EP-4740006-A1 - A THREE-DIMENSIONAL ELECTRODE STRUCTURE, A PROCESS FOR OBTAINING THEREOF AND AN ELECTROCHEMICAL BIOSENSOR OBTAINED USING THE THREE-DIMENSIONAL ELECTRODE STRUCTURE

EP4740006A1EP 4740006 A1EP4740006 A1EP 4740006A1EP-4740006-A1

Abstract

The invention relates to biosensors and three-dimensional electrodes, which can be used for manufacturing electrochemical biosensors. The claimed process for obtaining of a three-dimensional electrode comprising the steps of: (i) providing a solid silicone-based formative bed, defining the three-dimensional electrode dimensions; (ii) introducing a UV curable polymer into the formative bed; (iii) positioning a mask over the formative bed filled with the UV curable polymer; wherein the mask comprises predefined patterns corresponding to a lattice structure, the patterns having areas that are transparent to UV light to define cells and supporting pillars of the lattice, and areas that are non-transparent to UV light to block UV exposure; (iv) exposing the UV curable polymer to UV light through the mask at a defined optical angle, such that the angled UV light exposure through the transparent areas of the mask defines the formation of cells and leaning supporting pillars of the lattice, while the areas of the mask that are non-transparent to UV light block the UV exposure; (v) removing the uncured portions of the UV curable polymer by a solvent, thereby revealing the three-dimensional lattice structure comprising cells and supporting pillars; (vi) subjecting the element obtained at a previous step to a treatment selected from thermal, chemical, physical, or radiation-based methods to impart a hydrophilic surface characteristic to the element; (vii) depositing a conductive layer onto the element's three-dimensional lattice structure, via an electroless plating by a deposition of at least 50 nm of a base or noble metal or metal alloy via reduction of metal ions in presence of a reducing agent.

Inventors

RIMSA, Roberts
PAIDERE, Gunita
CIPA, Janis
ZUTIS, Edmunds
MOZOLEVSKIS, Gatis

Assignees

Latvijas Universitates Cietvielu fizikas instituts

Dates

Publication Date: 20260513
Application Date: 20241010

Claims (7)

1. A process for obtaining of a three-dimensional electrode comprising the following steps: (i) providing a solid silicone-based formative bed, defining the three-dimensional electrode dimensions; (ii) introducing a UV curable polymer into the formative bed, wherein the UV curable polymer is a thiol-ene polymer or a thiol-ene based polymer, more preferably, a off- stoichiometry thiol-ene polymer, or a off-stoichiometry thiol-ene based polymer; (in) positioning a mask over the formative bed filled with the UV curable polymer; wherein the mask comprises predefined patterns corresponding to a lattice structure, the patterns having areas that are transparent to UV light to define cells and supporting pillars of the lattice, and areas that are non-transparent to UV light to block UV exposure; Civ) exposingthe UV curable polymerto UV light through the mask ata 40-70-degree optical angle, such that the angled UV light exposure through the transparent areas of the mask defines the formation of cells and 40-70-degrees leaning supporting pillars of the lattice, while the areas of the mask that are non-transparent to UV light block the UV exposure; (v) removing the uncured portions of the UV curable polymer by a solvent, thereby revealing the three-dimensional lattice structure comprising cells and supporting pillars; (vi) subjecting the element obtained at a previous step to a treatment selected from thermal, chemical, physical, or radiation-based methods to impart a hydrophilic surface characteristic to the element; (vii) depositing a conformal, conductive layer onto the element’s three-dimensional lattice structure, via an electroless plating by a deposition of at least 50 nm of a base or noble metal or metal alloy via reduction of metal ions in presence of a reducing agent.
2. The process according to claim 1, further comprising, after the step (ii) and before the step (hi), the step (ii) of placing an electrically conductive wire to a location within the formative bed where the UV curable polymer is introduced, wherein the electrically conductive wire is in electrical communication with the conductive layer of the lattice structure after step (vii).
3. The process according to claim 1, further comprising, after the step (vii), the step (viii) of connecting an electrically conductive wire to the conductive layer of the lattice structure, ensuring electrical communication between the wire and the lattice structure.
4. The process according to any preceding claims, further comprising the step of performing gold electroplating of the lattice structure of the electrode obtained.
5. The process according to any preceding claims, wherein the mask areas that are transparent to UV light and corresponding to a lattice structure, are designed such that the angled exposure of UV light through the transparent areas results in formation of cells with dimensions in the range of 5-500 pm.
6. A process for obtaining an electrochemical biosensor, comprising the following steps: (i) providing the three-dimensional electrode structure obtained according to any of claims 1-5 and providing a primary antigen, capable of selective capturing of biological entities; the primary antigen modified with conjugated enzyme, which is capable to produce an electroactive product; (ii) attaching the primary antigen to the electrode surface; (hi) introducing the primary antigen, attached to the electrode surface to an environment comprising target biological entities to be captured by the primary antigen; thereby creating of a detectable signal through generation of an electroactive product resulting from enzymatic activity upon binding of the target biological entities to the primary antigen.
7. A process for obtaining an electrochemical biosensor, comprising the following steps: [i] providing the three-dimensional electrode structure obtained according to any of claims 1-5 and providing a primary antigen, capable of selective capturing of biological entities; (ii) attaching the primary antigen to the electrode surface; (in) introducing the primary antigen, attached to the electrode surface to an environment comprising target biological entities to be captured by the primary antigen; (iv) providing a secondary antigen modified with conjugated enzyme and capable of selective attachment to the same target biological entities as the primary antigen; wherein the conjugated enzyme is capable to produce an electroactive product; (v) introducing the secondary antigen to the target biological entities captured by the primary antigen for attachment of the secondary antigen to the target biological entities, and for creation of a detectable signal through generation of an electroactive product resulting from enzymatic activity upon binding of the secondary antigen to the biological entities captured by the primary antigen.

Description

A three-dimensional electrode structure, a process for obtaining thereof and an electrochemical biosensor obtained using the three-dimensional electrode structure Technical Field [001] The invention relates to biosensors and three-dimensional electrodes, which can be used for sensing biomolecules electrochemically. Background Art [002] Electrochemical biosensors are based on, typically, a two or three electrode system, where working electrode has a functional layer in the form of an affinitybased chemical compound that upon reacting with the analyte creates a charge that can be detected on the surface of the working electrode directly or through a mediating and/or amplifying layer. This effectively means that the electrode surface area is key determinant factor for sensitivity of the biosensor given a fixed sensing stack. Subsequently, increased surface area can lead to enhanced sensitivity of the biosensors. [003] To date most of the 3D structured electrodes have been focused on nanostructuring of the surface of the electrode, which indeed can yield higher surface area, however, this area is not actively engaged with the liquid due to limited perfusion of liquid within the nanostructures, where the liquid penetration is diffusion dependent [1, 2] and cannot be used for rapid detection subsequently. Moreover, true control of the shape and the subsequent surface area of the nanostructures on these electrodes is further limited by the process and its’ repeatability. [004] Whereas microstructured electrodes to date have been predominantly focused around microstructuring SU-8, which in essence is 2.5D structuring, hence limiting the surface area increase only via the lateral center to center spacing of the structured pillars [3], This process, although, giving high control over center to center spacing of the pillars, has limitations since slanted pillars will have higher surface area than straight pillars. Furthermore, ifthese pillars are integrated into microfluidic channels, there will be different interaction times between liquid at the edges of the channels and the middle, due to Poiseuille flow distribution along the length of the pillars. Contrary, 3D pillars present similarly distributed electrode density across the volume of the microchannel, subsequently all the surface area of liquid is interacting in the same way. [005] There is known a synthetic paper [4] manufactured with a method comprising the steps of: (a) providing at least two types of photo-polymerizable monomers, [b] exposing the volume to a three-dimensional light pattern to induce a polymerization reaction, and [c] removing uncured monomer to create an open microstructure. The volume comprises at least one monomer comprising at least two thiol groups and at least one monomer comprising at least two carbon-carbon double bonds, where the ratio (rl) between the number of thiol groups and the number of carbon-carbon double bonds fulfills one of: 0.5^rl^0.9 and l.l^rl^2. [006] There is known a method of preparing a functionalized electrode array [5] comprising deposition of a conductive material onto the surface of a substrate by droplet-based printing of particles comprising an electrically-conductive material. The surface of the conductive material is functionalized with a binding reagent that binds to an analyte. [007] The PDMS structures disclosed in [4 and 6] indicate that gold attachment to the OSTE surface via thiol groups is not possible. It was experimentally proven. This occurs because the thiol groups (-SH) present in the OSTE polymer react with the allyl groups (H2C=CH3-J in the PDMS material, makingthe thiol groups unavailable on the surface. [008] There is a known method of plating non-conductive surfaces, where metal ions in a solution are reduced by a reducing agent to form a metal coating on a surface without external electric current referred to as electroless plating [7], The method comprises a salt containing the desired metal ions, for example, silver (Ag+), and a reducing agent, for instance, glucose, where the metal ions (e.g. Ag+] are reduced to their metallic state (e.g. Ag] at a temperature below 80°C, leading to a uniform coating. Herein, the reaction can be performed using base or noble metals, for instance, copper, palladium, cobalt, and alloys to deposit a conductive layer typically thicker than 50 nm to achieve optimal conductivity [8], Summary of the Invention [008] The goal of the invention is to overcome the drawbacks of the prior art and provide a process for obtaining of a three-dimensional electrode and a process for obtaining an electrochemical biosensor. [009] The set goal is reached by the claimed process for obtaining of a three- dimensional electrode comprising the steps of: (i) providing a solid silicone-based formative bed, defining the three-dimensional electrode dimensions; (ii) introducing a UV curable polymer into the formative bed; the UV curable polymer is a thiol-ene polymer or a thiol-ene