EP3295162A1

EP3295162A1 - Method for producing a device for electrochemically detecting molecules by means of redox-cycling, and device therefor and use thereof

Info

Publication number: EP3295162A1
Application number: EP16726763.2A
Authority: EP
Inventors: Alexey YAKUSHENKO; Bernhard Wolfrum; Nouran Yehia ADLY HASSAN; Andreas Offenhäusser
Original assignee: Forschungszentrum Juelich GmbH
Current assignee: Forschungszentrum Juelich GmbH
Priority date: 2015-05-08
Filing date: 2016-04-09
Publication date: 2018-03-21
Also published as: JP2021004898A; US20180088072A1; JP2020144146A; WO2016180385A1; US10739298B2; DE102015005781A1; JP2018514777A; CN107533029A

Abstract

The invention relates to a method for producing a device for electrochemically detecting molecules by means of redox-cycling, and to a device therefore and use thereof. A porous dielectric layer, which can absorb redoxactive molecules, is arranged between two electrode layers and is eventually biofunctionalised. The individual layers are applied, preferably, by means of a ink jet printing method.

Description

Method for producing a device for the electrochemical detection of molecules by means of redox cycling, and device for this and their use

The invention relates to a method for producing a device for the electrochemical detection of molecules by means of redox cycling, and to a device for this purpose and their use.

State of the art

Redox cycling is an electrochemical process in which electrochemically active molecules are oxidized and reduced several times. These reactions take place between two closely spaced electrodes. For this purpose, oxidizing and reducing potentials are applied to the electrodes so that the molecules are directly oxidized or reduced on contact with the electrodes. If the molecule then diffuses to the other electrode, the reverse process (reduction / oxidation) takes place. As a result of this iterative process, charge transport between the electrodes takes place through each individual molecule, which leads to an amplification of the measurable total signal.

In the prior art, such sensors are manufactured by means of optical lithography or electron beam lithography. Several designs for redox cycling sensors and methods of making them have been published:

From Goluch et al. (2009) a sensor with laterally adjacent electrodes is known. These so-called "interdigitated electrodes" have a distance in the range of nanometers to micrometers between the individual fingers. The method of production is electron beam lithography, which provides elaborate lift-off and etching processes.

Wolfrum et al. (2008), Kätelhön et al. (2010) and Zevenbergen et al. (2011) are known in the Z-axis, that is to say electrodes arranged one above the other, which have a nano-scaled gap as a so-called "nanocavity" or "nanochannel" in between. The production takes place with electron beam lithography or optical Lithography comprising several etching steps and the removal of, inter alia, a chromium sacrificial layer.

Hüske et al. (2014) disclose in the Z-axis, that is superimposed electrodes, which have a nano-scaled dielectric between the two electrodes. The electrodes are produced by optical lithography and electron beam lithography. The manufacturing process comprises several deposition and etching steps, but also a so-called "soap assembly" step by anodization of aluminum.

From Gross et al. (2015) are known in the Z-axis, that is to say superimposed electrodes which have a microscale gap. The electrodes are made by bonding two separate electrodes with a thick-film dielectric in between.

Disadvantages in the prior art are: a. Elaborate manufacturing processes using photolithography or electron beam lithography, which does not allow economically meaningful scaling of production. b. High production costs, or non-scalable production process. c. In all previous designs is for the detection of biomolecules such. As antigens, antibodies, DNA, etc. post-modification or assembly with recognition molecules such. Antibodies, aptamers and so on are needed by the electrodes and / or an intermediate layer. These are time consuming and costly. d. Insufficient sensitivity in microscale redox cycling sensors. e. Mechanical instability in very small columns.

f. Etched scratch or several chemical steps during fabrication. G. Some steps in existing methods are difficult to reproduce. Further disadvantages of the methods are: 1. Zu Goluch et al. 2009: The process leads to very high production costs due to the electron beam lithography used. In addition, there is delamination of the individual fingers at very small lateral distances. At longer distances there is insufficient sensitivity due to low efficiency of redox cycling.

2. To Wolfrum et al., Kätelhön et al. 2010 and Zevenbergen et al .: These methods also lead to high production costs due to the optical or electron beam lithography used. In addition, several lithography processes with very good alignment are a prerequisite for the functioning of the sensors. Mechanical instability occurs with very small gaps and / or laterally extended gaps. In addition, etching steps for the removal of the sacrificial layer are necessary, which allow the formation of the intermediate layer after the etching.

3. To Hüske et al. 2014: It is a difficult reproducible manufacturing process due to the variations in the anodization steps. Alternatively, electron beam lithography is used in the process, resulting in very high manufacturing costs. Several chemical steps or washing steps are necessary in the fabrication.

4. Zu Gross et al. 2015: It is a difficult reproducible production process with very low redox cycling efficiency.

Object of the invention

The object of the invention is to provide an inexpensive and rapid method for producing a device for the electrochemical detection of molecules by means of redox cycling. The method is intended to provide mechanically stable devices reproducibly and quickly at a low cost. Another object of the invention is to provide a related device for the electrochemical detection of molecules by redox cycling and to show their potential uses. Solution of the task

The object is achieved by the method according to claim 1 and the device and the use according to the independent claims. Advantageous embodiments of this result from the claims relating thereto.

Description of the invention

The method for producing a device for the electrochemical detection of analytes by means of redox cycling is characterized by the following steps: a) a first, electrically conductive electrode is arranged on a substrate, b) a first dielectric layer is permeable to redox-active molecules c) on the dielectric layer, a second, electrically conductive electrode is arranged with conductor track, wherein at least one of the steps a) to c) with a printing method of electrically conductive and / or electrically insulating particles.

Advantageously, this provides a method in which at least one of steps a) to c) is carried out with a printing method. The printing process is advantageous fast and inexpensive and very easy to reproduce.

The method for producing the redox cycling sensor thus comprises in particular but not exclusively a choice of conductive and / or insulating, printable particles with which the electrode and / or the dielectric layer can be arranged in a structured manner one above the other. In particular, but not exclusively, ink jet printing, aerosol jet process, screen printing, gravure printing, offset printing, nanoimprint printing or hot stamping is used. Combinations of coating and ablation steps that apply the same layers can be performed in combination with various coating techniques such as slot-die, laser ablation, and so on. Printable particles in the sense of the invention, preferably nanoparticles, are contained, for example, in an ink or in a paste or in another carrier medium for the particles.

As a substrate z. As glass, silicon or a polymer can be selected. Various polymers are particularly suitable, such as e.g. Polyethylene naphthalate, polyerylene terephthalate, polyimide, polymethyl methacrylate, polycarbonate, and so on.

According to step a), a first electrode of conductive particles, e.g. gold, silver, platinum, carbon, a conductive polymer, e.g. Poly (3,4-ethylenedioxythiophene) polystyrene sulfonates, polyaniline, polypyrols or the like and a conductor z. B. arranged by ink jet printing method or another printing method on the substrate and transferred to a finished structure. The first electrode has either no or only very small pores. The first electrode should be conductive and preferably show good electrochemical properties, that is z. B. have a fast electrode kinetics with standard redox mediators and be as resistant to electrode contamination by adsorption and / or corrosion.

According to step b), a redox-active-molecule-permeable dielectric layer having access to the introduction of the redox-active molecules into the dielectric layer is arranged on the first electrode. Step b) can be carried out in various ways. The access can z. Example, be prepared by in step b) a porous dielectric layer is disposed on the first electrode, in which the pores lead up to the surface of the first electrode. The dielectric layer is preferably printed on the first electrode. The size of the nanoparticles for the dielectric layer should be selected larger than any pores present in the first electrode, so that the nanoparticles for the structure of the dielectric do not penetrate into the pores of the first electrode. The nanoparticles are in turn contained in an ink, paste or other carrier medium. Then, optionally in step c), a porous second, electrically conductive electrode, preferably with a conductor track, can be arranged on the porous dielectric layer, with the pores leading to the surface of the dielectric layer. Then the pores of the second electrode and the dielectric form the access for the redox-active molecule. By applying a redox-active molecule in solution on the surface of the second electrode, this passes through the pores of the second electrode and the pores of the dielectric layer to the surface of the first electrode. The electrodes themselves have no electrical contact with each other.

The size of the nanoparticles for the second electrode should be selected to be larger than the pores present in the dielectric so that the nanoparticles for the construction of the second electrode do not penetrate into the pores of the dielectric. The nanoparticles are in turn contained in an ink, paste or other carrier medium.

In an advantageous embodiment of the invention, therefore, the method for producing a device for the electrochemical detection of redox-active molecules by redox cycling, characterized by the steps: a) on a substrate, a first, electrically conductive electrode is arranged, b) on the first electrode a porous dielectric layer is arranged, in which the pores lead to the surface of the first electrode, c) on the dielectric layer, a porous second, electrically conductive electrode is arranged, in which the pores lead to the surface of the dielectric layer at least one of the steps a) to c) is carried out with a printing method of electrically conductive and / or electrically insulating particles.

It is understood that the electrodes must be made contactable. Thus, a porous dielectric layer is arranged at least on the first electrode, in which the pores lead to the surface of the first electrode. The pores in the dielectric layer are subsequently filled with the molecule to be converted, or analyte or redox mediator. It is conceivable to arrange the second electrode without a pore system. Then the access of redox-active molecules to the dielectric layer z. B. by a lateral access. Such access may alternatively be provided by a single z. B. needlepoint-like opening in the second electrode can be set.

According to step b) of the method according to the invention, an ink with dielectric nanoparticles is preferably printed on the active region of the first electrode. This ink contains nanoparticles, such as polymethylmethacrylate, polystyrene, silica, titania, or the like, as a functional material.

The nanoparticle size in the ink for the dielectric layer should not be chosen smaller in the active region, that is to say the range of the conversion of the molecule at the first electrode, but should be chosen to be larger than any pores in the first electrode itself, so that the nanoparticles of the ink of the dielectric does not penetrate into the pores of the first electrode.

In the case of a printed ink, this can be advantageously sintered in the subsequent z. As thermal, photonic, by UV or similar energy input, so that the nanoparticles only partially melt and form a homogeneous dielectric layer with uniformly distributed pores of defined size, which extend to the surface of the first electrode and expose them.

Alternatively, the dielectric above the first electrode can also not be sintered, so that the nanoparticles remain unchanged and in this way the porosity remains ensured down to the surface of the first electrode.

It is particularly advantageously possible to use sol-gel inks for the production of the dielectric nanoporous layer of the sensor in step b). The material or the ink for the porous dielectric layer should be prepared so that after deposition, for example by means of ink-jet printing, this layer dries out and / or hardens and has the desired porosity, so that together menhängende pores are present in the layer. The layer has access to the liquid, e.g. B. on the pores. Such a layer can be made by the use of sol-gel materials and a sol-gel ink. For this you can z. As tetramethyl orthosilicate (TMOS), tetraethyl orthosilicate (TEOS), tetraisopropyl ortho-silicate (TPOS) as silicate for the sol-gel use. But other materials such as aluminum (2-propylate), aluminum (2-butoxide), zirconium propylate, titanium ethylate, titanium (2-propylate) and so on are useful. As the solvent or active material for the condensation reaction of the sol gel, different materials having OH groups can be used. Among others: water, alcohols (eg ethanol, methanol, etc.), different glycols like ethylene glycol, diethylene glycol, trithylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol and so on and also polyglycols with different chain lengths of for example 200, 300 , 400 or more monomer units such as polyethylene glycol, polypropylene glycol, and also glycerol can be used. In addition to the above-mentioned solvents or their mixtures, the ink may also contain additives such as surface tension modifiers, tackifiers, adhesion promoters, binders, and the like.

The sol-gel formation of a silica gel on the point-like applied dielectric follows the steps described in the publication "The Sol-Gel Preparation of Silica Gels" (Buckley, AM, Greenblatt, M. 1994. Journal of Chemical Education Volume 71, No. 7, 599-602) and the contents thereof, in particular for the preparation of the sol gel, are hereby incorporated by reference into this patent application, which also achieves the object of the invention in an advantageous embodiment of the invention For this purpose, an ink with conductive nanoparticles can advantageously be printed on a nanoporous dielectric layer and laterally further to form conductor tracks, so that this second electrode can also be contacted. For example, gold, platinum, carbon, a conductive polymer, such as poly (3,4-ethylenedioxythiophene ) polystyrene sulfonate, polyaniline, or the like, as the active material. The nanoparticle size in the second The electrode is preferably larger than the pores in the underlying dielectric layer. This advantageously has the effect that the nanoparticles of the second electrode can not penetrate into the pores of the dielectric and can form a short circuit with the lower electrode. In the present case, only the region which serves as a reservoir for the molecule between the electrodes, but not optionally existing passivation layers outside the active region, is referred to as the dielectric or dielectric layer. The pore system in the dielectric is the reservoir for the molecule in solution. In the reservoir, the redox reaction of the molecule takes place.

For the arrangement of the two electrodes identical or non-identical materials such. As inks can be used, provided that they can be applied with a suitable voltage, which allows the redox reaction of the molecule.

The dielectric in the active region of the device is preferably made of dielectic polymers such. As polyimide, polymethylmethacrylate, other acrylic-based polymers, polyvinylphenol, ceramic materials and oxides such. Silicon dioxide, titanium dioxide, silicon nitride and so on.

It is particularly advantageous to use an ink-jet printing method for arranging at least one of the two electrodes and / or additionally the dielectric layer. Ink jet printing is fast and inexpensive with high reproducibility of the electrodes and dielectric layers to be arranged and small dimensions.

In the case of inkjet printing z. As an ink printed with conductive and / or insulating particles. The particles are optionally sintered, z. As thermal, photonic, by UV or similar energy input. This advantageously has the effect that a homogeneous conductive and / or insulating layer is formed. It is also conceivable to use colloidally dissolved gold or carbon or even dissolved polymers as inks.

Generally, in the case of printed ink, it is optionally sintered, e.g. B. thermal, photonic, UV or similar energy input. This advantageously acts to form a homogeneous conductive or insulating layer or an area with many nanopores. The second, upper electrode has no ohmic or electrical contact with the lower, first electrode. This causes the two electrodes to form a pair of electrodes for the redox cycling process.

The active region of the device thus generally provides for the arrangement of at least one electrode and / or the dielectric by a printing process which quickly and inexpensively provides corresponding sensors for redox cycling.

The active region of the device is the region in which the reaction of the redox-active molecule takes place locally at the two electrodes above and below the dielectric layer or the reservoir. This part, the reservoir, of the dielectric layer is preferably limited in its area to approximately 1 μm ² to 1 cm ² . Preferably, the range in size is between 100 pm ² to 1 mm ² . The thickness of the dielectric is preferably from 10 nm to 1000 nm. In an advantageous embodiment of the invention, all steps a) to c) are carried out by a printing process, preferably by inkjet printing. This has the advantageous effect of providing a particularly inexpensive and at the same time fast and reproducible method for producing the sensor.

In addition, all steps a) to c) can be carried out particularly advantageously by a printing process, preferably by inkjet printing, in which particles are printed, wherein smaller particles than in step b) and / or in step b) are smaller in step a) Particles are printed as in step c). An ink jet printing method is advantageous because it has a particularly high throughput with high reproducibility and accuracy. In the printable inks or other starting materials, such. B. printable pastes contained particles are then in the active area of the device of step a) after step c) increasingly larger.

The method can then be carried out in such a way that in step a) of the method, inks are selected which have smaller particles than in step b) and / or in step b) of the method, inks are selected which have smaller particles than in step c). , In the context of the invention, pores can be produced in the second electrode which are larger than the pores in the dielectric layer and / or pores in the dielectric layer which are larger than pores in the first electrode, if they have pores at all , In general, the method may be characterized by the choice of an ink with conductive particles of gold, platinum, silver, carbon or conductive polymers, such as poly (3,4-ethylenedioxythiophene) polystyrene sulfonate, polyaniline for the preparation of the two electrodes.

The method can advantageously have at least one sintering process of printed conductive and / or insulating particles.

In a further embodiment of the invention, a passivation layer for passivating the first electrode can be arranged between the first electrode and the second electrode. This passivation layer advantageously also prevents that there is no electrical contact between the electrodes. The passivation layer may preferably have a cutout for the dielectric.

Further intermediate layers of dielectric nanoparticles can be arranged on the first electrode in the active region, eg. B. of polymers such. As polyimide, polymethyl methacrylate, other acrylic-based polymers, polyvinyl and also of ceramic materials and oxides such. For example, silica, titania, silicon nitride, and so on, but also from porous hydrogels can be arranged.

In an advantageous embodiment of the invention, a biofunctionalized ink for the arrangement of the dielectric layer on the first electrode in the active region is selected or arranged on this. This advantageously has the effect that the sensor can be used for the indirect detection of the biological or biochemical molecules.

By selecting an ink with insulating particles for the production of the dielectric layer to which antibodies, receptors, DNA, enzymes zyme or other biomolecules are bound, is advantageously causes the complementary biomolecules, z. As antigens, can be detected indirectly or electrochemically active products of enzyme activity can be detected directly. In the case of a bound antibody, the indirect detection is carried out with the aid of a redox mediator. The complementary antigens partially block the pores and thereby reduce the redox cycling current of the redox mediator. Reduction of the redox cycling current quantitatively corresponds to the blocked area, that is, the concentration of the antigen. In the case of a bound enzyme, a substrate is processed, thereby producing redox-active products. These redox-active products can be reduced and oxidized between the two electrodes of the redox cycling sensor, thereby amplifying the signal. In this case, the redox cycling current corresponds to the concentration of the enzyme substrate.

The advantage of this latter method is the fact that the biofunctionality takes place even before printing by choosing a suitable ink, so that time-consuming and costly post-treatments are avoided.

The device according to the invention for the electrochemical detection of molecules by means of redox cycling thus has a first, electrically conductive electrode on a substrate. On the first electrode, a dielectric layer permeable to redox-active molecules is disposed with access to introduce the redox-active molecules into the dielectric layer. On this dielectric

Layer is disposed a second, electrically conductive electrode without electrical contact to the first electrode.

At the electrodes, the redox reaction of a molecule such. An analyte, wherein the dielectric layer is the reservoir for the molecule in solution.

According to the invention, at least one of the two electrodes consists of printed electrically conductive particles and / or the dielectric layer consists of printed electrically insulating particles.

On the first electrode, a porous dielectric layer is arranged, in which pores in the dielectric layer up to the surface of the first electrode füh- The access of the redox-active substance into the conversion region can then take place directly via the surface of the second electrode as well as the pore system of the second electrode and of the pore system of the dielectric.

In an advantageous further embodiment of the invention, a porous second, electrically conductive electrode without electrical contact to the first electrode is arranged on the dielectric layer.

In the device, the two electrodes of the device are working electrodes in a potentiostat which are in contact with a reference electrode and / or a counterelectrode. The device according to the invention for the electrochemical detection of molecules by means of redox cycling then preferably has a first, electrically conductive electrode on a substrate. On the first electrode, a porous dielectric layer is arranged, in which the pores in the dielectric layer lead to the surface of the first electrode. On this dielectric layer, a porous second, electrically conductive electrode is arranged without electrical contact with the first electrode. The redox reaction of the molecule or of an analyte or of a redox mediator takes place at the two electrodes. The dielectric layer is a reservoir for the molecule in solution. At least one of the two electrodes consists of printed electrically conductive particles and / or the dielectric layer consists of printed electrically insulating particles. This region of the device identifies the so-called active region of the device which serves to convert the molecule or analyte or redox mediator.

In the device, the conductive particles of the second electrode are advantageously larger than the pores in the dielectric layer below and / or the insulating particles of the dielectric layer are larger than the pores in the first electrode underneath. This also causes the electrochemical properties of the electrodes and the dielectric to be preserved.

The two electrodes of the device represent the working electrodes in a potentiostat, and are contacted with a reference electrode and / or a counterelectrode. The reference electrode defines the voltages applied to the working electrodes. put potentials which are above and below the redox potential of the molecule or analyte and / or redox mediator so that it is alternately reduced and oxidized at the two electrodes. The resulting current flow is indicated by measuring the current with a counter electrode. Particularly advantageously, the dielectric layer in the active region of the sensor has an area between at least 1 pm ² to at most 1 cm ² . As a result, the generation of a radial mass transport to the electrodes and quickly achievable steady-state signals is advantageously effected.

Preferably, the range in size is between 100 pm ² to 1 mm ² . The thickness of the dielectric is preferably from 10 nm to 1000 nm.

The active region of the sensor or active material is thus the region of the electrodes and of the dielectric at which the redox cycling of the molecule is carried out. The active material of the dielectric is thus the region of the dielectric which is arranged directly between the active regions of the electrodes and serves as a reservoir for the molecule in solution or the analyte / redox mediator in solution.

The object of the invention is also achieved by the sensitive detection of redox-active molecules by means of the at least partially printed redox cycling sensor. In the context of the invention it has been recognized that for the realization of such

Sensors a special arrangement of the electrodes is necessary, in which the electrodes are very close to each other. The efficiency of the redox cycling or the amplification of the signal per molecule depends on the square of the distance between the electrodes. For this reason, in the case of the devices according to the invention for redox cycling, a nanoscale distance between the first and second electrodes is sought which allows the most sensitive detection.

It is understood that the sensor can be used by introducing a solution with a molecule or analyte or redox mediator as a redox-active molecule and for detecting the redox reaction. This is in solution and is applied to the pores of the second electrode above the porous dielectric layer in Reservoir applied. Or it is z. B. by a lateral or other vertical access to the dielectric. The molecule diffuses between the pores of the dielectric layer back and forth. The applied voltage at the first electrode and the second electrode drives the reduction and oxidation of the molecule or analyte (redox mediator) at the two electrodes above and below the reservoir and generates a detectable current flow.

If the redox-active molecule via another access z. B. is introduced a lateral or vertical access to the dielectric, no pore system of the second electrode is necessary. The invention is not limited to this. Within the scope of the invention, it has been recognized that conventional ink for use in a printing process can be used to make a device for the electrochemical detection of analytes by redox cycling. This is preferably biofunctionalized.

In step b), a biomodified, dielectric ink can thus advantageously be present on the active region of the first electrode after step a). In general, this ink contains in step b) nanoparticles, z. Polymethyl methacrylate, polystyrene, silica, titania, and so on which are optionally loaded with antibodies, DNA, aptamers, or the like, as the active material. The nanoparticle size should not be smaller than possibly existing pores in the first electrode, so that the nanoparticles do not penetrate into the electrode layer. Biofunctionalized ink should only be sintered to such an extent (thermally, photonically, UV or by another method) that the biochemical units, such as antibodies, DNA, aptamers and the like, do not lose their biological recognition properties and, in particular, do not denature. When using this optional step, a sintering method must be used which does not damage the biological material. Here, in particular, to pay attention to a low temperature according to the biological property of the layer.

Dielectric nanoparticles of an ink are preferably biofunctionalized with the following elements: • Full antibodies (eg: total IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgD, IgA, IgA1, IgA2, IgE) either recombinant or human, mouse, rat, goat, rabbit or porcine antibodies.

Antibodies bound to protein A, protein G and protein L as a biorecognition element.

• Fragment antibody (Fab 'fragment), F (ab') generated enzymatically (eg cysteine, papain, pepsin, ficin, bromelain) or by photonic activation.

• single-chain fragment variable (scFv).

• Artificial baroreceptors such as B. aptamers and Molecularly imprinted polymers (MIPs).

Enzymes that are either electrochemically active (such as glucose oxidase) and convert a substrate into a redox-active molecule that can then be amplified by redox cycling.

The material of the substrate is preferably selected from glass, silicon, various polymers such. Polyethylene naphthalate, polyetherethylene terephthalate, polyimide, polymethylmethacrylate, polycarbonate, and so on.

The electrodes are preferably made of gold, platinum, silver, carbon in various forms (carbon nanoparticles, graphite, graphene, carbon nanotubes, diamond, etc.), conductive polymers such as carbon nanoparticles. As poly (3,4-ethylenedioxythiophene) polystyrene sulfonates, polyaniline etc.

It is thus also possible to indirectly detect redox-active molecules and analytes by means of redox cyclings. This detection can be carried out by the immobilization of antibodies, DNA, aptamers, etc. on one or both of the electrodes and / or on the dielectric or the dielectric layer. When adding an analyte containing not only redox-active molecules, but also the containing complementary antigens, DNA, etc., these will specifically bind to the immobilized antibody, DNA, aptamers and so on. As a result, the surface available for the electrochemical reactions is reduced. Alternatively, the diffusion path of the redox-active molecule is prolonged by the blockade caused by the specific bonds. This leads to a change in the redox cycling current, which can be measured with a potentiostat. Since the blocked surface scales with the concentration of complementary antigens, DNA, etc., such a sensor can be used as a quantitative immunosensor.

When using immunosensors as a point-of-care diagnostic, their price is an important criterion. For disposable sensors, the price per piece should be below 1 € in most cases. In today's manufacturing process for the redox cycling sensors this price is not achievable. In addition, today's manufacturing processes always require a post-modification step for the immobilization of the antibodies, which further increases the cost per piece.

The method presented here, however, solves the problem by selecting a biomodified nanoparticle-containing ink from the outset in the arrangement of the dielectric layer, which in the subsequent, ie z. B. after sintering, has their biological function.

As a result, a device for the electrochemical detection of molecules by redox cycling is again provided, in which a first, electrically conductive electrode is arranged on a substrate, on the first electrode a biofunktionalisierte, porous dielectric layer is arranged, in which the pores to lead to the surface of the first electrode, and on the dielectric layer, a preferably porous second, electrically conductive electrode is disposed, wherein the nanoparticles of the second electrode, and the ink for the second electrode are larger than the pores in the dielectric. Alternatively, the second electrode is pore-free and access of the redox-active molecule is via another access to the dielectric, e.g. B. lateral. In general, the term pores in the present patent application is defined as follows. Pores in the dielectric layer and / or in the electrodes are preferably not pinhole-like or prickly.

The pores are due to the printing process and in particular due to an ink jet printing process and due to an optionally subsequent sintering, preferably of a spongy nature. The production method according to the invention thus requires sponge-like pore systems in the dielectric or in the electrode (s).

The pores should be distributed as equally as possible in the electrode (s) and / or the dielectric. The pores in one of these layers can either be ordered, e.g. B. by a plurality of channel-shaped, continuous pores (variety greater than 2 pores) or by hexagonal arrangement of the nanoparticles and formation of the pores. The pores can also be disordered (eg spongy porosity). The particles from the upper layer should preferably always be larger than the pores of the underlying layer.

The pore size of the first electrode is preferably 0 to 50 nm in diameter.

The pore size in the dielectric is preferably 10 to 1000 nm in diameter.

The pore size of the second electrode is preferably 100 to 10,000 nm in diameter. In the second electrode, as an alternative to a pore system, only a single large, z. B. annular opening may be arranged as access for the redox-active molecule.

Specifically, a porous dielectric may consist of a spongy scaffold of optionally partially melted and non-conductive particles traversed by pores leading from the surface of the dielectric to the opposite surface to the first electrode.

Optionally, in particular, a porous electrode, in particular a porous second electrode on the dielectric, may be optional from a spongy framework partially melted and conductive particles, which is traversed by pores, which lead from the surface of the second electrode to the opposite surface of the dielectric. An access of the molecule is then possible by applying a solution with molecules on the surface of the second electrode and the pore system of the second electrode and the dielectric up to the first electrode. A redox-active molecule, which is applied to the surface of the second electrode of such a sensor, thus passes through the pore system of the second electrode and the pore system of the dielectric to the surface of the first electrode and can be implemented alternately. It can be reduced and oxidized according to the applied voltages on the surfaces of the two electrodes.

If another access leads to the molecule according to the main claim in the dielectric, z. B. a lateral access z. B. on the passivation layer no pores in the second electrode in the active region are necessary. The use of the at least partially printed redox cycling sensors is in the range of the direct detection of chemical analytes, in particular for the detection of antigens.

EXEMPLARY EMBODIMENTS

In the following, the invention will be explained in more detail with reference to exemplary embodiments and the attached FIGURE, without thereby limiting the invention.

It shows:

FIG. 1: A method according to the invention.

Figure 2: A device according to the invention. First embodiment:

A sensor with sponge-like pores in the second electrode and in the dielectric is produced by means of the method described above. Step a): A gold ink is selected as the material for the first electrode 2a. The conductive structures of the gold ink are printed on a polyethylene naphthalate (PEN) substrate 1 with an ink jet printer and then sintered at 125 ° C for 1 hour. In this way, a first electrode 2a is formed on the substrate 1, which either has no pores or pores with a maximum size of 20 nm.

Shown in FIG. 1 is a right-hand region of the electrode 2 a, which defines the active measuring range for the conversion of the redox-active substance (not shown). In addition, a left-hand portion of sintered gold ink 2b is shown on the substrate 1 to which a voltage is applied and thus constitutes a trace. The region 2a of the first electrode runs out of the image plane to the right and is contacted with a potentiostat to apply voltage.

Passivation: A polyimide ink is chosen. With the aid of this ink, approximately 100 × 100 μm ² recesses 5 ^* are defined in the ink-jet printing as electrode regions, as shown in the right-hand part of FIG. 1 (active region). The polyimide ink is arranged to passivate the first electrode 2a. As a result, the right active region of the first electrode 2a is passivated by the region 5 ^* . The passivation ink 3a, 3b is thereby printed as a passivation layer around the later dielectric 5, so that an area 5 * for the later dielectric 5 is recessed.

In addition, with polyimide also a part of the conductor 2b made of gold is passivated. Thus, in the left-hand inactive region of FIG. 1, the polyimide layer 3c is arranged on the printed circuit 2b such that the printed conductor is partially exposed on the side facing the first electrode 2a and a step-shaped arrangement of polyimide 3c and printed circuit 2b results on substrate 1.

The passivation layers 3a, 3b and 3c are arranged in a single process step. It goes without saying that the regions of the first electrode 2a and the printed conductor 2b lying in the image depth are completely passivated.

Step b) Non-bio-modified polystyrene nanoparticle ink with nanoparticles of 100 nm size is deposited in the recessed area 5 ^{* of} the passivation 3a, 3b in FIG active area of the sensor arranged by ink jet printing. Due to its porosity, this dielectric 5 or this layer 5 forms a reservoir for the molecule or the analyte / redox mediator in solution and to be reacted. This layer has dimensions of about 100 m × 100 μm at a height of 500 nm.

The dielectric 5 is sintered at 115 ° C for 5 min, so that a homogeneous nanoporous layer 5 formed by the partial fusion of the particles.

In the dielectric, the pore size is about 30 nm in diameter.

Step c): Carbon ink with 300-400 nm carbon nanoparticles is selected as the second, upper electrode 4a and placed on the passivation 3a, 3b and the dielectric 5. The ink is also partially printed on the passivation layer 3a, 3b as well as on the dielectric 5 in the region of the first electrode 2a and also in the inactive region of the sensor shown on the left in FIG. 1 and further contact points for the second electrode in FIG Form area 4b over the conductor 2b. The ink is sintered at 125 ° C for 1 hour.

In the second electrode, the pore size is about 100 nm in diameter.

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

As a result, via the conductor 2b, a voltage z. B. above the oxidation potential of the molecule or analyte / Redoxmediators be applied to the active region of the second electrode 4a, which leads to the oxidation of the molecules / the analyte at the electrode. Accordingly, a voltage which is below the reduction potential of the analyte and thus enables the alternating redox cycling process is applied to the active region of the first electrode 2a. However, the detection can also be carried out the other way round, so that the Reduction potential on the electrode 4a and the oxidation potential is applied to the electrode 2a.

A molecule or analyte such. B. Ferrocendimethanol is brought in the form of a solution on the electrode (oxidized or reduced). The lower, first electrode in region 2a and the upper, second electrode 4a are respectively contacted and set to an oxidizing potential of +600 mV and reducing potential of 0 mV against an Ag / AgCl reference electrode. The detection of the analyte in different concentrations is carried out by measuring the redox cycling current at the oxidizing and / or reducing electrode. Second Embodiment: Use of the Printed Redox Cvclinq Sensor for the Detection of Ovalbumin.

A second sensor with sponge-like pores in the dielectric and the second electrode is produced as follows using a method described above (FIG. 1): Step a) and c) and the passivation follow those in Embodiment 1.

Step a): A gold ink is selected as the material for the first electrode 2a. The gold ink conductive structures are printed on a polyethylene naphthalate (PEN) substrate 1 with an ink jet printer and then sintered at 125 ° C for one hour. In this way, a first electrode 2a is formed on the substrate 1, which either has no pores or pores with a maximum size of 20 nm.

Shown in FIG. 1 is a right-hand region of the electrode 2 a, which defines the active measuring range for the conversion of the redox-active substance (not shown). In addition, a left-hand area of sintered gold ink 2b is shown on the substrate 1, which serves for the application of voltage and thus represents a conductor track. The region 2a of the first electrode runs out of the image plane to the right and is contacted with a potentiostat to apply voltage. Passivation: A polyimide ink is chosen. With the aid of this ink, approximately 100 × 100 μm electrode areas are defined as recess 5 * in inkjet printing, as shown in the right-hand part of FIG. 1 (active area). The polyimide ink is arranged to passivate the first electrode 2a. As a result, the right active region of the first electrode 2a is passivated by the region 5 *, the later reservoir. The passivation ink 3a, 3b is printed as a passivation layer around the later dielectric 5, so that an area 5 ^* for this dielectric 5 is recessed.

With the aid of this ink, in turn, as in exemplary embodiment 1, approximately 100 μm × 100 μm electrodes, conductor tracks and contact points for the sensor are defined.

Step b): 100 nm polystyrene nanoparticle ink, that is, a polystyrene nanoparticle ink with 100 nm nanoparticles is used. The polystyrene nanoparticles are equipped with anti-ovalbumin antibody and used as a dielectric 5 or intermediate layer between the two electrodes 2a and 4a. The ink is printed in the area 5 ^* which has been recessed by the passivation layer 3a, 3b to define the active area of the electrodes 2a, 4a. The dielectric 5 is heated at 40 ° C for 30 min so that the solvents evaporate but the biological material is not damaged and maintains its function.

The pore size corresponds approximately to that of the first embodiment.

Step c): Carbon ink with 300-400 nm carbon nanoparticles is arranged as upper electrode 4a on the passivation layer 3a, 3b and the dielectric 5. net. The ink is thereby printed on the passivation layer in the region of the first electrode 2a and also beyond to form the contact points in the region 4b via the conductor 2b and then photonically sintered so that the biological material of the dielectric 5 is not damaged. The pore size corresponds approximately to that of the first embodiment.

The inactive area thus otherwise corresponds to the inactive area of the first exemplary embodiment, and also the contacting of the first electrode 2a and the generated printed conductor 2b below the contact area 4b are identical.

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

As a result, via the conductor 2b, a voltage z. B. above the oxidation potential of the molecule or analyte / Redoxmediators be applied to the active region of the second electrode 4a above the dielectric 5, resulting in the oxidation of the molecules / the analyte at the electrode. Accordingly, a voltage is applied to the active region of the first electrode 2a which lies below the reduction potential of the analyte and thus enables the redox cycling process. The detection can also be performed in reverse, so that the reduction potential is applied to the electrode 4a and the oxidation potential to the electrode 2a. A solution with ovalbumin and a redox mediator such. B ferrocene dimethanol is placed on the surface of the second electrode 4a. The lower electrode 2a and the upper electrode 4a are respectively contacted 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 be done by measuring the redox cycling current. At higher concentrations of ovalbumin, the redox cycling current will decrease because the available electrochemical surface also decreases with concentration.

FIG. 2 shows a schematically illustrated device in the active region, which is simplified compared with FIG. 1, and the cyclic conversion of an analyte at a first electrode 22a or bot. and a second electrode 24a or top. El. and their arrangement in the potentiostat.

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

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

The object is achieved by a design having arranged in the Z-axis superimposed electrodes having a nano-scaled dielectric between the electrodes, wherein the electrodes and / or the dielectric is completely printed. There are advantageously no etching steps in the method. This is achieved by the fact that the three layers (first first lower conductive electrode, second dielectric layer, third second upper conductive electrode) show different porosity. Each additional layer has larger particles than the underlying layer, so that the top layer 4a can not flow into the underlying layer 5 during deposition from the liquid phase (e.g., ink jet printing) and out of layer 5, not into layer 2a.

For the exemplary embodiments, an inkjet printer OJ300 from UniJet (Korea) was used.

The substrate - Teonex (PEN) was obtained from DuPont-Teijin Films (England). The gold ink - Au25 was obtained from UT Dots (USA).

Polymer inks such as polyimide (PI) PMA-1210P-004 were obtained from Sojitz (Japan). Polystyroinanoparticle ink was mixed by itself from 200 nm polystyrene beads from Polysciences (USA).

Karbontinte 3800 was obtained from the company Methode (USA). Further embodiments:

These relate to the use of the sol-gel inks for the construction of the nanoporous dielectric. For example, in the first embodiment in step b), the nanoporous dielectric can be provided as follows: In step b), a non-bio-modified sol-gel-based ink is prepared. For this, TMOS 1: 1: 1 (parts by weight) is mixed with deionized water and glycerol in a 100 ml bottle and stirred for one hour with magnetic stirrer on a magnetic disk at room temperature. Thereafter, a 100 mM solution of hydrochloric acid in 500: 1 (sol-gel: acid, parts by weight) for the start of the condensation reaction given to it. The sol-gel ink will be in the recessed area

5 ^{* of} the passivation 3a, 3b are arranged in the active region of the sensor by ink jet printing. The dielectric 5 or the layer 5, 25 forms by its porosity after hydrolysis and curing, the reservoir for the molecule in solution and to be reacted or the analyte / redox mediator. This layer has dimensions of about 100 pm x 100 pm at a height of about 500 nm.

The dielectric 5, 25 is sintered at room temperature for 60 minutes, so that a homogeneous nanoporous layer 5 is formed by the condensation reaction in the printed sol-gel layer. In the dielectric, the pore size is then about 20-40 nm in diameter. It is understood that other sol-gel materials that undergo acid-catalyzed and / or base-catalyzed condensation reaction and hydrolysis may be used by those skilled in the art. Bibliography:

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

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

Hüske M., Stockmann R., Offenhäusser A., Wolfrum B. (2014). Redox cycling in nanoporous electrochemical devices. Nanoscale 6, 589-598 Gross A.J., Holmes S., Dale S.E.C., Smallwood M.J., Green S.J., Winlove C.P., Benjamin N., Winyard P.G., Brands F. (2015). Nitrites / nitrates detection in serum based on dual-plate generator-collector currents in a microtrench. Talanta 131: 228-235

Claims

Patent claims

1. A method for producing a device for the electrochemical detection of molecules by means of redox cycling, characterized by the steps:

On a substrate (1) a first, electrically conductive electrode (2a) is arranged,

On the first electrode (2a) a redox-active molecules permeable dielectric layer (5) is arranged with an access for introducing the redox-active molecules into the dielectric layer,

On the dielectric layer (5), a second, electrically conductive electrode (4a) is arranged, wherein at least one of the steps a) to c) is carried out with a printing method of electrically conductive and / or electrically insulating particles.

2. Method according to the preceding claim,

characterized in that

in step b) a porous dielectric layer (5) is arranged on the first electrode (2a), in which the pores lead to the surface of the first electrode (2a).

3. The method according to any one of the preceding claims,

characterized in that

in step c) on the dielectric layer (5) a porous second electrically conductive electrode (4a) is arranged with conductor track, wherein the pores lead to the surface of the dielectric layer (5).

4. The method according to any one of the preceding claims,

characterized in that

all steps a) to c) are carried out with a printing process.

5. Method according to one of the preceding claims,

characterized in that

a passivation layer (3a, 3b) for passivating the first electrode (2a) is arranged between the first electrode (2a) and the second electrode (4a), wherein the passivation layer (3a, 3b) has a recess for the dielectric layer (5) ,

6. The method according to any one of the preceding claims,

marked by

at least one sintering step of printed particles.

7. The method according to any one of the preceding claims,

characterized in that

in step a) smaller particles than in step b) and / or in step b) smaller particles than in step c) are printed.

8. The method according to any one of the preceding claims,

in which

Pores may be produced in the second electrode (4a) which are larger than the pores in the dielectric layer (5) and / or pores in the dielectric layer (5) which are larger than the pores in the first electrode (2a ).

9. The method according to any one of the preceding claims,

characterized in that

in step c) conductive particles are printed for the second electrode (4a) which are larger than the pores in the dielectric layer (5) and / or in step b) insulating particles for the dielectric layer (5) are printed, which are larger are as the pores in the first electrode (2a).

10. The method according to any one of the preceding claims,

marked by

an ink-jet printing method for arranging at least one of the two electrodes (2a, 4a) and / or the dielectric layer (5) and / or the passivation layer (3a, 3b).

11. The method according to any one of the preceding claims,

marked by

Choice of a biofunctionalized ink for arranging the dielectric layer (5) on the first electrode (2a).

12. Method according to the preceding claim,

marked by

Choice of an ink with insulating particles, to which enzymes, antibodies, receptors, or other biomolecules are bound for the production of the dielectric layer (5).

13. The method according to any one of the preceding claims,

marked by

Choice of an ink with conductive particles of gold, platinum, silver, carbon or conductive polymers, such as. Example, poly (3,4-ethylenedioxythiophene) polystyrenes sulfonates, polyaniline for the preparation of at least one of the two electrodes (2a, 4a).

14. The method according to any one of the preceding claims,

characterized in that

a sol-gel ink in step b) is used to construct the dielectric layer (5).

15. An apparatus for electrochemical detection of molecules by redox cycling, wherein

a first, electrically conductive electrode (2a) is arranged on a substrate (1) and on the first electrode (2a) a redox-active-molecule-permeable dielectric layer (5) with access for introduction of the redox-active molecules into the dielectric layer is arranged, and a second, electrically conductive electrode (4a) without electrical contact to the first electrode (2a) is arranged on this dielectric layer (5) and wherein the redox reaction of the molecule can take place at the electrodes (2a, 4a), the dielectric layer (5 ) is a reservoir for the molecule in solution and at least one of the electrodes (2a, 4a) made of printed electrically conductive Particles and / or the dielectric layer (5) consists of printed electrically insulating particles.

16. Device according to previous claim,

in which

on the first electrode (2a) a porous dielectric layer (5) is arranged, lead in the pores in the dielectric layer (5) to the surface of the first electrode (2a).

17. Device according to one of the preceding claims,

characterized in that

on the dielectric layer (5) a porous second, electrically conductive electrode (4a) without electrical contact to the first electrode (2a) is arranged.

18. Device according to one of the preceding claims,

in which

the two electrodes (2a, 4a) of the device are working electrodes in a potentiostat which are contacted with a reference electrode and / or a counterelectrode.

19. Device according to one of the preceding claims,

characterized in that

the dielectric layer (5) has an area between at least 1 μm ² to at most 1 cm ² .

20. Use of a device according to one of the preceding claims, characterized by

introducing a solution containing redox-active molecules into the dielectric layer (5) permeable to the redox-active molecules and applying a voltage to the electrodes (2a, 4a) which causes the alternating reduction and oxidation of the molecule at the electrodes.

21. Use of a device according to the preceding claim,

in which

the redox-active molecules are introduced via an access into a porous dielectric layer (5).

22. Use of a device according to one of the preceding claims, wherein

the access of the redox-active molecules into the dielectric takes place via pores of the second electrode (4a).

23. An ink for use in a printing process for producing a device for the electrochemical detection of analytes by redox cycling according to any one of the preceding claims.

24. Ink according to the preceding claim,

marked by

a sol-gel ink which cures after application to the active region (5 *) and forms the nanoporous layer (5).