Abstract
Methods and devices for electrochemically controlled degradation of metals particles using halide biochemistry allowing in-situ control and being suitable for analyte detection and quantification.
Claims
1. A method for electrochemically controlled degradation of metals comprising the following steps: A) metal particles free floating or adsorbed on or in a substrate and being conjugated to biomolecules having the ability to recognize and bind a specific analyte, B) contacting the metal particles with a solution not degrading the metal particles and containing an inactive precursor which remains inactive until step C, C) applying an electrochemical potential to the solution containing the inactive precursor and thereby generating a solution that degrades the metal particles, D) detecting or quantifying electrically or electrochemically the degraded metal particles.
2. The method according to claim 1, wherein the inactive precursor is a halide.
3. The method according to claim 1, wherein at least two electrodes are used for the application of the electrochemical potential for electrochemical generation of the solution that degrades the metals.
4. The method according to claim 1, wherein the metal particles are adsorbed on or in a substrate and wherein the substrate is a non-conductive substrate or a conductive substrate with a non-conductive coating.
5. The method according to claim 1 wherein the detection electrode is made of indium tin oxide.
6. (canceled)
7. The method according to claim 1, used to detect presence of an analyte and/or quantify an analyte within a lateral flow assay which may be operated as competitive or sandwich assay and/or used to simultaneously detect presence of multiple analytes in a single experiment.
8. The method according to claim 1, wherein step D) comprises plating of metal ions caused by degradation due to application of a plating potential to a detection electrode and subsequently determination of electric current caused by electrochemical dissolution of plated metal ions.
9. A device for electrochemically controlled degradation of metal particles suitable for detection or quantification of analytes in solution, comprising: at least two electrodes, metal particles conjugated to biomolecules having an ability to recognize and bind a specific analyte, where the metal particles are free floating or adsorbed on or in a substrate being not directly electrically connected by an electrochemical circuit, and a solution containing an inactive precursor, wherein application of an electrochemical potential to the solution containing the inactive precursor is suitable for generating a solution that degrades the metal particles which are then detected or quantified.
10. The device according to claim 9, wherein the inactive precursor is a halide.
11. The device according to claim 9, comprising three electrodes, wherein a first and second of said three electrodes are suitable for application of an electrochemical potential for electrochemical generation of a solution that degrades metals and a third of said three electrodes is a reference or control electrode, or comprising at least four electrodes, wherein said at least four electrodes comprise at least two working electrodes, one counter electrode and one reference or control electrode, or comprising at least two sets of electrodes, wherein each set of electrodes comprises at least one detection electrode, at least one dissolution electrode, at least one counter electrode, and at least one reference electrode.
12. The device according to claim 9, wherein the metal particles are adsorbed on or in a substrate and wherein the substrate is a non-conductive substrate.
13. The device according to claim 9, wherein the detection electrode is made or indium tin oxide.
14. The device according to claim 9, containing electrodes for detection of the dissolved metal being different than the electrodes for application of the electrochemical potential for the electrochemical generation of a solution that degrades metals and/or containing several electrodes for detection of the dissolved metal arranged within several detection areas, wherein each detection area refers to a single analyte or to a specific analyte concentration.
15. A device containing at least two electrodes, including a working electrode made of indium tin oxide, suitable for quantification or detection of gold nanoparticles conjugated to a biomolecule wherein the method for quantification comprises the following steps: a. degradation of the gold nanoparticles conjugated to a biomolecule, b. quantifying an amount of degraded gold with anodic stripping voltammetry on the indium tin oxide working electrode, wherein the degradation and quantification are performed in presence of iodide.
16. A method for the quantification of gold nanoparticles conjugated to a biomolecule comprising the following steps: degradation of gold nanoparticles conjugated to a biomolecule, quantifying the amount of degraded gold with anodic stripping voltammetry on an indium tin oxide working electrode, wherein the degradation and quantification are performed in presence of iodide.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0094] These and further features of the invention are described hereinafter with reference to the drawings, wherein:
[0095] FIGS. 1A-1B show a schematic overview of a method according to the invention and a device for the method.
[0096] FIGS. 2A-2D show a schematic overview of a method according to the present invention suitable for detection of an analyte and an assay chamber for the detection and quantification of analytes.
[0097] FIGS. 3A-3D show a schematic overview (top view) of a device suitable to perform a method according to the present invention arranged as lateral flow assay.
[0098] FIGS. 4A-4D show a schematic overview (side view) of a device suitable to perform a method according to the present invention arranged as lateral flow assay.
[0099] FIG. 5 shows a top view of an alternative electrode design for the device shown in FIGS. 3A-3D.
[0100] FIGS. 6A-6C show a top view of another alternative electrode design for the device shown in FIGS. 3A-3D.
[0101] FIG. 7 shows a top view of alternative designs for the membrane shape of the device shown in FIGS. 3A-3D.
[0102] FIGS. 8A-8B show an exemplary design of an assay suitable including multiple detection areas configuring for a different target concentration range.
[0103] FIGS. 9A-9B show the device used to acquire the measurements shown in FIGS. 10A-14B.
[0104] FIGS. 10A-10B show light microscopy images of gold nanoparticles on a transparent indium tin oxide electrode insulated by a layer of bovine serum albumin showing the successful etching of the electrically insulated particles.
[0105] FIGS. 11A-11B show in a plot experimental results of the setup as in FIGS. 9A-9B. The particle scattering intensity is plotted against time with applied potential cycles with and without potassium iodide in solution.
[0106] FIG. 12 shows in a plot experimental results of the setup as in FIGS. 9A-9B, with a constant potential applied and varying amount of potassium iodide.
[0107] FIG. 13 shows in a plot experimental results of the setup as in FIGS. 9A-9B, with a constant amount of potassium iodide and varying applied potential.
[0108] FIGS. 14A-14B illustrate a proof-of-concept experimental device for biosensing applications of the method according to the invention that was used to acquire the experimental measurements shown in FIGS. 15A-15B.
[0109] FIGS. 15A-15B show in a plot experimental results of the setup as in FIGS. 14A-14B, with the quantitative readout current after the dissolution of varying amounts of gold particles.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0110] The following more detailed description of the embodiments of the method is a representative of exemplary embodiments of the technology, wherein similar parts are designated by same numerals throughout.
[0111] FIGS. 1A-1B show a schematic overview of a method according to the invention and an etching chamber used for the method. Within a chamber three electrodes, a counter electrode 102, a working electrode 106 and a reference electrode 104, are arranged. The setup includes further a power supply 100 and conductors to the electrodes (101, 103, 105). A solution containing I″ is filled in the chamber and has contact to the electrodes and at least one metal particle (107 such a s gold). As long as the power supply is off, no etching occurs. Activation of the power supply (FIG. 1 B) leads to the local generation of I.sup.3− from I.sup.− (108; or analogous reactions using other halide molecules) and to the efficient etching of the metal particle. In a first reaction 121 elemental Iodine 122 is formed and in a second reaction 123 this elemental iodine reacts with the gold particle, resulting: AuI.sub.2 124. This reaction might also be used to dissolve Ag, Pt or other metal particles. The counter and reference electrodes may be two separate electrodes or fused to one electrode.
[0112] FIGS. 2A-2D show a schematic overview of a method according to the present invention suitable for detection of an analyte and an assay chamber for the detection and quantification of analytes. Assay chamber for the detection and quantification of molecules. In FIG. 2A) the assay for the binding of the analyte molecule 204 is completed. The analyte 204 has been bound to the receptor molecule 203 (e.g. capture IgG) and the metal particle 206 has been conjugated to a secondary receptor molecule 205 (e.g. reporter IgG). During the binding assay the secondary receptor molecule 205 as bound to the analyte 204. In FIG. 2 B), the etching reaction is initiated leading to the dissolution of the metal particle 206 into metal ions 220. The etching reaction is carried out when an electrochemical potential is applied using the dissolution electrode 201 and counter electrode 207. The application of a plating potential to the detection electrode 202 (counter electrode 208) leads to the plating of the metal ions on the detection electrode 202 (electro plated metal ions 240). The electrochemical dissolution of the plated metal particles 240 generates an electric current proportional to the amount of plated metal and thus proportional to the amount of analyte molecules 204 present in the sample.
[0113] FIGS. 3A-3D show a schematic overview (side view and top view) of a device suitable to perform a method according to the present invention arranged as lateral flow assay suitable for the detection of molecules in liquid samples. FIG. 3A) shows a side view. The sample is deposited on the sample pad 300 and wicks through the pads and filter membrane 303 towards the collection pad 306 and the read-out device 307. The electrodes and the sample carrier strip are separated by an insulator 317.
[0114] FIG. 3 B) shows a top view of an exemplary lateral flow assay. The substrate bottom 308 supports a sample pad 300, and a conjugation pad 302, separated by a filter membrane 301. A sample carrier strip 303 includes a detection area 304 and a control area 305. At the end a collection pad is arranged.
[0115] FIG. 3 C) shows a top view without the pads and the membrane revealing the underlying structures. Here one can see the arrangement of the electrodes, which are present twice, one set for the sample detection, one set for the control stripe. The test counter electrode 309 is located next to a test reference electrode 310 and followed by the test working electrode 311 (dissolution electrode). Next to it the test working electrode 312 (detection electrode) is located. The set for the control contains control counter electrode 313, control reference electrode 314, control working electrode (dissolution electrode) 315 and control working electrode (detection electrode) 316. FIG. 3 D) shows another top view without the pads, membranes and insulating layers revealing the underlying structures.
[0116] FIGS. 4A-4D show a schematic overview (side view) of alternative devices suitable to perform a method according to the present invention arranged as lateral flow assay and being an alternative design of the device shown in FIGS. 3A-3D. One difference is a substrate top 400 containing the test working electrode 312 (detection electrode).
[0117] FIG. 5 shows an alternative electrode design, wherein a control counter electrode 313 and a test reference electrode 310 are arranged concentric around the test working electrode 312 (detection electrode).
[0118] FIGS. 6A-6C show alternative designs for the detection electrode suitable for devices according to the present invention. The detection electrode 312 may be one area or may be separated to several areas by insulators 317.
[0119] FIG. 7 shows a top view of alternative designs for the membrane shape of the device shown in FIGS. 3A-3D or FIGS. 4A-4D. The thickness of the membrane may decrease to the middle where the detection area 304 and the control area 305 are located (concentration of the analyte solution).
[0120] FIGS. 8A-8B show an exemplary design of an assay suitable including multiple detection areas configuring for a different target concentration range for an increased overall dynamic range or for an improved quantification accuracy or for multiplexed quantification of different molecules. Different detection areas (800, 801, 802) can be located on the sample carrier strip 303 one after the other and being followed by one control area 305, as shown in FIG. 8A. Alternatively, the sample carrier strip 303 may be separated by multi-channel barriers 806, 807 into different channels (e.g. located in parallel) wherein each channel contains one each detection area (800, 801, 802) and one control area (803, 804, 805). It is preferred that the individual detection areas (800, 801, 802) and control areas (803, 804, 805) show a staggered arrangement.
[0121] FIG. 9A shows a setup used for proof of concept experiments. Indium tin oxide coated microscopy slides (shown in FIG. 9B) were coated with bovine serum albumin and subsequently coated with gold nanoparticle (40 nm). The slides were further mounted in a flow cell providing an open well that allows in situ imaging using dark field microscopy).
[0122] After addition of a 150 mM NaCl solution, the gold nanoparticles show a constant intensity while applying 6 cycles of cyclic voltammetry from −0.2 to 1.4 V (see FIG. 10 (a) and FIG. 11 (a)). After adding a 3 mM solution of KI in 150 mM NaCl, the Au-nanoparticles remain unchanged before an electrochemical potential is applied, but they are etched very quickly while the applied potential reaches 1.2 V as illustrated in FIG. 10 (b) and FIG. 11 (b). The same setup (FIG. 9A-9B) was used to measure the dissolution kinetics depending on the used amount of potassium iodide in FIG. 12, and on the applied overpotential in FIG. 13.
[0123] FIG. 14A-14B shows another setup used for a proof of concept experiment, namely a proof-of-concept embodiment of an electrochemical lateral flow assay with a three electrodes setup and a lateral flow assay strip. A line of adsorbed gold nanoparticles was added to said strip and is shown in FIG. 14A. The same setup after the electrochemical dissolution of the gold nanoparticles is illustrated in FIG. 14 B. The gold nanoparticles were functionalized with biotin and the detection/quantification line is functionalized with streptavidin, which specifically binds the biotin on the gold nanoparticles. The corresponding readout current using cyclic voltammetry of the test strip described above is shown in FIG. 15 (a), after the dissolution of the gold nanoparticles (circles) and a control experiment without gold nanoparticles (crosses). FIG. 15 (b) shows the linear response of the peak currents of a dilution series of the gold nanoparticles.
