Method for Producing a Device for Electrochemical Detection of Molecules by Way of Redox Cycling, Device Therefor and the Use Thereof

Abstract

The invention relates to a method for producing a device for the electrochemical detection of molecules by way of redox cycling, to a device therefor, and to the use thereof. A porous dielectric layer is present between two electrode layers, which is able to receive redox-active molecules and may be biofunctionalized. The individual layers are preferably applied by way of an ink jet printing method.

Claims

1. A method for producing a device for the electrochemical detection of molecules by way of redox cycling, characterized by the following steps: a) disposing a first electrically conductive electrode on substrate; b) disposing a dielectric layer permeable by redox-active molecules on the first, electrode, including an access for introducing redox-active molecules into the dielectric layer; and c) disposing a second electrically conductive electrode on the dielectric layer, wherein at least one of the steps a) to c) is carried out by way of a method of printing electrically conductive and/or electrically insulating particles.

2. The method according to claim 1, wherein in step b), a porous dielectric layer is disposed on the first electrode, in which the pores extend to the surface of the first electrode.

3. A method according to claim 1, wherein in step c), a porous second electrically conductive electrode (4a), comprising a conductor track, is disposed on the dielectric layer, in which the pores extend to the surface of the dielectric layer.

4. A method according to claim 1, wherein all of the steps a) to c) are carried out using a printing method.

5. A method according to claim 1, wherein a passivation layer for the passivation of the first electrode is disposed between the first electrode and the second electrode, wherein the passivation layer comprises a recess for the dielectric layer.

6. A method according to claim 1, comprising at least one step of sintering printed particles.

7. A method according to claim 1, wherein smaller particles are printed in step a) than in step b) and/or smaller particles are printed in step b) than in step c).

8. A method according to claim 1, wherein pores are produced in the second electrode that are larger than the pores in the dielectric layer and/or pores are produced in the dielectric layer that are larger than the pores in the first electrode.

9. A method according to claim 1, wherein in step c), conductive particles are printed for the second electrode that are larger than the pores in the dielectric layer and/or in step b) insulating particles are printed for the dielectric layer that are larger than the pores in the first electrode.

10. A method according to claim 1, comprising an ink jet printing method for the arrangement of at least one of the two electrodes and/or the dielectric layer and/or the passivation layer.

11. A method according to claim 1, further comprising selecting a biofunctionalized ink for disposing the dielectric layer on the first electrode.

12. The method according to claim 11, further comprising selecting an ink comprising insulating particles, to which enzymes, antibodies, receptors or other biomolecules are bound, for producing the dielectric layer.

13. A method according to claim 1, further comprising selecting an ink comprising conductive particles made of gold, platinum, silver, carbon or conductive polymers, such as poly(3,4-ethylenedioxythiophene)polystyrene sulfonate, polyaniline, for producing at least one of the two electrodes.

14. A method according to claim 1, wherein a sol gel ink is used in step b) for creating the dielectric layer.

15. A device for the electrochemical detection of molecules by way of redox cycling, wherein: a first, electrically conductive electrode is disposed on a substrate, a dielectric layer permeable by redox-active molecules is disposed on the first, electrode, including an access for introducing the redox-active molecules into the dielectric layer, and a second electrically conductive, electrode, having no electrical contact with the first electrode, is disposed on this dielectric layer, wherein the redox reactions of the molecule can take place at the electrodes wherein the dielectric layer is a reservoir for the molecule present in solution, and at least one of the electrodes is composed of printed electrically conductive particles and/or the dielectric layer is composed of printed electrically insulating particles.

16. The device according to the claim 15, wherein a porous dielectric layer is disposed on the first electrode in which pores in the dielectric layer extend to the surface of the first electrode.

17. A device according to claim 15, wherein a porous second electrically conductive electrode, having no electrical contact with the first electrode, is disposed on the dielectric layer.

18. A device according to claim 15, wherein the two electrodes of the device are working electrodes in a potentiostat, which are in contact with a reference electrode and/or a counter electrode.

19. A device according to claim 15, wherein the dielectric layer has a surface area between at least 1 m2 and no more than 1 cm2.

20. Use of a device according to claim 15, wherein an introduction of a solution comprising redox-active molecules into the dielectric layer permeable by redox-active molecules, and the application of a voltage to the electrodes, causes the alternating reduction and oxidation of the molecules at the electrodes.

21. Use of a device according to claim 20, wherein the redox-active molecules are introduced via an access into the porous dielectric layer.

22. Use of a device according to claim 20, wherein the access of the redox-active molecules into the dielectric takes place via pores of the second electrode.

23. Ink for use in a printing method for producing a device for the electrochemical detection of analytes by way of redox cycling according to claim 1.

24. The ink according to the claim 23, by comprising a sol gel ink, which cures after application into the active region and forms the nanoporous layer.

Description

IN THE DRAWINGS

[0114] FIG. 1 shows a method according to the invention; and

[0115] FIG. 2 shows a device according to the invention.

FIRST EXEMPLARY EMBODIMENT

[0116] A sensor comprising sponge-like pores in the second electrode and in the dielectric is produced by way of the above-described method.

[0117] Step a): A gold ink is selected as the material for the first electrode 2a. The conducting structures made of the gold ink are printed onto a polyethylene naphthalate (PEN) substrate 1 using an ink jet printer, and are then sintered at 125 C. for 1 hour.

[0118] In this way, a first electrode 2a is formed on the substrate 1, which has either no pores or pores having a maximum size of 20 nm.

[0119] FIG. 1 shows a right region of the electrode 2a, which defines the active measuring region for the reaction of the redox-active substance (not shown). Furthermore, a left region made of sintered gold ink 2b on the substrate 1 is shown, to which a voltage is applied and which thus represents a conductor track. The region 2a of the first electrode extends to the right out of the image plane and is contacted with a potentiostat to apply voltage.

[0120] Passivation: An ink made of polyimide is selected. Using this ink, recesses 5* measuring approximately 100100 m.sup.2 are defined as electrode regions by way of ink jet printing, as shown in the right part of FIG. 1 (active region). The polyimide ink is disposed thereon so as to passivate the first electrode 2a. In this way, the right active region of the first electrode 2a is passivated around the region 5*. The passivation ink 3a, 3b is printed as a passivation layer around the later dielectric 5, so that a region 5* is recessed for the later dielectric 5.

[0121] In addition, a portion of the conductor track 2b made of gold is passivated by way of polyimide. In the left inactive region of FIG. 1, the polyimide layer 3c is thus disposed on the conductor track 2b in such a way that the conductor track is partially exposed on the side facing the first electrode 2a, and a step-like arrangement of polyimide 3c and conductor track 2b is produced on the substrate 1.

[0122] The passivation layers 3a, 3b and 3c are disposed in a single method step. It goes without saying that the regions of the first electrode 2a and of the conductor track 2b lying in the depth of the image are completely passivated.

[0123] Step b) Non-biomodified polystyrene nanoparticle ink comprising nanoparticles 100 nm in size is disposed in the recessed region 5* of the passivation 3a, 3b in the active region of the sensor by way of ink jet printing. This dielectric 5 or this layer 5, due to the porosity thereof, forms a reservoir for the molecule present in solution and to be reacted, or for the analyte/redox mediator. This layer has dimensions of approximately 100 m100 m at a height of 500 nm.

[0124] The dielectric 5 is sintered at 115 C. for 5 minutes, so that a homogeneous nanoporous layer 5 is formed as a result of the partial fusion of the particles.

[0125] The pore size in the dielectric is approximately 30 nm in diameter.

[0126] Step c): Carbon ink comprising carbon nanoparticles of 300 to 400 nm in size is selected as the second, top electrode 4a and disposed on the passivation 3a, 3b and the dielectric 5. The ink is also partially printed onto the passivation layer 3a, 3b and onto the dielectric 5 in the region of the first electrode 2a, and moreover in the inactive region of the sensor, which is shown on the left in FIG. 1, so as to form further contact points for the second electrode in the region 4b via the conductor track 2b. The ink is sintered at 125 C. for 1 hour.

[0127] The pore size in the second electrode is approximately 100 nm in diameter.

[0128] In addition to the active region for the redox reaction in the right part of FIG. 1, a further particularly advantageous embodiment of the method and of a device thus produced is shown in the left part of FIG. 1. This left region is the so-called inactive region of the sensor. The inactive region comprises the conductor track 2b made of gold, which extends out of the image plane to the left (not shown). The conductor track 2b is contacted with a potentiostat (not shown).

[0129] In this way, a voltage, such as above the oxidation potential of the molecule or analyte/redox mediator, can be applied via the conductor track 2b to the active region of the second electrode 4a, which results in oxidation of the molecule/analyte at the electrode. Correspondingly, a voltage is applied to the active region of the first electrode 2a, which is below the reduction potential of the analyte and thus enables the alternating redox cycling process. The detection, however, can also just as well be carried out conversely, so that the reduction potential is applied to the electrode 4a, and the oxidation potential is applied to the electrode 2a.

[0130] A molecule or an analyte, such as ferrocenedimethanol, is applied to the electrode in the form of a solution (oxidized or reduced). The bottom, first electrode in the region 2a and the top, second electrode 4a are accordingly brought in contact and set to an oxidizing potential of +600 mV and a reducing potential of 0 mV with respect to an Ag/AgCl reference electrode. The detection of the analyte in various concentrations takes place by measuring the redox cycling current intensity at the oxidizing and/or reducing electrode.

SECOND EXEMPLARY EMBODIMENT: USE OF THE PRINTED REDOX CYCLING SENSOR FOR THE DETECTION OF OVALBUMIN

[0131] A second sensor comprising sponge-like pores in the dielectric and the second electrode is produced as follows by way of an above-described method (FIG. 1):

[0132] Steps a) and c) and the passivation follow exemplary embodiment 1.

[0133] Step a): A gold ink is selected as the material for the first electrode 2a. The conducting structures made of the gold ink are printed onto a polyethylene naphthalate (PEN) substrate 1 using an ink jet printer, and are then sintered at 125 C. for one hour.

[0134] In this way, a first electrode 2a is formed on the substrate 1, which has either no pores or pores having a maximum size of 20 nm.

[0135] FIG. 1 shows a right region of the electrode 2a, which defines the active measuring region for the reaction of the redox-active substance (not shown). Furthermore, a left region made of sintered gold ink 2b on substrate 1 is shown, which is used for the application of the voltage and thus represents a conductor track. The region 2a of the first electrode extends to the right out of the image plane and is contacted with a potentiostat to apply voltage.

[0136] Passivation: An ink made of polyimide is selected. Using this ink, electrode regions approximately 100100 m in size are defined as the recess 5* by way of ink jet printing, as shown in the right part of FIG. 1 (active region). The polyimide ink is provided so as to passivate the first electrode 2a. In this way, the right active region of the first electrode 2a is passivated around the region 5*, this being the subsequent reservoir. The passivation ink 3a, 3b is printed as a passivation layer around the later dielectric 5, so that a region 5* is recessed for this dielectric 5.

[0137] In addition, a portion of the conductor track 2b made of gold is passivated by way of polyimide. In the left inactive region of FIG. 1, the polyimide layer 3c is thus disposed on the conductor track 2b in such a way that the conductor track is partially exposed on the side facing the first electrode 2a, and a step-like arrangement of polyimide 3c and conductor track 2b is produced on the substrate 1.

[0138] The passivation layers 3a, 3b and 3c are disposed in a single method step. It goes without saying that the regions of the first electrode 2a and of the conductor track 2b lying in the depth of the image are completely passivated.

[0139] Using this ink, as in exemplary embodiment 1, approximately 100 m100 m electrodes, conductor tracks and contact points for the sensor are again defined.

[0140] Step b): 100 nm polystyrene nanoparticle ink, which is to say a polystyrene nanoparticle ink comprising nanoparticles of 100 nm in size, is used. The polystyrene nanoparticles are equipped with anti-ovalbumin antibodies and used as a dielectric 5 or intermediate layer between the two electrodes 2a and 4a. The ink is printed in the region 5*, which was recessed by the passivation layers 3a, 3b so as to define the active region of the electrodes 2a, 4a. The dielectric 5 is heated at 40 C. for 30 minutes, so that the solvents evaporate, but the biological material is not damaged and preserves the function thereof.

[0141] The pore size corresponds approximately to that of the first exemplary embodiment.

[0142] Step c): Carbon ink comprising carbon nanoparticles of 300 to 400 nm in size is disposed as the top electrode 4a on the passivation layer 3a, 3b and the dielectric 5. The ink is printed onto the passivation layer in the region of the first electrode 2a, and also beyond, so as to form the contact points in the region 4b via the conductor track 2b, and is then sintered, so that the biological material of the dielectric 5 is not damaged.

[0143] The pore size corresponds approximately to that of the first exemplary embodiment.

[0144] The inactive region thus otherwise corresponds to the inactive region of the first exemplary embodiment, and the contacting of the first electrode 2a and of the generated conductor track 2b beneath the contact region 4b is also identical.

[0145] In addition to the active region for the redox reaction in the right part of FIG. 1, a further particularly advantageous embodiment of the method and of a device thus produced is thus shown in the left part of FIG. 1. This left region is the so-called inactive region of the sensor. The inactive region comprises the conductor track 2b made of gold, which extends out of the image plane to the left (not shown). The conductor track 2b is contacted with a potentiostat (not shown).

[0146] In this way, a voltage, such as above the oxidation potential of the molecule or analyte/redox mediator, can be applied via the conductor track 2b to the active region of the second electrode 4a above the dielectric 5, which results in oxidation of the molecule/of the analyte at the electrode. Correspondingly, a voltage is applied to the active region of the first electrode 2a which is below the reduction potential of the analyte and thus enables the redox cycling process. The detection can also be carried out conversely, so that the reduction potential is applied to the electrode 4a, and the oxidation potential is applied to the electrode 2a.

[0147] A solution comprising ovalbumin and a redox mediator, such as ferrocenedimethanol, is applied to the surface of the second electrode 4a. The bottom electrode 2a and the top electrode 4a are accordingly brought in contact and set to an oxidizing potential of +600 mV and a reducing potential of 0 mV against an Ag/AgCl reference electrode. The detection of the analyte in different concentrations will take place by measuring the redox cycling current intensity.

[0148] At higher concentrations of ovalbumin, the redox cycling current intensity will decrease, since the available electrochemical surface also decreases with the concentration.

[0149] FIG. 2 shows a schematically illustrated device, which is simplified compared to FIG. 1, in the active region, the cyclical reaction of an analyte at a first electrode 22a or Bot. El. and a second electrode 24a or Top. El. and the arrangement thereof in the potentiostat.

[0150] The porous dielectric 25, which serves as the reservoir for the analyte/redox mediator present in solution, is disposed between the two electrodes. Voltages are applied to the porous second electrode 4a and the first electrode 2a, which drive the cyclical redox reaction. The generated current is correspondingly measured against the counter electrode.

[0151] According to the invention, a nanoscale redox cycling sensor is thus produced only by way of printing technologies, without additional etching steps or sacrificial layers, and optionally also by way of biomodification without further steps.

[0152] The object is achieved by a design comprising electrodes disposed in the Z axis on top of one another, which comprise a nanoscale dielectric between the electrodes, wherein the electrodes and/or the dielectric are completely printed. Advantageously, there are no etching steps in the method. This is achieved in that the three layers (1. first bottom conducting electrode; 2. dielectric layer; 3. second top conducting electrode) have differing porosities. Every further layer has larger particles than the layer disposed beneath, so that the layer 4a lying on top, upon deposition from the liquid phase (such as ink jet printing), cannot flow into the layer 5 lying at the bottom, and cannot flow from layer 5 into layer 2a.

[0153] For the exemplary embodiments, an OJ300 ink jet printer from UniJet (Korea) was used.

[0154] The Teonex (PEN) substrate was obtained from DuPont-Teijin Films (England).

[0155] The Au25 gold ink was obtained from UT Dots (USA).

[0156] Polymer inks such as polyimide (PI) PMA-1210P-004 was obtained from Sojitz (Japan). Polystyrene nanoparticle ink was mixed from 200 nm polystyrene beads from Polysciences (USA).

[0157] Carbon ink 3800 was obtained from Methode (USA).

[0158] Further Exemplary Embodiments:

[0159] These relate to the use of the sol gel inks for creating the nanoporous dielectric. In the first exemplary embodiment in step b), for example, the nanoporous dielectric can be provided as follows.

[0160] In step b), a non-biomodified sol gel-based ink is prepared. For this purpose, TMOS 1:1:1 (percent by weight) is mixed with deionized water and glycerol in a 100 ml flask and stirred for one hour at room temperature using a magnetic stirrer on a magnetic plate. Afterwards, a 100 mM solution of hydrochloric acid at 500:1 (sol gel:acid, percent by weight) is added for starting the condensation reaction. The sol gel ink is disposed in the recessed region 5* of the passivation 3a, 3b in the active region of the sensor by way of ink jet printing. The dielectric 5 or the layer 5, 25, due to the porosity thereof, forms the reservoir for the molecule present in solution and to be reacted, or for the analyte/redox mediator, after hydrolysis and curing. This layer has dimensions of approximately 100 m100 m at a height of approximately 500 nm.

[0161] The dielectric 5, 25 is sintered at room temperature for 60 minutes, so that a homogeneous nanoporous layer 5 forms as a result of the condensation reaction in the printed sol gel layer. The pore size in the dielectric is then approximately 20 to 40 nm in diameter.

[0162] It goes without saying that a person skilled in the art can also use other sol gel materials that are subject to an acid-catalyzed and/or base-catalyzed condensation reaction and hydrolysis.

LIST OF REFERENCES

[0163] Goluch E. D., Wolfrum B., Singh P. S., Zevenbergen M. A. G., Lemay S. G. (2009). Redox cycling in nanofluidic channels using interdigitated electrodes. Anal Bioanal Chem 394:447-456

[0164] Wolfrum B., Zevenbergen M., Lemay S. (2008). Nanofluidic redox cycling amplification for the selective detection of catechol. Anal Chem 80, 972-977

[0165] Ktelhn E., Hofmann B., Lemay S. G., Zevenbergen M. A. G., Offenhusser A., Wolfrum B. (2010). Nanocavity Redox Cycling Sensors for the Detection of Dopamine Fluctuations in Microfluidic Gradients. Anal Chem 82, 8502-8509

[0166] Zevenbergen M. A. G., Singh P. S., Goluch E. D., Wolfrum B. L., Lemay S. G. (2011). Stochastic sensing of single molecules in a nanofluidic electrochemical device. Nano Lett. 11, 2881-2886

[0167] Hske M., Stockmann R., Offenhusser A., Wolfrum B. (2014). Redox Cycling in nanoporous electrochemical devices. Nanoscale 6, 589-598

[0168] Gross A. J., Holmes S., Dale S. E. C., Smallwood M. J., Green S. J., Winlove C. P., Benjamin N., Winyard P. G., Marken F. (2015). Nitrite/Nitrate detection in serum based on dual-plate generator-collector currents in a microtrench. Talanta 131:228-235