Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
  • G01N33/5438—Electrodes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
  • G01N33/587—Nanoparticles

Definitions

• the present invention relates to electrochemical assays in particular such assays utilising metal nanoparticles as an electrochemical label.
• Prior art assays involving metal labels such as those described in WO 2005/121792 require chemical oxidants in order to dissolve the metal nanoparticles.
• a problem with this is that the oxidant can interfere with the electrochemical profile of the scan by disrupting the baseline of the scan.
• the assay described in WO 2005/121792 relies on the formation of metal ions in order for the metal to be transferred to an electrode surface electroanalytically. This is problematic when analysing biological samples. Proteins and other molecules in a biological sample solution typically bind to the metal ions rendering them electrochemically inactive.
• the metal ions can also be rendered electrochemically inactive by chelation/coupling with the chemical oxidate. Accordingly, the sensitivity of the signal is reduced.
• WO 2004/016160 discloses use of a chemical redox cleavant to release an organic polymer nanoparticle from a nanoparticle-labelled analyte. Electrochemical flow cytometry is then used to detect redox products (such as metal ions) released by the redox cleavant from the polymer nanoparticle.
• the present invention seeks to overcome one or more of the above problems.
• a method for determining the presence or amount of a metal-labelled analyte in a sample comprising the steps of: adding a release agent to the metal labelled analyte to release the metal label from the analyte, the release agent and the metal label together forming a charged species, applying a potential to bring the charged species to an electrode, applying- a positive potential to the charged species to form metal ions, and carrying out quantitative determination procedure to determine the presence or amount of the metal-labelled analyte.
• the metal ions are formed at the electrode surface. There is therefore little opportunity for the metal ion to be deactivated before it is measured.
• the charged species is typically insoluble. ' ⁇
• the release agent includes a charged component, the charged component of the release agent and the metal nanoparticle together forming the charged species.
• the charged species may be brought to an electrode by applying a positive potential, which also results in the formation of the metal ions.
• the charged species is preferably negatively charged, although in some embodiments it may be positively charged.
• the release agent may be any salt or chemical that is able to form a charged layer on the surface of the metal label and thus enable it to be moved under an electrical potential.
• the release agent may comprise a thiol with a charged unit.
• the thiol acts to denature or. destroy the binding between the metal label and the analyte.
• the charged unit provides a charge to the metal label.
• suitable release agents include ammonium thiocyanate. and potassium . thiocyanate. Instead of thiocyanate, the release agent may comprise thiosulphate or a charged thiol chain.
• the release agent may provide ions to form the charged species.
• suitable-release agents include NaCl or HCl, which are able to provide chloride ions to form the charged species. NaCl and HCl are particularly useful as release agents because they do not form a complex with the metal ions after oxidation.
• the release agent is preferably in a concentration of about IM or more. This range is preferred as below this molarity the silver sensitivity is not as good.
• the method may further include labelling the analyte with the metal label wherein the analyte is incubated with a binding moiety capable of binding to the analyte and which is labelled with the metal label.
• the analyte may be incubated with a further binding moiety capable of binding to the analyte and which is secured to a solid support.
• the solid support may be mobile or fixed, it may be magnetised, it may be a particle that may be larger or smaller than the metal nanoparticle so as to allow filtering of unbound from bound metal nanoparticles, it may be a charged particle in order-to enable electrical separation, it may be a porous structure, it may be a two- or three- dimensional surface or structure. '
• the metal label is silver or gold, for example a silver nanoparticle or a gold nanoparticle.
• the metal label is a particulate or a nanoparticle label such as a silver nanoparticle.
• the analyte can be labelled indirectly wherein the label is attached to a species or binding moiety which is in turn bound to the analyte.
• the quantitative determination procedure may be a voltammetric method, such as anodic stripping voltammetry (ASV). " ⁇ . _
• Figures 2 and 3 show the anodic stripping voltammetry (ASV) scan results for standard ⁇ solutions OfAgNO 3 plus IM NH 4 SCN (l-50ppm Ag"); ' .
• Figures 4 and 5 show the anodic stripping voltammetry (ASV) scan results- for standard solutions OfAgNO 3 plus IM NH 4 SCN (0.01-0.8 ppm Ag + ); .
• Figures 6 and 7 shows the concentration of Ag + in the presence of an oxidant ferricyanide;
• FIGS 8 to 11 show the concentration of Ag + in the presence/absence of ferricyanide
• Figures 12 and 13 show the absorbance of silver nanoparticle with and without NH 4 SCN;
• Figures 14a to f show the effect of 80% and 100% silver nanoparticle solution on size distribution;
• Figures 15 and 16 compare results with KSCN and NH 4 SCN;
• Figures- 17 and 18 compare the influence, of pre-treatment time on an embodiment of the method
• Figure 19 shows results for assays for myoglobin to determine the amount of antibody " required for maximum sensitivity of the assay
• Figures 20 to 23 show the results for further embodiments of the method.
• Figures 24 and 25 illustrate negative results where the measured species is uncharged. . . • - .
• the assay determines . the presence or amount of an.analyte of interest (such as an antibody, a mimotope or a nucleic acid strand) and one embodiment is schematically illustrated in ' •
• an.analyte of interest such as an antibody, a mimotope or a nucleic acid strand
• a first binding moiety 10 which is capable of binding to an analyte 12 of interest is attached to a solid phase 14 (surface or magnetic particle).
• a second binding moiety 16 which is capable of binding to a different region on the analyte
• a metal label 18 typically a particulate label.
• a sample solution comprising the analyte 12 is added to the solid support 14.
• the analyte 12 binds to the first binding moiety 10.
• the support 14 is then incubated with a solution comprising the labelled second binding moiety 16, which binds to the analyte 12.
• a "sandwich" occurs between the substrate and the silver particle 18 thus capturing the particle 18.
• Unbound particles are then removed (such as by washing). This ensures such particles are not oxidised.
• silver nanoparticles 18 are used as an electrochemical label.
• the silver nanoparticle 18 gives a molecular amplification of the electrochemical signal, as each 40nm silver nanoparticle contains approximately 10 s silver ions. Thus the sensitivity - of the -ass-ay is enhanced..
• silver nanoparticles 18 are easily oxidised electrically to form silver ions. Some other metal nanoparticles require harsh chemical oxidants to form metal ions.
• ammonium thiocyanate 20 is introduced 100 and removes the silver nanoparticle 18 from its biocomplex and forms a layer (which may be a monolayer) chemically bound around the silver nanoparticle resulting in a negatively charged nanoparticle 22.
• the charged nanoparticle 22 can be migrated 200 under an electrical potential to the surface of a positive electrode.
• the silver nanoparticle at that electrode then dissolves under the positive potential 300 to form silver ions 26.
• the silver ions 26 are then measured by accumulation stripping voltammetry.
• a small proportion of the silver ions 26 may be in the form of a complex with a chelating agent where the release agent is capable of chelating the silver ions .
• release agents 20 such as a thiol with a charged unit, for example thiosulphate
• Non-thiolate molecules such as NaCl and HCl could also be used.
• Ammonium thiocyanate also forms complexes with other metal ions and the ammonium thiocyanate positions the electrochemistry of these metal ions away from the electrochemistry of the silver ions complex thus removing spectroscopic interference.
• Standard solutions OfAgNO 3 were prepared (1.25, 6.25, 12.5, 25, 37.5, 50 and 62.5ppm AgNO 3 ). To each 160 ⁇ l of standard solution, 40 ⁇ l of 5M NH 4 SCN were added. Subsequently, 50 ⁇ l of each of the AgNO 3 and NH 4 SCN solutions were, in turn, applied onto an electrode and ASV conducted.
• the ASV parameters were as follows: a), +0.4V for l ⁇ s (pre-treatment) - '
• a 'mix' solution of 5MNH 4 SCN, 0.25M K 3 Fe(CN) 6 and 0.05M K 4 Fe(CN) 6 was prepared. Two solutions of 160 ⁇ l silver nanoparticles were prepared. 40 ⁇ l of 5M NH 4 SCN were added to the first silver solution. 40 ⁇ l of the mix solution were added to the second solution. Once prepared, 50 ⁇ l of each solution were added in turn onto an electrode and ASV conducted.
• NH 4 SCN dissolves silver nanoparticle
• spectroscopic methods can be used.
• the absorbance of silver nanoparticle with and without NH 4 SCN can be measured from 500 to 300 run.
• Silver nanoparticle (40nm) has an absorption peak at around 400nm and we can see that after adding NH 4 SCN this peak vanishes. It may mean that silver nanoparticle dissolves but according to literature the mechanism of vanishing of this peak is different: SCN " is coupled to the surface of silver particles as a monolayer and silver nanoparticles form aggregates. This is why absorbance of silver nanoparticle at 400 nm decreases but a new absorbance peak is obtained at around 550nm, so the experiment was repeated with a wider range of wavelengths.
• the UV spectrum shows a decrease in signal at 400nm and a broad peak increase in signal at around 650 nm indicating an increase in size of the nanoparticle.
• KSCN also forms electroactive complexes with Ag + and with silver nanoparticle generates current peaks.
• the second method was to keep the pre-treatment time the same (10s) and vary the potential of the pre-treatment.
• the current signal from Ag + is potential-independent; the signal from silver nanoparticle • is potential dependent.
• the potential has no effect at all on the silver ions but it strongly affects silver nanoparticle.
• Silver nanoparticles and antibody conjugation 1. Spin down 10 ml of silver nanoparticle (British Biocell) in 5 x 2-ml-tubes ' (13 200- rpm, 5 mins, 4°C).
• the potential is referenced to the reference electrode.
• the reference electrode is typically a D2 carbon ammonium thiocyanate reference electrode.
• the concentration of coating antibody does not greatly affect the assay.
• the sensitivity was very good for varying amounts of 4E2 antibody and the calibration curves are very similar (5, 10 and 15 ⁇ g/ml) as shown in Figure 19.
• too high concentration of coating antibody can negatively influence the assay. This is very much dependent on the antibody used and the surface to which it is attached.
• the sensitivity obtained is good, with a plateau reached at about 50ng myoglobin/ml. This sensitivity is similar or even better than the classical ELISA based on enzymatic detection of analyte.
• a release agent such as a thiol with a charged unit is added to the solid phase analyte silver nanoparticle complex.
• suitable release agents include ammonium thiocyanate and potassium thiocyanate.
• a thiosulphate could be used.
• Thiols are preferred as they bind to silver more effectively than other agents.
• the thiol weakens the antibody interaction so that the silver nanoparticle is released from the complex.
• the charged thiol also forms a layer around the silver nanoparticle making the particle charged, and allowing it to be electrically migrated to the electrode where the measurement takes place.
• Measurement can be conducted using ASV.
• ASV is an analytical technique that involves preconcentration of a metal phase onto an electrode surface and selective oxidation of each metal phase species during an anodic potential sweep.
• the silver-thiol charged nanoparticle is electroactive and stable.
• the electroactive charged nanoparticle can be electrochemically plated onto an electrode as a pre- concentrating step. Once at the electrode, the silver nanoparticle is electrochemically dissolved by oxidising under a positive potential.
• the ions are plated on the electrode by reversing the potential to a negative potential.
• the silver plate is then electrochemically stripped off the electrode (by once again applying a positive potential) giving a stronger silver peak signal.
• the amount of silver ions stripped off the electrode can be related to the number of silver nanoparticles used to give a signal which relates to the number of analytes captured on the solid phase of the assay.
• Metal nanoparticles are commercially available and may be conjugated to binding moieties ' by known methods.
• the binding moiety may be anything capable of binding to the analyte to be detected e.g. protein, peptide, antibody or, fragment thereof, nucleic acid.
• the first binding moiety need not be immobilized on a solid support.
• silver nanoparticles were prepared as described above with respect to Example 4 (without ferricyanide) except that IM thiosulphate was used as the release agent.
• the silver nanoparticle-thiosulphate species was measured by ASV.
• Example 11 The experiment of Example 11 was repeated, but IM NaCl was used as the release agent. .
• the results illustrating that NaCl is a suitable release agent are shown in Figure 21. " ⁇
• Example 13 The experiment of Example 13 was repeated, but IM thiocyanate was used as the release agent. The results are shown in Figure 23.
• ASV was carried out an gold and silver nanoparticles in the absence of a charged release agent.
• Figures 24 (gold) and 25 (silver) illustrates that no peak is observed when the nanoparticles are uncharged.
• two different analytes within a same sample can be detected by using both gold and silver nanoparticles.
• gold nanoparticles are attached to a first binding moiety that recognises a first analytei Gold ions and silver ions give different distinguishable peaks when measured on the same electrode by ASV.
• kits may be provided.
• the kit may comprise a metal label for binding to an analyte, at least one release agent for releasing the metal agent once bound to an analyte ' and for forming a charged nanoparticle with the metal label, a device having at least one zone for binding the metal label to an analyte and at least two electrodes.
• the kit may contain a binding moiety capable of binding to the ' analyte of interest and labelled with a metal label.
• the device may be a multiwell plate.

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