2 except for that the antenna. In this example, the coil does not comprise a coiled wire used as an antenna. Instead, the electronic circuit component comprises an antenna for the data and power transmission.

(22) FIG. 4 illustrates the working principle of the potentiostat in the electronic circuit component in the sensor according to the present invention. The potentiostat consists of a differential input amplifier (OpAmp) and a transimpedance amplifier (TIA). The differential input amplifier compares the potential between the working (WE) and reference (RE) electrodes to adjust the required working bias potential. For this purpose, the voltage between the working and the reference electrodes may be amplified and applied to the counter electrode as an error signal. Thus the voltage between working and reference electrodes is maintained to be constant. The transimpedance amplifier is connected to the working electrode and converts the cell current into a voltage (Out). The transimpedance amplifier keeps the potential of the working electrode at virtual ground.

(23) FIGS. 5-8 illustrate various examples of the configuration of the biosensor according to the present invention.

(24) In FIG. 5, the coil consists of four wires coiled in parallel. The wires are connected to the respective points of the electronic circuit component so that they respectively function as: counter electrode 1, working electrode 2, reference electrode 3 and antenna 4.

(25) In FIG. 6, the coil consists of three wires coiled in parallel. The wires are connected to the respective points of the electronic circuit component so that they respectively function as: counter electrode 1, working electrode 2, reference electrode 3. In this embodiment, the wire which functions as a reference electrode also functions as an antenna.

(26) In FIG. 7, the coil consists of five wires coiled in parallel. The wires are connected to the respective points of the electronic circuit component so that they respectively function as: counter electrode 1, working electrode 2, reference electrode 3, antenna 4 and spacer 5.

(27) In FIG. 8, the coil consists of four wires coiled in parallel. The wires are connected to the respective points of the electronic circuit component so that they respectively function as: counter electrode 1, working electrode 2, reference electrode 3 and spacer 5. In this embodiment, the wire which functions as a reference electrode 3 also functions as an antenna.

(28) An example of the process according to the invention is described referring to FIG. 9.

(29) Four wires are wound on a mandrel in parallel, as shown in FIG. 9(a). In this example, three wires are made of a platinum-plated stainless steel and are uncoated. The remaining one wire is a silver plated stainless steel and is coated with a silver chloride layer.

(30) Subsequently, the mandrel with the coiled wires are placed in a phosphate buffered saline (PBS) of EDOT, glucose oxidase and PEG as shown in FIG. 9(b). Two platinum-lated stainless steel wire and one Ag/AgCI wire are connected to an external electronic circuit so that a three electrode electrochemical cell capable of an electropolymerisation process is formed. Electric current is passed through the uncoated platinum-plated stainless steel wire acting as the working electrode. For example, the potential is cycled from 0.3V between 0.2-1.2 V at a scan rate of 0.1 V/s for 30 cycles. EDOT polymerizes at the surface of the wire and forms a coating thereon of PEDOT comprising glucose oxidase. A coil in which only one of the wires is coated with PEDOT comprising glucose oxidase is thus obtained.

(31) The mandrel is taken out of the solution and the excess solution is removed by wiping. The mandrel is then placed in a second buffer solution of EDOT, lactate dehydrogenase and additives. Again, a three electrode electrochemical cell capable of an electropolymerisation process is formed, but using the remaining uncoated Pt wire as the working electrode. Electric current is passed through the working electrode. EDOT polymerizes at the surface of the wire and forms a coating thereon of PEDOT comprising lactate dehydrogenase.

(32) A coil in which one of the wires is coated with PEDOT comprising glucose oxidase and another one of the wires is coated with PEDOT comprising lactate dehydrogenase is thus obtained. The mandrel is taken out of the solution and the excess solution is removed by wiping. The coil of four wires is removed from the mandrel.

(33) The coil is subsequently cut into a number of coils having a suitable length, e.g. 1 cm, as shown in FIG. 9(c). The four wires of the coil of the suitable length are connected to an electric circuit component such that they function as follows:

(34) The wire coated with PEDOT comprising glucose oxidase and the wire coated with PEDOT comprising lactate dehydrogenase: working electrode

(35) The uncoated wire: counter electrode

(36) The silver plated stainless steel with a silver chloride layer: reference electrode

(37) After the connections are made, the electronic circuit component is encapsulated with a biocompatible resin. The other side of the coil is also provided with an end capping of the biocompatible resin. The result is shown in FIG. 9(d).

(38) The assembly of the coil and the electronic circuit component is coated with a polysaccharide. A sensor encapsulated in a polysaccharide hydrogel is thus obtained.

EXAMPLES

Example 1: Preparation of a Pt Working Electrode with an Electroconductive Layer of PEDOT, Without a Top Layer (Non-Parallel Wires)

(39) An (enzyme) working electrode was prepared by dispersing 3,4-ethylenedioxythiophene (EDOT) (10.sup.2 M) in phosphate buffered saline (PBS), GOX (110 U/mL) was added and was allowed to dissolve without agitation.

(40) A three electrode electrochemical system was used whereby a platinum coiled wire (0.01 mm diameter) functions as the working electrode (WE), a platinum coiled wire functions as the counter electrode (CE) and a coiled Ag/AgCl/saturated KCl functions as the reference electrode (RE). The wires were not coiled in parallel, but existed as separate components. The electrodes formed an electrochemical cell for an electropolymerisation process.

(41) The WE was ultrasonically cleaned in ultra-pure water before use. The electrodes were placed in the EDOT/GOx/PBS solution and the potential was cycled between 0.2 and 1.2V/s for 15 cycles. The resulting coated electrode was washed with fresh PBS solution and could be used directly resulting in an immobilized GOx PEDOT matrix onto the platinum wire (Pt/PEDOT/GOx).

Example 2: Sensing Function

(42) A glucose calibration curve was made using the three electrode setup obtained by Example 1.

(43) Glucose PBS solutions from 0.00 to 0.40 mM with steps of 0.05 mM and from 0.50 to 8.00 mM with steps of 0.5 mM were prepared.

(44) Amperometry was performed for the different solutions. One potential step of 600 mV vs Ag/AgCl/saturated KCl was applied and the current was measured for 600 s, while the solution was stirred continuously. The average current and the standard deviation between 60 and 600 seconds was calculated and plotted against the glucose concentration.

(45) It was observed that the current was linearly proportional to the glucose concentration in a range between 0 and 10 mM. The calibration curve produced for the low concentration range (0 to 0.7 mM) is shown in FIG. 10. Symbols represent individual measurement points and the straight line represents the least squares fit to these points. Additional examples demonstrating the detection of glucose at hyperglycemic, physiological and hypoglycemic concentrations are shown in FIG. 11. The region between 0 mM and 3.9 mM represents the hypoglycemic region, the region between 3.9 mM and 5.5 mM represents the normal glucose region and the region above 5.5 mM represents the hyperglycemic region.

Example 3: Preparation of a Pt Working Electrode with an Electroconductive Layer of PEDOT Without a Top Layer (Non-Parallel Wires)

(46) An (enzyme) working electrode was prepared by, dispersing 3,4-ethylenedioxythiophene (EDOT) (0.01M) in phosphate buffered saline (PBS) containing PEG8000 (0.001M). To the EDOT solution was added GOx (5312.7 U) which was allowed to dissolve without agitation.

(47) A three-electrode electrochemical system was used: consisting of coiled working electrode (WE), diameter 1.5 mm, a coiled platinum wire as counter electrode (CE) and a Ag/AgCl/saturated KCl reference electrode (RE). The electrodes formed an electrochemical cell for an electropolymerisation process.

(48) The WE was precleaned by sequential washing in H.sub.2SO.sub.4, ultrapure water and finally in PBS. The electrodes were placed in the EDOT/GOx/PEG/PBS solution and the potential was cycled from 0.3V between 0.2-1.2 Vat a scan rate of 0.1 V/s for 30 cycles.

(49) A coiled platinum wire coated with a conductive coating of PEDOT comprising GOx was thus obtained.

Example 4: Sensing Function

(50) Example 2 was repeated, but the working electrode was replaced by the electrode obtained by example 3. Calibration curves were produced showing that the measured current through the working electrode was substantially proportional to the glucose concentration, as shown in FIG. 12.

Example 5: Parallel Wires

(51) Examples 1-4 are repeated, except that the wires used as the WE, CE and RE are coiled in parallel. No substantial difference is noted in the sensing behavior between the examples wherein the wires not coiled in parallel are used (Examples 1-4) and the examples wherein the wires coiled in parallel are used (Example 5).

Example 6: Parallel Wires with a Top Layer

(52) Examples 1-4 are repeated, except that the wires used as the WE, CE and RE are coiled in parallel and the coil is dip coated with a solution of Nafion (tetrafluoroethylene-perfluoro-3,6-dioxa-4-methyl-7-octenesulfonic acid copolymer) (10 wt % in water) and allowed to dry at room temperature. A coil encapsulated in Nafion is obtained. No substantial difference is noted in the sensing behavior between Examples 5 and 6.

Example 7: Preparation of a Pt Working Electrode with an Electroconductive Layer of PEDOT (Parallel Wires)

(53) A wire of platinum plated stainless steel, diameter 0.152 mm, may be coated with a copolymer of BMA and NVP. Another wire of platinum plated stainless steel, diameter 0.152 mm, may be coated electrochemically with PEDOT/GOx which again may be coated with the copolymer of BMA and NVP. A silver plated stainless steel wire, diameter 0.152 mm, may be coated with a silverchloride layer which may also be coated with the copolymer. These three wires may be coiled in parallel around a mandrel. The formed coil then have an outer diameter of 0.87 mm. After removal of the mandrel the coil may be cut into pieces of 1 cm in length. One of the ends of the coil may be closed with a drop of UV-curable polymer. The three wires at the other end of the coil may then be connected to an electronic circuit.

(54) Calibration curves are produced according to the procedure similar to Example 2. The measured current through the working electrode is substantially proportional to the glucose concentration.

Example 8

(55) The coil made according to the examples 1-7 is connected to an electronic circuit component to form the biosensor. The electronic signal obtained from the sensor may be transmitted by an antenna system and received by an external device, for example mounted in a pair of glasses. This may in turn amplify the signal and transmit it to another device, for example an insulin pump.

Example 9

(56) Two platinum wires of diameter 0.127 mm were provided to be used as the working and counter electrodes in the following steps. The platinum wires were cleaned prior to use by sequential washing in H.sub.2SO.sub.4, ultrapure water and finally in PBS.

(57) A reference electrode was constructed as follows: an electrochemical cell was created with a silver wire of diameter 0.127 mm used as a working electrode in a saturated solution of KCI (3.4 g KCl, 10 ml MilliQ). A potential of 6V against the reference electrode was then applied for 2 times 50 s. The electrode was then kept overnight in the electrolyte solution, followed by a potentiometric measurement (zero current).

(58) The three wires obtained as described above were coiled in parallel around a non conductive mandrel as shown in FIG. 9b.

(59) To a stirred solution of phosphate buffered saline (PBS, 10 mL, pH 7.4) at room temperature, was added EDOT (20 L, 210.sup.2 m), followed after 5 min by addition of polyethylene glycol (PEG, 30 mg, average Fw=6000 g/mol, 510.sup.4 m). The resulting solution was stirred for another 5 min followed by addition of glucose oxidase (7 mg, Aspergillus niger, 270 U/mg material, BBI enzymes) and stirred gently.

(60) The parallel coiled wires and mandrel were immersed in the PBS solution containing EDOT and glucose oxidase. Cyclic voltametry (0-1.2V, 40 cycles, 0.05 V/s, against reference electrode) was used to electropolymerise EDOT on the surface of the working electrode. The parallel coiled wires were removed from the mandrel. After this, the electrodes were cut to the appropriate length.

(61) A top coating was applied to the parallel coiled wires. The parallel coiled wires were coated in a mixture of chitosan (2 mL, 1% in MilliQ/AcOH (99:1)) and glutaraldehyde (20 uL, 25% in water) by dip-coating. The coating was allowed to dry for 2 h at room temperature and the sensor system was then suitable for use.

Example 10

(62) A glucose calibration curve was made using the parallel coiled electrode set-up obtained by Example 9.

(63) Glucose PBS solutions from 0.00 to 1 mM with steps of 0.25 mM and from 1.0 to 5.0 mM with steps of 1 mM were prepared.

(64) Amperometry was performed for the different solutions. One potential step of 500 mV vs Ag/AgCl was applied and the current was measured for 150 s without stirring. The average current and the standard deviation between 60 and 150 seconds was calculated and plotted against the glucose concentration.

(65) The result is shown in FIG. 13. It was observed that the current was linearly proportional to the glucose concentration in the range 0 and 5 mM.