In-situ controlled dissolution of metals using electrochemistry

Abstract

The present invention is in the field of metal dissolution suitable for biosensing applications. The present invention refers to a method and device for electrochemically controlled decomposition of metal particles using the biochemical properties of halides, which allows in-situ control and is suitable for the detection and quantification of analytes.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention is in the field of metal dissolution suitable for biosensing applications. The present invention refers to a device and method for dissolving metal particles using the biochemistry of halides, which allows in-situ control and is suitable for analyte detection and quantification.

[0002] [Background of the invention] The present invention includes a novel method for electrochemically controlled decomposition of metals without directly connecting them to an electrical circuit. Traditional methods for decomposing metals include dissolution in solution by directly applying a potential / current, thereby directly inducing an electrochemical dissolution reaction. Alternatively, metals can be decomposed by adding an etching solution, such as an acid or KI / I2 solution. The present invention enables the decomposition of metals that are not directly connected or that can be connected by an electrical potential, without the direct addition of an etching solution. Instead, metals are decomposed by locally generating active etching molecules from inert, and therefore non-etching, precursor molecules. This approach enables electrically controlled etching (controlled etch start, etch stop, and etch rate) of metals that are not or cannot be directly electrically connected, such as metal particles immobilized on a non-conductive substrate or freely floating in solution. The technology described herein can therefore dissolve such metal particles without the need for rinsing steps or toxic substances. Furthermore, this technology facilitates analyte detection in bioassays.

[0003] [Summary of the Invention] Therefore, the object of the present invention is to A) Providing metal flakes or particles either floating freely or adsorbed on or within a substrate B) contacting the metal with a solution containing an inert precursor C) applying an electrochemical potential to a solution containing an inert precursor, thereby producing a solution that decomposes the metal. The present invention provides a method for electrochemically controlled decomposition of metals, comprising:

[0004] The invention includes a method or device used to decompose metals or metal particles. The term "metal flakes or metal particles" refers to metal flakes or metal alloys, respectively. The invention is suitable for decomposing metals, metal salts, or metal alloys. Preferred metals can be selected from the group consisting of gold, iron or iron oxide, silver, platinum, cadmium, tellurium, lead, indium, zinc, copper, aluminum, germanium, niobium, strontium, vanadium, titanium, chromium, mercury, gallium, and palladium. The metal salt is preferably a metal oxide, sulfide, or nitrate, and can be selected from the group consisting of iron oxide, silicon oxide, zinc oxide, iron sulfite, gold(III) oxide (AuO), zinc sulfide, and cadmium-zinc sulfide.

[0005] Suitable metal particles include colloidal gold nanoparticles, europium nanoparticles, magnetic particles such as FeO particles or maghemite (FeO, γ-FeO), selenium nanoparticles, silver nanoparticles, platinum nanoparticles, palladium nanoparticles, copper nanoparticles, molybdenum nanoparticles, tungsten nanoparticles, titanium nanoparticles, aluminum nanoparticles, zinc oxide nanoparticles, zinc sulfide nanoparticles, cadmium sulfide nanoparticles, lead sulfide nanoparticles, and gallium arsenide nanoparticles. Metal particles that can be used as labels in bioassays are preferred. In one embodiment, the metal particles are colloidal particles. Therefore, the metal particles should be detectable at very low concentrations and retain their metal particle properties after binding with biorecognition molecules. Gold nanoparticles and silver nanoparticles are most preferred.

[0006] In step B) of the present application, the metal is contacted with a solution containing inert precursors. These solutions do not decompose the metal, so the initial etch rate is zero. Inert precursors can be molecules that cannot decompose or etch the metal, but can be converted (transformed) into active substances or molecules that can decompose or etch the metal. The inert precursors are preferably halide ions. Halide ions are negatively charged halogen atoms. Halide ions are iodides (I - ), fluoride (F - ), chloride (Cl - ), bromide (Br - ) and astatine (At - ). The solution may also contain a mixture of two or more different halide ions. A preferred halide ion is iodide. Halide ions, especially iodide, are usually added by dissolving a halide salt in the solution. A preferred salt is an iodide salt. The iodide salt may be selected from the group comprising: KI, NaI, LiI, HI, RbI, CsI, CuI, AgI, AuI, AtI, IBr, ICI, or TiI.

[0007] The inert precursor may be present or used in an amount sufficient to decompose the total amount of metal used. The appropriate concentration depends on different characteristics such as the volume, size of the electrode, and the amount of metal to be decomposed. The amount of inert precursor may range from 1 μM to 1 M, preferably 0.1 to 100 mM, preferably 0.5 to 50 mM, and even more preferably 1 to 20 mM.

[0008] Step B) of the method can be performed by deposition of dried inert precursor molecules onto a substrate. In another embodiment, the inert precursor can be adsorbed onto a respective substrate. In a second substep, the dried inert precursor is dissolved. The precursor can be dissolved by pipetting a solvent into the method setup. Suitable solvents are water (HO) or aqueous solutions (buffer solutions, salt solutions, acidic or basic solutions, organic solvent-containing solutions, oil-containing emulsions, or hydrogels, etc.). The inert precursor can also be dissolved by a fluid sample containing the analyte to be detected (saliva, blood, urine, tears, sweat, environmental samples, milk, etc.). The added solvent or sample can transport the inert precursor and / or its activated product and / or metal particles to the location where the metal is dissolved. Alternatively, the solution containing the inert precursor can be introduced by pipetting or microfluidic injection. The solution can also be present in the setup, and in step B), metal pieces or metal particles are added to the solution.

[0009] Step C) of the method according to the present invention refers to "applying an electrochemical potential to a solution containing an inert precursor, thereby generating a solution that decomposes the metal." To apply an electrochemical potential to a solution containing an inert precursor, at least two electrodes are placed in contact with the solution and a sufficient overpotential is applied to the electrodes. This can be done by attaching or turning on an external power source connected to the electrodes or by adding an electrochemical reaction to generate the required overpotential. Thus, one embodiment of the present invention refers to a method in which at least two electrodes are used to apply an electrochemical potential for electrochemically generating a solution that decomposes the metal. These electrodes may be a working electrode and a counter electrode.

[0010] Preferred embodiments include other electrodes, such as a reference electrode. Preferred embodiments include several working electrodes or sets of working and counter electrodes. Different working electrodes can be used as decomposition electrodes (first working electrode) or detection electrodes (second working electrode). The electrode sets can be configured to allow for multidetection assays, enabling multiplexed detection. Thus, a device according to the present invention may include at least five electrodes: at least two pairs of electrodes for applying an electrochemical potential to electrochemically generate a solution that decomposes the metal, and one electrode that is a reference or control electrode. The device may also include multiple (more than two, preferably more than ten) sets of electrodes, each set including at least a counter electrode, a reference electrode, a dissolution electrode, and a detection electrode. The set may also include two counter electrodes, two reference electrodes, two dissolution electrodes, and one detection electrode.

[0011] Thus, the present invention refers to a method in which the metal is not initially directly electrically connected to an electrochemical circuit. The decomposition of the metal is initiated and controlled by the electrochemical generation of active decomposition or etching molecules. In other words, the method allows for the electrochemically controlled decomposition of metals as an in-situ controlled reaction.

[0012] The term "producing a metal-decomposing solution" refers to the conversion of inert precursor molecules to molecules in solution that can decompose, etch, or dissolve the metal (active metal-decomposing molecules). This conversion or reaction is driven by an electrochemical reaction on an electrode, resulting in the production of an active metal-dissolving solution.

[0013] With iodide as an inert precursor in solution, electrochemical reactions on the electrode convert iodide to iodine and triiodide, resulting in the decomposition of the metal. This local generation of an active metal decomposition solution allows for electrochemically controlled etching of the metal, even when the metal is not directly connected to an external overpotential. The decomposition reaction kinetics can be further controlled by using different concentrations of inert precursor molecules, such as iodide (or another halide), in the solution. Furthermore, the amount of active metal decomposition molecules can be precisely controlled electrochemically by an externally applied power source. Larger and multiple overpotentials applied for longer periods generate larger amounts of active metal decomposition molecules, resulting in faster decomposition of the metal (etch rate) and metal decomposition further away from the electrode (etch location). Furthermore, because reaction rates can vary significantly for different materials, the electrode material can be used as a control factor. Desired metal decomposition parameters, reaction rates and amounts at a given location can thus be achieved by using appropriate concentrations of active metal decomposition molecules, such as iodide, and by applying appropriate external power functions, including but not limited to constant potential or current, potential or current steps, potential or current ramps, pulsed potential or current, or combinations thereof, appropriate to the electrode material.

[0014] A suitable anodic potential for a single indium tin oxide working electrode and iodide precursor may be 0.5 to 2 volts (relative to a carbon reference electrode) for 0.1 to 60 seconds at 1 to 50 mM KI. The process can be controlled more precisely by controlling multiple electrodes of different potentials and / or different materials. This allows for avoidance of excessive production of certain chemical products, such as iodine, which can result in a reduction in the dissolved chemistry by triggering a cathodic reaction at the second electrode. A suitable combination of two electrodes is indium tin oxide at a potential ranging from -1.1 V to -0.1 V and carbon at a potential ranging from +1 V to +0.2 V (relative to a carbon reference electrode) for 10 to 2000 seconds at 0.1 mM to 5 M KI. Furthermore, such a setup has the advantage of allowing simultaneous dissolution and deposition of metals. Depending on the application, deposition can be useful for metal detection.

[0015] Metals decomposed by this method or device can then be detected by known metal detection techniques, including, but not limited to, anodic stripping voltammetry, impedance spectroscopy, or resistance changes after metal plating of electrodes or resistive elements (e.g., nanowires) in a circuit.

[0016] The described invention can be used for electrochemical quantification of the amount of metal contained in metal particles. Such results can be used to quantify either the size or number of metal particles. An application of the quantification of metal particles using the described invention is a molecular sensing method or device in which molecules are quantified by binding to metal particles and subsequently quantifying the metal particles using the described method or device.

[0017] The described method is particularly suitable for detecting analytes in biosensing applications. It can be used as a detection and / or quantification method in assays that use biomolecular reporter molecules to detect analytes. Thus, the method of the present invention can be used in connection with immunoassays, e.g., lateral flow assays (LFAs), one-pot assays, and generally as a detection method in microfluidic assays. In general, the method of the present invention can be used for detecting and / or quantitating analytes in solution. Analytes can be any type of molecule, such as peptides, polypeptides, proteins, lipids, polysaccharides, polynucleotides, metabolites, hormones, toxins, or drug molecules.

[0018] The analyte may be present in any type of sample. The majority of biosensor applications are in clinical analysis, including the detection of various clinical analytes in whole blood, serum, saliva, sweat, tears, plasma, urine, cells, tissues, and other biological samples. Further suitable samples are: water, fuel, food, beverages, and plant extracts.

[0019] The metal particles can be directly or indirectly bound to molecules capable of binding to the analyte so that the analyte can be detected and / or quantified. Biosensor applications can be based on the principle of sandwich assays (generally used for larger analytes) or competitive assays (generally used for smaller analytes). Biosensor applications can also be designed as multiplex assays. Biosensor applications can be used to simultaneously detect the presence of multiple analytes in a single experiment. The number of analytes measured within one assay can be more than 3, more preferably more than 10, or even more than 100.

[0020] The steps of the bioassay must be carried out under conditions suitable for allowing binding between the relevant molecules. These conditions are known to those skilled in the art and refer, for example, to temperature, time, pH value. One condition is that the relevant molecules must not change their structure (e.g., proteins must not be denatured).

[0021] Thus, one embodiment of the present method involves decomposition of metal particles bound to biomolecules. The biomolecules may be proteins, polypeptides, oligopeptides, aptamers, polynucleotides, polycarbohydrates, e.g., oligosaccharides, or lipids. The biomolecule may be any molecule capable of recognizing and binding to a specific analyte. Examples include receptors, ligands, peptides, polypeptides, proteins, polysaccharides, polynucleotides, antibodies, and antibody fragments. Suitable antibody fragments include: Fab fragments, Fab' fragments, F(ab')2 fragments, Fd fragments, Fd' fragments, Fv fragments, dAb fragments, scFv (single-chain) fragments, isolated CDR regions, dsFv diabodies, single-chain antibodies, and combinations thereof. Protocols for labeling metal particles, such as those based on NHS-ester modification, are well known in the art.

[0022] In immunoassays, it may be suitable for metal particles and / or inert precursors to be adsorbed onto or within a substrate. Therefore, the substrate is preferably a non-conductive substrate. The substrate may be a glass plate, a plastic plate such as a multi-well plate (microtiter plate), a microchannel wall, a porous three-dimensional substrate (e.g., paper, cellulose nitrate membrane, or glass fiber membrane), or a non-conductively coated electrode. The substrate may be capable of liquid transport.

[0023] Using the method of the present invention, it is possible to quantify the analyte in the sample. To be able to quantify the analyte, the amount of dissolved metal must be quantified. Therefore, the present invention further provides an additional D) Electrically or electrochemically detecting and / or quantifying the dissolved metals It means an embodiment containing:

[0024] Detection of the dissolved metal is accomplished using the same or a different electrode than that used to drive the decomposition reaction. Anodic stripping voltammetry, impedance spectroscopy, or similar techniques, such as plating electrode resistance change, are suitable techniques for quantifying the dissolved metal.

[0025] The step "electrically or electrochemically quantifying the decomposed metal" may involve plating the decomposed metal (metal ions) onto an electrode, e.g., a plating electrode or a detection electrode, by application of a plating potential followed by dissolution of the plated metal. The subsequent dissolution of the plated metal generates a current proportional to the amount of plated metal and, therefore, proportional to the amount of analyte present in the sample. Thus, one embodiment of the present invention refers to a method in which step D) comprises plating of decomposition-induced metal particles by application of a plating potential to a detection electrode, followed by measurement of the current caused by the electrochemical dissolution of the plated metal particles. Metal plating can occur during or after application of the decomposition trigger potential.

[0026] One aspect of the present invention is A1) providing metal pieces or particles bound to molecules specific for the analyte in solution; A2) adding the solution of A1) to a setup where another molecule specific for the analyte is bound to the electrode and binds the analyte. A3) Binding metal particles to the specimen B) contacting the metal with a solution containing an inert precursor C) applying an electrochemical potential to a solution containing an inert precursor, thereby producing a solution that decomposes the metal. D) Quantifying the dissolved metals electrically or electrochemically The present invention also refers to a bioassay comprising the method of the present invention, which may comprise:

[0027] Another aspect of the present invention is a method for producing a semiconductor device comprising: A1) providing metal pieces or particles bound to molecules specific for the analyte in solution; A2) adding the solution of A1) to a setup in which another molecule specific for the analyte is bound to an electrode. A2') Binding the analyte to a molecule specific for the analyte and bound to an electrode A3) Binding metal particles to the analyte (via molecules specific to the analyte and bound to the metal particles). B) contacting the metal with a solution containing an inert precursor C) applying an electrochemical potential via electrodes to the solution containing the inert precursor, thereby producing a solution that decomposes the metal. D) Quantifying the dissolved metals electrically or electrochemically The present invention also refers to a bioassay comprising the method of the present invention, which may comprise:

[0028] The above steps may be performed in a different chronological order or may be performed simultaneously.

[0029] One aspect of the present invention is A1) Providing a setup containing a substrate with at least two electrodes in close proximity to molecules specific for the analyte and bound to the substrate. A2) adding to the setup of A1) metal pieces or particles that are bound to another molecule specific for the analyte. A3) Adding an inert etching precursor to the setup B1) Adding a solution containing an analyte (e.g., a sample) to the setup of A3). B2) Dissolving metal particles and an inert precursor in a solution B3) Binding the analyte to the metal particles B4) Binding the analyte to the molecules on the substrate B5) contacting the solution with the electrodes of the setup of A3) C) applying an electrochemical potential via electrodes to the solution containing the inert precursor, thereby producing a solution that decomposes the metal. D) Quantifying the dissolved metals electrically or electrochemically The present invention also refers to a bioassay comprising the method of the present invention, which may comprise:

[0030] The chronological order of the above steps may be changed or may be performed simultaneously. Proximity therefore means that the distance between at least one electrode and the analyte-specific molecule bound to the substrate is 10 mm or less, preferably less than 3 mm, and even more preferably less than 1 mm.

[0031] Another aspect of the present invention is a method for producing a semiconductor device comprising: A1) Providing metal pieces or particles that are bound to molecules specific for the analyte and floating freely in solution. A2) adding a specimen (e.g., a sample) to the solution of A1). A3) Adding the solution from A2) to a setup on which a known amount of analyte is immobilized. A3) Binding metal particles to the immobilized analyte B) contacting the metal with a solution containing an inert precursor C) applying an electrochemical potential to a solution containing an inert precursor, thereby producing a solution that decomposes the metal. D) Quantifying the dissolved metals electrically or electrochemically The present invention also refers to a bioassay comprising the method of the present invention, which may comprise:

[0032] Another aspect of the present invention is a method for producing a semiconductor device comprising: A1) providing a setup containing a substrate with at least two electrodes in close proximity to a known amount of analyte molecules bound to the substrate, which are specifically bound to metal particles via analyte-specific binding molecules; A2) Adding an inert etching precursor to the setup B1) adding a solution containing an analyte (e.g., a sample) to the setup of A2) B2) Dissolving an inert precursor in a solution B3) Replacing the substrate-bound analytes with free-floating analytes and dissociating a portion of the metal particles from the substrate. B4) contacting the solution with the electrodes of the setup of A3) C) applying an electrochemical potential via electrodes to the solution containing the inert precursor, thereby producing a solution that decomposes the remaining metal. D) Quantifying the dissolved metals electrically or electrochemically The present invention also refers to a bioassay comprising the method of the present invention, which may comprise:

[0033] The present invention may also include at least one washing step, in which unbound analyte molecules and / or unbound metal particles are washed away. Furthermore, the present invention may be suitable for blocking non-specific binding of different components of the assay, such as metal particles. Those skilled in the art will know or can determine when such washing or blocking steps are necessary.

[0034] A preferred assay for practicing the methods of the present invention is a lateral flow assay. Lateral flow assays are a subset of assays that combine various reagents and process steps into a single assay strip, thereby providing a sensitive and rapid means for detecting analytes. In a lateral flow assay, the inactive precursor can be dried onto the conjugate pad or an additional separate pad.

[0035] In a typical method, a liquid sample potentially containing an analyte is added to a porous carrier, which may consist of an arrangement of several components. More specifically, a measurement time, e.g., 5 seconds, or a measurement volume, e.g., 2 drops, is introduced into a designated area at the sampling end (also called the "proximal end") of the porous carrier. The liquid sample then migrates through the porous carrier toward the dry end (also called the "distal end"). While migrating through the porous carrier, the sample mobilizes metal particles that act as labels reversibly (temporarily) immobilized in the porous carrier. Alternatively, the metal particles may be added together with the sample solution. While the analyte interacts with the bound metal particles, the liquid sample and the mobilized metal particles continue to migrate through the porous carrier toward the detection zone, where a reagent that binds to the same analyte is fixed or immobilized, usually in the form of a line (which may be a dot). If an analyte is present in the liquid sample, a "sandwich" of mobilized metal particles:analyte:immobilized reagent is formed, and the resulting concentration of metal particles results in a visible line appearing in the detection zone, indicating a positive result. Alternatively, the metal particles are first decomposed using the methods of the present invention to become detectable. In either case, the metal particles, and therefore the analyte, can be quantified using the methods for decomposition of metals of the present invention.

[0036] Therefore, the inert precursors are also dissolved by the sample fluid and transported to the detection zone. To quantify the adsorbed metal particles, the particles are first dissolved using the method of the present invention, followed by detection of the dissolved metal molecules by anodic stripping voltammetry, impedance spectroscopy or electrode resistance changes using the same or a different electrode.

[0037] After the detection zone, there may be a control zone, where another reagent is immobilized. This reagent binds directly to the bound particles. If the test is performed properly, the reagent acts as a control. The control can be analyzed optically or by the methods of the invention described.

[0038] A second aspect of the present invention is directed to a device for carrying out the above method. Accordingly, one embodiment of the present invention is directed to a device for electrochemically controlled decomposition of metals, comprising at least two electrodes, metal flakes or particles either free-floating or adsorbed on or in a substrate not directly electrically connected to the electrochemical circuit, and a solution containing an inert precursor, wherein application of an electrochemical potential to the solution containing the inert precursor is suitable to produce a solution that decomposes the metal.

[0039] Preferably, the inert precursor is a halide. Moreover, all aspects disclosed in relation to the above method should also apply to the device. The device may further include leads to a power source and to the electrodes. Moreover, it is preferred that the device further includes a reference electrode. Thus, one embodiment of the device includes three electrodes: two electrodes for applying an electrochemical potential for electrochemical generation of a solution that decomposes the metal, and one electrode is a reference electrode.

[0040] The metal flakes or particles can be adsorbed on or within a substrate, where the substrate is a non-conductive substrate or a conductive substrate coated with a non-conductive coating. The device may include a housing and / or backing containing the substrate and the electrodes. A recommended example of a device is the use of a three-electrode setup including a working, counter, and reference electrode. The working electrode may be made of any conductive material or combination of conductive materials, such as carbon, graphene, carbon nanotubes, gold, silver, silver chloride, platinum, boron-doped diamond, mercury droplets, bismuth, or indium tin oxide. The counter electrode may be made of any conductive material or combination of conductive materials, such as carbon, graphene, carbon nanotubes, gold, silver, silver chloride, platinum, boron-doped diamond, mercury droplets, bismuth, or indium tin oxide. The reference electrode may be made of any conductive material or combination of conductive materials, such as carbon, graphene, carbon nanotubes, gold, silver, silver chloride, platinum, boron-doped diamond, bismuth, mercury droplets, or indium tin oxide.

[0041] Another embodiment of the present invention refers to a device containing at least one or a first electrode pair for dissolved metal and a second electrode pair used to apply an electrochemical potential for electrochemical generation of a solution that decomposes the metal. For the purpose of rapidly decomposing metal particles and accurately measuring the amount of dissolved metal using the described method or device, it is not necessary, but beneficial, to use an electrode setup that includes more than two electrodes, including more than one working electrode. One (subset) of working electrode(s) can be used to effect the dissolution chemistry, while another (subset) of working electrode(s) can be used to detect the dissolved metal and may not be involved in effecting the dissolution chemistry.

[0042] The device according to the present invention is preferably suitable for quantifying an analyte in a solution. Therefore, the device can be designed in a typical setup of a bioassay, such as a lateral flow assay. The device may have a single assay chamber, allowing for a so-called one-pot assay.

[0043] The most accurate quantification of dissolved metal can be achieved by using micro- or nano-sized sensing working electrode(s). To maximize the signal-to-noise ratio, best results can be obtained by using multiple micro- or nano-sized sensing working electrodes positioned and connected to maximize the amount of dissolved metal captured during plating while simultaneously minimizing the background current generated.

[0044] In certain embodiments, the dynamic range can be expanded by multiple detection regions configured for different analyte concentration ranges. Each region is a reporter for a different drug concentration range, so that the overall dynamic range of the assay or device is increased due to the combination of the individual concentration ranges. This effect can be achieved by multiple detection regions with constant or varying concentrations of analyte-binding molecules. Alternatively, the electrochemical decomposition time of the metal particles can be varied in different regions, from short to long. This allows for a large dynamic range while maintaining rapid test results when long decomposition times are not required. A third approach is to use different concentrations of an inert precursor in different detection regions. This can be achieved by multiplexing in separate, isolated channels. Thus, one embodiment refers to a device of the present invention that includes multiple detection regions, which can be configured differently, e.g., so that the dynamic range of each region falls within a different concentration range, increasing the combined overall dynamic range of the device or improving quantification accuracy. It is also possible to quantify several different analytes. Thus, one embodiment refers to a device of the present invention that is suitable for multiplexed detection of different analytes. Multiple measurements can be performed sequentially, both in time or space, adjacent to each other or overlapping each other, or by using different metals or different electrodes.

[0045] The present invention also contemplates devices that utilize at least one reference region: one or more electrochemical references, such as positive or negative control standards or a combination thereof, using electrochemical or optical readout methods, or additionally introduced electroactive species.

[0046] Another embodiment of the present invention is: At least two electrodes Metal particles capable of binding to biomolecules as defined above an inert precursor as defined above, which may be dissolved in a solution; A kit for electrochemically controlled decomposition of metals comprising:

[0047] These kits may further contain solutions for dissolving inactive precursors, analyte-binding molecules, blocking buffers, control or standard reagents. In molecular biology or medical diagnostics, a kit is a package containing all the materials necessary to perform a particular method or single step. Standard chemicals present in any standard molecular biology, chemistry, or medical laboratory are typically not included. Nevertheless, some of these standard chemicals may be essential for properly performing the assays or methods of the present invention. It should be understood that all materials are provided in amounts sufficient to properly perform the desired reactions in the majority of scientific, diagnostic, and industrial applications. Often, but not always, these materials are provided in ready-to-use or nearly ready-to-use, pre-prepared solutions. There may also be combinations of different materials already added together. An additional advantage is that such kits are pre-tested. Therefore, the operator does not need to re-prove the feasibility of the diagnostic method, eliminating at least some control experiments. Kits are therefore very popular tools in research, diagnostic, and industrial laboratories. [Brief explanation of the drawings]

[0048] [Figure 1] 1 shows a schematic diagram of a method and a device for the method according to the invention; [Figure 2] 1 shows a schematic diagram of a method according to the present invention suitable for detecting an analyte and an assay chamber for detecting and quantitating the analyte. [Figure 3] 1 shows a schematic diagram (top view) of a device suitable for carrying out a method according to the invention arranged as a lateral flow assay. [Figure 4] 1 shows a schematic diagram (side view) of a device suitable for carrying out a method according to the invention arranged as a lateral flow assay. [Figure 5] FIG. 4 shows a top view of an alternative electrode design for the device shown in FIG. 3. [Figure 6] FIG. 4 shows a top view of another alternative electrode design for the device shown in FIG. 3. [Figure 7] FIG. 4 shows a top view of an alternative electrode design for the membrane geometry of the device shown in FIG. 3. [Figure 8] An exemplary design of a suitable assay is shown, which includes multiple detection zones that represent different target concentration ranges. [Figure 9] 15 shows the device used to obtain the measurements shown in FIGS. 10 to 14. [Figure 10] 1 shows an optical microscope image of gold nanoparticles on a transparent indium tin oxide electrode insulated by a layer of bovine serum albumin, demonstrating successful etching of electrically isolated particles. [Figure 11] The experimental results of the setup shown in Figure 9 are plotted as particle scattering intensity versus time, with and without potassium iodide in solution, including applied potential cycles. [Figure 12] The plot shows experimental results for a setup like that of Figure 9, where a constant potential is applied and the amount of potassium iodide is varied. [Figure 13] The plot shows the experimental results for a setup like that shown in Figure 9, where the amount of potassium iodide is kept constant and the applied potential is varied. [Figure 14] 16 illustrates a proof-of-concept experimental device for biosensing applications of the method according to the present invention that was used to obtain the experimental measurements shown in FIG. 15. [Figure 15] Experimental results of a setup such as that shown in FIG. 14 are plotted, including quantitative readout currents after dissolution of various amounts of gold particles.

[0049] DESCRIPTION OF THE PREFERRED EMBODIMENTS The following more detailed description of embodiments of the present methods is representative of exemplary embodiments of the technology, with like parts numbered throughout.

[0050] Figure 1 shows a schematic diagram of the method according to the invention and the etching chamber used in the method. Three electrodes are arranged in the chamber: a counter electrode 102, a working electrode 106 and a reference electrode 104. The setup further comprises a power supply 100 and leads to the electrodes (101, 103, 105). - A solution containing I is filled into the chamber and is in contact with the electrode and at least one metal particle (such as 107 gold). As long as the power supply is off, no etching occurs. Upon activation of the power supply (Figure 1B), I - From I3 - This results in the local generation of iodine (108; or similar reactions with other halide molecules) and efficient etching of the metal particles. In a first reaction 121, elemental iodine 122 is formed, which reacts with the gold particles in a second reaction 123 to give AuI 124. This reaction can also be used to dissolve Ag, Pt, or other metal particles. The counter and reference electrodes can be two separate electrodes or fused into one electrode.

[0051] FIG. 2 shows a schematic diagram of a method according to the present invention suitable for analyte detection and an assay chamber for analyte detection and quantification. In FIG. 2A, an assay for binding analyte molecules 204 has been completed. The analyte 204 is bound to a receptor molecule 203 (e.g., a capture IgG), and metal particles 206 are bound to a second receptor molecule 205 (e.g., a reporter IgG). During the binding assay, the second receptor molecule 205 bound to the analyte 204. In FIG. 2B, an etching reaction is initiated, dissolving the metal particles 206 to produce metal ions 220. The etching reaction is carried out when an electrochemical potential is applied using the dissolution electrode 201 and counter electrode 207. Application of a plating potential to the detection electrode 202 (counter electrode 208) results in plating of metal ions on the detection electrode 202 (electroplated metal ions 240). Electrochemical dissolution of the plated metal particles 240 generates a current proportional to the amount of plated metal and therefore proportional to the amount of analyte molecules 204 present in the sample.

[0052] Figure 3 shows a schematic diagram (side view and top view) of a device suitable for carrying out the method according to the invention arranged as a lateral flow assay suitable for the detection of molecules in a liquid sample. Figure 3A shows a side view. The sample is deposited on a sample pad 300 and transported through the pad and filtration membrane 303 towards a collection pad 306 and a readout device 307. The electrodes and sample carrier strip are separated by an insulator 317.

[0053] Figure 3B) shows a top view of an exemplary lateral flow assay. The bottom surface 308 of the substrate supports a sample pad 300 and a conjugate pad 302, separated by a filtration membrane 301. A sample carrier strip 303 contains a detection region 304 and a control region 305. A collection pad is located at the end.

[0054] Figure 3C) shows a top view without the pads and membrane, revealing the underlying structure. Here, the electrode arrangement can be seen, which occurs twice: one set for sample detection and one set for the control stripes. Test counter electrode 309 is placed next to test reference electrode 310, followed by test working electrode 311 (dissolution electrode). Next to that is test working electrode 312 (detection electrode). The control set contains control counter electrode 313, control reference electrode 314, control working electrode (dissolution electrode) 315, and control working electrode (detection electrode) 316. Figure 3D) shows another top view without the pads, membrane, and insulating layer, revealing the underlying structure.

[0055] The figure shows a schematic diagram (side view) of an alternative device suitable for carrying out a method according to the invention arranged as a lateral flow assay, which is an alternative design to the device shown in Figure 3. One difference is the substrate upper surface 400, which contains the test working electrode 312 (detection electrode).

[0056] FIG. 5 shows an alternative electrode design in which a control counter electrode 313 and a test reference electrode 310 are arranged concentrically around a test working electrode 312 (the sensing electrode).

[0057] 6 is an alternative design of a sensing electrode suitable for a device according to the invention. The sensing electrode 312 may be one area or may be separated into several areas by insulators 317.

[0058] Figure 7 shows a top view of an alternative design for the membrane geometry of the device shown in Figure 3 or 4. The membrane thickness may decrease (analyte solution concentration) towards the center where the detection region 304 and control region 305 are located.

[0059] Figure 8 shows an exemplary design of a suitable assay including multiple detection regions constituting different target concentration ranges for increasing the overall dynamic range, improving quantification accuracy, or multiplexing quantification of different molecules. As shown in Figure 8A, different detection regions (800, 801, 802) may be arranged one after the other on the sample carrier strip 303, followed by one control region 305. Alternatively, the sample carrier strip 303 may be separated into different channels (e.g., arranged in parallel) by multi-channel barriers 806, 807, each containing one respective detection region (800, 801, 802) and one control region (803, 804, 805). Preferably, the individual detection regions (800, 801, 802) and control regions (803, 804, 805) exhibit a staggered arrangement.

[0060] Figure 9A shows the setup used for the proof-of-concept experiment. Indium tin oxide-coated microscope slides (shown in Figure 9B) were coated with bovine serum albumin and subsequently with gold nanoparticles (40 nm). The slides were further mounted in a flow cell providing open wells that allowed in situ imaging using dark-field microscopy.

[0061] After adding a 150 mM NaCl solution, the gold nanoparticles show a constant intensity during six cycles of cyclic voltammetry from -0.2 to 1.4 V (see Figures 10(a) and 11(a)). As illustrated in Figures 10(b) and 11(b), after adding a 3 mM solution of KI in 150 mM NaCl, the Au nanoparticles remain unchanged before the electrochemical potential is applied, but are etched very quickly once the applied potential reaches 1.2 V. Using the same setup (Figure 9), we measured the dissolution kinetics, which depend on the amount of potassium iodide used (Figure 12) and the applied overpotential (Figure 13).

[0062] Figure 14 shows another setup used for proof-of-concept experiments, namely, a proof-of-concept embodiment of an electrochemical lateral flow assay, including a three-electrode setup and a lateral flow assay strip. A line of adsorbed gold nanoparticles was added to the strip, as shown in Figure 14A. The same setup after electrochemical dissolution of the gold nanoparticles is illustrated in Figure 14B. The gold nanoparticles were functionalized with biotin, and the detection / quantification line was functionalized with streptavidin, which specifically binds the biotin on the gold nanoparticles. The corresponding readout currents using cyclic voltammetry of the above test strip are shown in Figure 15(a), after dissolution of the gold nanoparticles (circles), and for a control experiment without gold nanoparticles (crosses). Figure 15(b) shows the linear response of the peak current for a dilution series of gold nanoparticles.

Claims

1. A) Providing gold nanoparticles, either free-floating or adsorbed on or within a substrate, that are conjugated with biomolecules capable of recognizing and binding specific analytes. B) contacting said gold nanoparticles with a solution that contains iodide as an inert precursor that remains inactive until step C) and does not decompose said gold nanoparticles. C) applying an electrochemical potential to the solution containing the inert precursor via an electrode that converts the iodide to iodine and triiodide, thereby producing a solution that decomposes the gold nanoparticles. D) electrically or electrochemically detecting or quantifying the decomposed gold nanoparticles using a working electrode made from indium tin oxide.

1. A method for electrochemically controlled degradation of gold nanoparticles conjugated to biomolecules, comprising:

2. The method of claim 1, wherein at least two electrodes are used to apply an electrochemical potential to electrochemically generate the solution that decomposes the gold nanoparticles.

3. 3. The method of claim 1 or 2, wherein the gold nanoparticles are adsorbed on or in a substrate, the substrate being a non-conductive substrate or a conductive substrate with a non-conductive coating.

4. 4. The method of any one of claims 1 to 3, used for detecting the presence of an analyte and / or for quantifying an analyte in a lateral flow assay, which may be operated as a competitive or sandwich assay, and / or used to simultaneously detect the presence of multiple analytes in a single experiment.

5. 5. The method of claim 1, wherein step D) comprises plating gold ions caused by decomposition by application of a plating potential to a detection electrode and subsequent determination of the current caused by electrochemical dissolution of the plated gold ions.

6. Decomposing the gold nanoparticles bound to the biomolecules quantitating the amount of decomposed gold nanoparticles using anodic stripping voltammetry on an indium tin oxide working electrode. wherein the degradation and quantification is performed in the presence of iodide, and the gold nanoparticles are not degraded before the iodide is converted to iodine and triiodide. Method for quantification of gold nanoparticles conjugated to biomolecules.

7. at least two electrodes, at least one of which is an indium tin oxide working electrode; Gold nanoparticles conjugated to biomolecules capable of recognizing and binding specific analytes, either free-floating or adsorbed on or within a substrate that is not directly electrically connected to an electrochemical circuit; and A solution containing an inert precursor, which is an iodide wherein application of an electrochemical potential to the solution containing the inert precursor is suitable to produce a solution that decomposes the gold nanoparticles, which are then detected or quantified via an electrode that converts the iodide to iodine and triiodide, and wherein the indium tin oxide working electrode is used to detect or quantitate the decomposed gold nanoparticles. A device for electrochemically controlled decomposition of gold nanoparticles suitable for the detection or quantification of analytes in solution.

8. three electrodes, two of which are for applying an electrochemical potential for the electrochemical generation of a gold-decomposing solution, and one electrode is a reference electrode; or It comprises at least four electrodes, at least two working electrodes (a dissolution electrode and a detection electrode), one counter electrode and one reference electrode; or 8. The device of claim 7, comprising at least two sets of electrodes, each set comprising at least one detection electrode, at least one dissolution electrode, at least one counter electrode, and at least one reference electrode.

9. 9. The device of claim 7 or 8, wherein the gold nanoparticles are adsorbed on or in a substrate, the substrate being a non-conductive substrate.

10. A device as claimed in any one of claims 7 to 9, comprising an electrode for detecting dissolved gold which is not the same as that used for applying an electrochemical potential for the electrochemical generation of a solution that decomposes gold, and / or several electrodes for detecting said dissolved gold arranged in several detection regions, each detection region representing a single analyte or a particular analyte concentration.

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