GB2462062A - Electrochemical assay - Google Patents

Electrochemical assay Download PDF

Info

Publication number
GB2462062A
GB2462062A GB0812845A GB0812845A GB2462062A GB 2462062 A GB2462062 A GB 2462062A GB 0812845 A GB0812845 A GB 0812845A GB 0812845 A GB0812845 A GB 0812845A GB 2462062 A GB2462062 A GB 2462062A
Authority
GB
United Kingdom
Prior art keywords
metal
analyte
charged
release agent
label
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
GB0812845A
Other versions
GB0812845D0 (en
Inventor
Robert Porter
Mateusz Szymanski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SEC DEP FOR INNOVATION UNIVERS
UK Secretary of State for Innovation Universities and Skills
Original Assignee
SEC DEP FOR INNOVATION UNIVERS
UK Secretary of State for Innovation Universities and Skills
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SEC DEP FOR INNOVATION UNIVERS, UK Secretary of State for Innovation Universities and Skills filed Critical SEC DEP FOR INNOVATION UNIVERS
Priority to GB0812845A priority Critical patent/GB2462062A/en
Publication of GB0812845D0 publication Critical patent/GB0812845D0/en
Priority to GB0912670A priority patent/GB2458420B/en
Priority to EP08854118A priority patent/EP2220494B1/en
Priority to US12/744,645 priority patent/US8337692B2/en
Priority to CN200880125056.2A priority patent/CN101971029B/en
Priority to AT08854118T priority patent/ATE533054T1/en
Priority to PCT/GB2008/003931 priority patent/WO2009068862A1/en
Priority to NZ585851A priority patent/NZ585851A/en
Priority to JP2010534545A priority patent/JP5055433B2/en
Priority to AU2008328588A priority patent/AU2008328588B2/en
Publication of GB2462062A publication Critical patent/GB2462062A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • G01N33/54373Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438Electrodes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553Metal or metal coated
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585Chemical 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
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00Labels used in chemical analysis of biological material
    • G01N2458/30Electrochemically active labels

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Inorganic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A method for determining the presence or amount of a metal labelled analyte (12) in a sample is described. The method comprises the steps of adding a release (or cleaving) agent (20) to the metal labelled analyte (12) to release the metal label (18) from the analyte (12), the release agent (20) forming a charged stable species (22) with the metal label (18), applying a potential to bring the charged stable species (22) to an electrode (22), dissolving the charged stable species (22) under a positive potential to form metal ions (26), and carrying out a quantitative determination procedure such as anodic stripping voltammetry to determine the presence or amount of the metal labelled analyte (12). The charged species may be insoluble and negatively charged. The release agent may comprise a thiol with a charged unit, such as potassium thiocyanate or sodium thiocyanate, thiosulphate or charged thiol chain. Alternatively the release agent may comprise sodium chloride or potassium chloride to provide chloride ions to form the charged species. The metal label may be a particulate label or nanoparticle and may comprise a silver or gold nanoparticle.

Description

Electrochemical Assay 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 2005/12 1792 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 chelationlcoupling with the chemical oxidate. Accordingly, the sensitivity of the signal is reduced.
The present invention seeks to overcome one or more of the above problems.
According to a first aspect of the present invention there is provided a method for determining the presence or amount of a metal labelled analyte in a sample, the method 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, dissolving the charged species under a positive potential to form metal ions, and carrying out a quantitative determination procedure to determine the presence or amount of the metal labelled analyte.
Since metal ions are formed at the electrode surface, there is little opportunity for the metal ion to be deactivated before it is measured. It is therefore not necessary to form a complex between the metal ion and a chelating agent. Moreover, as an electrochemical potential is used to dissolve the metal label, it is not necessary to use a chemical oxidant and the problems associated with chemical oxidants are avoided.
The charged species is typically insoluble.
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.
Examples of suitable release agents include ammonium thiocyanate and potassium thiocyanate. Instead of thiocyanate, the release agent may comprise thiosuiphate or a charged thiol chain. Other suitable release ageiits include NaC1 or HCI, which are able to provide chloride ions to form the charged species. NaCl and HCI 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.
In a preferred embodiment the metal label is silver or gold, for example a silver nanoparticle or a gold nanoparticle. Preferably 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).
Preferred embodiments will now be described by way of example only and with reference to the drawings in which: Figure 1 schematically illustrates an embodiment of the invention; Figures 2 and 3 show the anodic stripping voltamnietry (ASV) scan results for standard solutions of AgNO3 plus 1M NH4 SCN (1-5oppm Ag); Figures 4 and 5 show the anodic stripping voltammetry (ASV) scan results for standard solutions of AgNO3 plus 1M NH4 SCN (0.0 1-0.8 ppm Ag); Figures 6 and 7 shows the concentration of Ag in the presence of an oxidant ferricyanide; Figures 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 NT-L1SCN; 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 NIH4SCN; 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; and 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 Figure 1. In a preferred embodiment, 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 12 is labelled with 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. In effect 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.
In a preferred einbodiment, silver nanoparticles 18 are used as an electrochemical label.
The silver nanoparticle 18 gives a molecular amplification of the electrochemical signal, as each 4Onm silver nanoparticle contains approximately 106 silver ions. Thus the sensitivity of the assay is enhanced. In addition, silver is preferred as it forms stable nanoparticles which can be used as a bio-nanolabel 18. Furthermore, silver nanoparticles 18 are easily oxidised electrically to form silver ions. Some other metal nanoparticles require harsh chemical oxidants to form metal ions.
In a preferred embodiment, 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.
Other release agents 20 (such as a thiol with a charged unit, for example thiosulphate) may be used. Non-thiolate molecules such as NaCI and HC1 could also be used.
Comparative Example 1 This Comparative Example explains the chemistry behind the formation of the electroactive complexes measured in the method disclosed in WO 2005/12 1792.
I. Measurements of complexed of silver ions (Ag) Silver ions in thepresence of NFJ4SCN form strong electroactive complexes: Ag + m(NT-I) + n(SCN)_>[Ag(NH)m(SCN)n]m Those complexes can be electrochemically measured using ASV. In this process silver complexes at negative potential are first deposited on the surface of carbon paste electrode: [Ag(N})m(SCN)nJm.n + e [Ag(N})m(SCN)n]m Then the potential is changed to positive value and silver complexes are stripped off the electrode giving a peak in the current.
[Ag(NH)m(SCN)n]m -[Ag(NH)m(SCN)n]ml + e 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 of AgNO3 were prepared (1.25, 6.25, 12.5, 25, 37.5, 50 and 62.5ppni AgNO3). To each 1 60tl of standard solution, 40il of SM NH4SCN were added.
Subsequently, 50xl of each of the AgNO3 and NH4SCN solutions were, in turn, applied onto an electrode and ASV conducted. The ASV parameters were as follows: a) +O.4V for I Os (pre-treatment) b) linear sweep +O.4V -) -1.6V at scan rate = 1V/s and step potential O.005V c) -1.6Vfor5s d) -1.2Vfor55s e) linear sweep -1.2V -* +O.1V at scan rate = 1V/s and step potential = 0.0 1V 2) Between each measurement the electrode was cleaned with 5M N}T4SCN and running ASV and then wiping with tissue.
Table 1
_______ _______ _______ _______ _______ _______ _______ ________
[ppm] 1 5 10 20 30 40 50 1 4.4OE-0 2.48E-06 5.34E-06 1.03E-05 1.39E-05 1.68E-05 2.34E-05 2 4.23E-0' 2.48E-06 4.86E-06 9.73E-06 1.62E-05 1.69E-05 2.1OE-05 3 3.9lE-0 2.51EL06 4.73E-06 1.07E-05 1.45E-05 i.77E-05 1.96E-05 1ean 4.18E-O 2.49E-06 4.97E-0( 1.02E-05 1.49E-05 1.71E-05 2.13E-05 St. Dev. 2.48E-08 1.94E-08 3.19E-0 4.8E-0 1.21E-06 4.61E-0 1.91E-06 CV% 5.93 0.78 6.42 4.69 8.17 2.69 8.96 The results are displayed in Figures 2 and 3. The area under the peak is proportional to the concentration of Ag in the range ito 5Oppm.
Comparative Example 2 The same experiment was carried out using lower concentrations of Agt
Table 2
[ppm] 0.01 0.025 0.05 0.1 0.25 0.5 0.8 1 no peak no peak no peak 1.2E-08 6.4E-08 1.6E-07 2.7E-07 2 no peak no peak no peak 1.7E-08 6.4E-08 1.5E-07 2.5E-07 3 no peak no peak no peak 5.1E-09 5.4E-08 1.5E-07 2.4E-07 Mean #DIVIO! #DIV/0! #DIV/0! 1.1E-08 6.1E-08 1.5E-07 2.5E-07 St. Dev. #DIV/0! #DIV/O! #DIV/O! 5.7E-09 5.9E-09 7.OE-09 i2E-08 CV% #DIVIO! #DIV/0! #DIV/0! 51.62 9.76 4.59 4.83 The results are displayed in Figures 4 and 5.
Comparative Example 3 Prior art assays, such as described in WO 2005/12 1792 use an oxidant (ferricyanate) to form silver ions for use in the methodology described in Examples 1 and 2. The following measurements were conducted in the presence of 0.05M K3Fe(CN)6 and O.O1M K4Fe(CN)6
Table 3
_______ _______ _______ _______ _______ _______ _______ _______ _______
ippm] 1 ________ 3 4 5 6 7 8 1 3.39E-0 7.31E-0 1.13E-06 1.57E-06 1.90E-06 2.07E-06 2.41E-06 2.73E-06 2 3.45E-0 7.34E-0 1.07E-06 1.50E-06 1.89E-06 2.20E-06 2.52E-06 2.79E-06 3 2.99E-0 7.34E-0 1.14E-06 1.47E-06 1.88E-06 2.23E-06 2.52E-06 2.83E-06 Vlean 3.28E-O 7.33E-0 1.12E-06 1.5lE-06 1.89E-06 2.17E-06 2.48E-06 2.78E-06 St. Dev. 2.5E-08 1.65E-09 4.07E-08 5.26E-08 1.37E-08 8.83E-08 6.36E-08 5.17E-08 CV% 7.633 131 0.225412 3.650072 3.47912 0.722371 4.074576 2.562306 1.859349 The results obtained are displayed in Figures 6 to 9. Whilst, the calibration curve for silver ions with and without ferricyanide is very similar, the shape of the scan results is different.
The following Examples illustrate the subject matter of preferred embodiments of the present invention.
Example 4
Electrochemical behaviour of silver nanoparticles with or without ferricyanide.
A mix' solution of SM NH4SCN, 0.25M K3Fe(CN)6 and 0.05M K4Fe(CN)6 was prepared.
Two solutions of 160tl silver nanoparticles were prepared. 4Otl of SM NH4SCN were added to the first silver solution. 4OtI of the mix solution were added to the second solution. Once prepared, 50pi1 of each solution were added in turn onto an electrode and ASV conducted.
Table 4
Silver 4anoparticle No Fe + Fe 1 2.23E-06 2.36E-06 2 2.06E-06 2.28E-06 3 1.93E-06 2.15E-06 4 1.37E-06 1.88E-06 1ean 1.9E-06 2.17E-06 St. Dcv 3.72E-0 2.12E-0 CV% 1i94O339.80704 The results are shown in Figure 10. Silver nanoparticles in the presence of thiocyanate generates similar but stronger peaks to the peaks generated by silver nanoparticles with the ferricyanide oxidant. However, the baseline is better in the case of silver nanoparticles without ferricyanide oxidant. The baseline is flatter and there is no current rise after the stripping peak (as in the case of the ferricyanide). Peak finding and integration or height determination are thus easier.
What is more (as shown in the next experiment) the area under the peak is proportional to silver nanoparticle concentration:
Table 5
Nanoparticle [%] 1 4 8 16 32 48 64 80 mo peak 1.19E-0 2.66E-0 5.21E-0 9.99E-0 1.70E-06 2.1OE-06 2.42E-06 2 io peak 1.18E-0 2.07E-0 4.31E-0' 9.57E-0 1.51E-06 1.88E-06 2.66E-06 3 io peak 8.77E-08 2.14E-0 4.23E-0 9.25E-07 1.44E-06 1.95E-0o 2.31E-06 4 o peak 6.31E-08 1.81E-0' 4.IOE-07 8.49E-0 1.29E-06 1.75E-06 2.21E-06 4ean #DIV/0! 9.70E-08 2.17E-0 4.46E-0' 9.32E-0 1.49E-06 1.92E-06 2.40E-06 St. Dcv. #DIV/0! 2.69E-08 3.54E-08 5.08E-08 6.32E-08 1.7E-0 1.46E-0 1.93E-0' CV% DIV/0! 27.77 16.32 11.40 6.78 11.41 7.58 8.04 These data are displayed in Figure 11.
Example 5
To check whether NH4SCN dissolves silver nanoparticle, spectroscopic methods can be used. The absorbance of silver nanoparticle with and without NH4SCN can be measured from 500 to 300 rim.
Silver nanoparticle (4Onm) has an absorption peak at around 400nm and we can see that after adding NH4SCN 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.
These data are displayed in Figures 12 and 13.
The UV spectrum shows a decrease in signal at 400nm and a broad peak increase in signal at around 650 nni indicating an increase in size of the nanoparticle.
Example 6
In this experiment, the size of silver particles in 100% silver nanoparticle solution and in 80% silver nanoparticle solution with 1M NH4SCN (filtered) was measured using Malvem zeta-sizer instrument. Figure 14a shows the results for a 100% silver nanoparticle solution.
The measurements were taken at t0 and a further three measurements were taken at t3 hours.
The measurements of silver nanoparticle solution show that the silver nanoparticle is stable in solution. This is shown in Figure 14b. After adding N}I4SCN the size of silver nanoparticles changes as shown in Figure 14c. After the ammonium thiocyanate was added it can be clearly seen from the results that the particles have aggregated and not dissolved at all (see Figure 14d).
After one hour, the size of aggregates seems to be growing (see Figures 1 4e and f).
Example 7
The indication from the data is that the ammonium thiocyanate forms a thiocyanate monolayer around the silver nanoparticle. Similar results can be obtained by changing the molecule but retaining the thiocyanate unit. In this experiment the measurement of silver nanoparticle and silver ions was prepared with NH4SCN as well as with KSCN:
Table 6
2.56ppm Ag+ 80% Silver Nanoparticle n_____ 1MKSCN IMNH4SCN 1MKSCN1MNH4SCN 1 4.51 E-06 4.78E-06: 3.64E-06 8.24E-06 2 5 1OE 06 501E 06 769E 06 5 iDE 06 3 5.62E-06 5.09E-06 4.94E-06 4.78E-06 4 5.78E-06 5.09E-06 7.89E-06 3.14E-06 5.34E-06 5.06E-06 9&4E-06 5.78E-06 6 1.05E-06 5.12E-06 7.85E-06 6.82E-06 7 5.04E-06 4.94E-06 8.09E-06 4.79E-06 8 5.39E-06 4.57E-06 7.93E-06 7.90E-06 9 5.27E-06 4.78E-06 6.79E-06 7.32E-06 5.64E-06 5.19E-06 9.88E-06 7.71E-06 Mean 5.47E-06 4.96E-06 7.45E-06 6.16E-06 St. 0ev. 6.64E-07 1.94E-07 1.94E-06 1.69E-06 CV% 12.13 3.90 26;06 27.43 The results are displayed in Figures 15 and 16.
We can see that KSCN also forms electroactive complexes with Ag and with silver nanoparticle generates current peaks.
Example 8
We have demonstrated above that the thiocyanate forms a monolayer on the surface of the silver nanoparticle to form a charged nanoparticle. This causes the nanoparticles to aggregate. The charged particle is migrated to the electrode under a positive potential. The following two experiments show how the silver nanoparticle may be measured by anodic stripping voltammetry. Firstly the influence of pre-treatment time (at U = +O.4\T) Oii measurements of silver ionsand silver nanoparticle was checked (see Table 7):
Table 7
_____ ______ 2.56ppm Ag+ in 1 M NH4SCN _______ ______ _______ ______ ______ ______ ______ time[s] 0 1 2 3 4 5 6 7 8 9 10 20 1 5.25E-06 5.83E-06 5.85E-06 5.83E-06 5.85E-06 5.81E-06 5.98E-06 5.67E-06 5.78E-06 4.69E-06 5.47E-06 5.41E-06 2 5.67E-06 5.76E-06 6.13E-06 6.29E-06 6.18E-06 6.07E-06 5.96E-06 5.88E-06 5.86E-06 5.91E-06 5.55E-06 5.64E-06 3 5.57E-066.1iE-06 5.83E-06 6.O1E-06 5.95E-06 5.84E-06 5.90E-06 5.61E-06 6.13E-06 5.91E-06 5.91E-06 5.73E-06 Mean 5.49E-06 5.90E-06 5.94E-06 6.04E-06 5.99E-06 5.91 E-06 5.95E-06 5.72E-06 5.92E-06 5.50E-06 5.64E-06 5.59E-06 St. Dev.2.22E-07 1.86E-07 1.70E-07 2.32E-07 t66E-07 1.45E-07 3.82E-08 1.42E-07 1.84E-07 7.04E-07 2.37E-07 1.65E-07 CV% 4.03624 3.15993 2.86715 3.84781 2.77693 2.45108 0.64229 2.48712 3.11015 12.7879 4.2055 2.94536 ____ 80% Silver Nanoparticle in 1M NH4SCN _______ ________ _______ _______ ________ ________ time [sI 0 1 2 3 4 5 6 7 8 9 10 20 40 - 1 1.16E-06 3.45E-06 4.4aE-06 3.86E-06 6.32E-06 5.72E-06 5.77E-06 7.04E-06 7.34E-06 8.03E-06 9.14E-06 9.12E-06 9.94E-06 2 1.55E-06 3.08E-06 3.48E-06 5.O1E-06 6.64E-06 6.12E-06 5.46E-06 7.63E-06 9.O1E-06 8.96E-06 1.06E-05 1.04E-05 3 1.33E-Q6 4.OOE-06 3.46E-06 3.52E-06 5.64E-06 7.29E-06 8.29E-06 6.47E-06 7.68E-06 8.76E-06 7.71E-06 1.02E-05 1.O1E-05 Mean 1.35E-06 3.51E-06 3.78E-06 3.69E-06 5.66E-06 6.55E-06 6.73E-06 6.32E-06 7.55E-06 8.60E-06 8.60E-06 9.97E-06 l.02E-05 St.Dev. 1.95E-07 4.62E-07 5.38E-07 2.37E-07 6.56E-07 7.91E-07 1.36E-06 8.02E-07 1.81E-07 5.07E-07 7.81E-07 7.63E-07 2.25E-07 CV% 14.5031 13.1592 14.2104 6.41866 11.5892 12.0772 20.2901 12.6885 2.39723 5.89705 9.08262 7.65 2.21985 The measurements of silver ions are tinie-independent, whereas measurements of silver nanoparticle are time-dependent. This demonstrates that the positive potential has an influence on the measurement signal. (See Figure 17).
As shown in Figure 18, the second method was to keep the pre-treatrnent time the same (lOs) 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. These data show that the electroactive species measured during the last step of ASV of silver nanoparticle is created during the pre-treatment time. The silver nanoparticle and its charged coverage is migrated to the electrode via a potential gradient.
The positive potential at the electrode dissolves the silver nanoparticle to silver ions. The silver ions can therefore be accumulated on the electrode and stripped off for measurement.
The skilled person would know how to do this.
Example 9
An electrochemical immunoassay was carried out as follows: Silver nanoparticles and antibody conjugation: 1. Spin down 10 ml of silver nanoparticle (British Biocell) in 5 x 2-mI-tubes (13 200 rpm, 5 mins, 4°C).
2. Discard the supernatants and combine all 5 pellets in one tube so that the final volume of silver nanoparticle is 1 ml.
3. Add an antibody solution (Hytest) to obtain desired concentration (-0.O5mg/ml).
4. Incubate for 40 mins on a roller mixer.
5. Centrifuge at 13 200 rpm, 5 mins, 4°C (salvage the nanoparticle from the supernatant if necessary).
6. Resuspend the pellet in I ml of blocking buffer (3% BSA).
7. Incubate for 40 mins on the roller mixer.
8. Spin down at 13 200 rpm, 5 mins, 4°C (salvage the nanoparticle from the supernatant if necessary).
9. Wash the pellet by resuspending it in 1 ml of 0.1 M borate buffer pH 7.5, then spin it down (13 200rpm, 5 mins, 4°C), (salvage the nanoparticle from the supernatant if necessary) and then again resuspend it in 1 ml of 0.1 M borate buffer pH 7.5.
10. Store at 4°C until used.
Electroimmunoassay: 1. Coat the plate with 50i11 of first antibody solution (lOpgIml in PBS) 2. Incubate overnight at 4°C (plate sealed).
3. Wash 3x with 200.tl of washing buffer.
4. Add 150 i.tl of blocking buffer (Pierce 1137536) and incubate 30 mins on a shaker.
1 5 5. Wash 3x with 200 tl of washing buffer.
6. Prepare the first concentration (the highest) of the analyte in wash buffer and perform serial dilutions in wash buffer.
7. Add 50 p1 of analyte solutions and incubate 30 mins on the shaker.
8. Wash 3x with 200 tl of wash buffer.
9. Dilute silver nanoparticle-antibody conjugate 5x in wash buffer and add 50 p1 of it to the wells. Incubate lh on the shaker.
10. Wash 3x with 200 tl of wash buffer.
11. Store at 4°C overnight (sealed and without wash buffer) 12. Add 50 tl of IM NT-I4SCN and shake on the mixer for lh.
13. Apply the solution from each well on the surface of carbon paste electrode and run anodic stripping voltammetry (ASV).
The steps of ASV are the following: a) Step: +0.4V for lOs (pre-treatment) b) Linear sweep: 0.OV -) -1.6V at scan rate lVfs and step potential 0.005V c) Step: -1.6V for 5s (nucleation) d) Step: -1.2 V for 55s (deposition [reduction] of electroactive species) e) Linear sweep: -1.2V -+O.1V at scan rate = 1V/s and step potential = 0.O1V (stripping [oxidation] of electroactive species). During this sweep a peak current is generated and the area under the peak (i.e. number of coulombs) is proportional to the concentration of electroactive species.
The potential is referenced to the reference electrode. The reference electrode is typically a D2 carbon ammonium thiocyanate reference electrode.
Example 10
Assays for myQglobin looking at the amount of anti-myQglobin Hytest 4E2 antibody required for the maximum sensitivity of the assay.
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 1gIml) as shown in Figure 19. However 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 5Ong 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. Examples of suitable release agents include ammonium thiocyanate and potassium thiocyanate. Alternatively, 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.
Instead of a "sandwich" or non-competitive immunoassay as described above, other formats could equally be used to capture the silver particles such as a competition immunoassay or a hapten-based assay. Such assays are well known.
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.
Example 11
Charged silver nanoparticles were prepared as described above with respect to Example 4 (without ferricyanide) except that 1M thiosuiphate was used as the release agent. The silver nanoparticle-thiosulphate species was measured by ASV.
The results are shown in Figure 20, and demonstrate that thiosulphate is a suitable release agent for use in this method.
Example 12
The experiment of Example 11 was repeated, but 1M NaCI was used as the release agent.
The results illustrating that NaCI is a suitable release agent are shown in Figure 21.
Example 13
The experiment of Example 11 was repeated but gold nanoparticles were used instead of silver nanoparticles. 1M HCI was used as the release agent. The results are shown in Figure 22.
Example 14
The experiment of Example 13 was repeated, but 1M thiocyanate was used as the release agent. The results are shown in Figure 23.
Example 15
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.
In a modification, two different analytes within a same sample can be detected by using both gold and silver nanoparticles. In this case, gold nanoparticles are attached to a first binding moiety that recognises a first analyte. Gold ions and silver ions give different distinguishable peaks when measured on the same electrode by ASV.
A kit 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. Optionally, 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 niultiwell plate.

Claims (25)

  1. CLAIMS1. A method for determining the presence or amount of a metal labelled analyte in a sample, the method 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 a quantitative determination procedure to determine the presence or amount of the metal labelled analyte.
  2. 2. A method as claimed in claim 1, wherein the charged species is insoluble.
  3. 1 A method as claimed in claim 1 or 2, wherein the charged species is brought to an electrode by applying a positive potential, the positive potential also resulting in the formation of the metal ions.
  4. 4. A method as claimed in claim 1, 2 or 3, wherein the charged species is negatively charged.
  5. 5. A method as claimed in any preceding claim wherein the release agent comprises a thiol with a charged unit.
  6. 6. A method as claimed in any preceding claim, wherein the release agent is ammonium thiocyanate or potassium thiocyanate.
  7. 7. A method as claimed in any preceding claim, wherein the release agent is a thiosuiphate or a charged thiol chain.
  8. 8. A method as claimed in any preceding claim, wherein the release agent is able to provide ions to form the charged species.
  9. 9. A method as claimed in claim 8, wherein the release agent is able to provide chloride ions to form the charged species.
  10. 10. A method as claimed in claim 9, wherein the release agent is NaCI or HCI.
  11. 11. A method as claimed in any preceding claim, further comprising the step of 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.
  12. 12. A method as claimed in claim 11, further comprising the step of incubating the analyte with a further binding moiety capable of binding to the analyte and which is secured to a solid support.
  13. 13. A method as claimed in claim 12, wherein the solid support is mobile or fixed.
  14. 14. A method as claimed in claim 13, wherein the mobile solid support is magnetised
  15. 15. A method as claimed in claim 13 or 14, wherein the metal label is a metal nanoparticle and wherein the solid support is a particle that is larger or smaller than the metal nanoparticle so as to allow filtering of unbound from bound metal nanoparticles.
  16. 16. A method as claimed in any of claims 13, 14 or 15, wherein the solid support is a charged particle in order to enable electrical separation.
  17. 17. A method as claimed in claim 13, wherein the fixed solid support is a three-dimensional surface or structure.
  18. 18. A method as claimed in claim 17, wherein the fixed solid support is a three-dimensional porous structure.
  19. 19. A method as claimed in claim 10, wherein the fixed solid support is a two-dimensional surface or structure.
  20. 20. A method as claimed in any preceding claim wherein the metal label is a particulate label or a nanoparticle.
  21. 21. A method as claimed in claim 19, where the metal label is a silver nanoparticle or a gold nanoparticle.
  22. 22. A method as claimed in claim 21, wherein the metal label is a silver nanoparticle.
  23. 23. A method as claimed in any preceding claim, wherein the quantitative determination procedure is a voltammetric method.
  24. 24. A method as claimed in claim 23, wherein the voltammetric method is anodic stripping voltammetry.
  25. 25. A method substantially as hereinbefore described with reference to, and as illustrated in, the accompanying drawings.AMENDMENTS TO THE CLAIMS HAVE BEEN FILED AS FOLLOWS:-CLAIMS1. A method for determining the presence or amount of a metal-labelled analyte in a sample, the method 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 an insoluble charged species, applying a potential to bring the charged species to an electrode, applying a positive potential to the charged species to dissolve the metal label to form metal ions, and carrying out a quantitative determination procedure to determine the presence or amount of the metal labelled analyte.2. A method as claimed in claim 1, wherein the release agent forms a charged layer on the surface of the metal label to enable it to be moved under an electrical potential.3. A method as claimed in claim 1 or 2, wherein the charged species is brought to an S...: electrode by applying a positive potential, the positive potential also resulting in the formation of the metal ions. S...: . . A method as claimed in claim 1, 2 or 3, wherein the charged species is negatively charged. S... * . . S. S2'3' 5. A method as claimed in any preceding claim wherein the release agent comprises a *..S thiol with a charged unit.6. A method as claimed in any preceding claim, wherein the release agent is ammonium thiocyanate or potassium thiocyanate.7. A method as claimed in any preceding claim, wherein the release agent is a thiosulphate or a charged thiol chain.8. A method as claimed in any preceding claim, wherein the release agent is able to provide ions to form the charged species.9. A method as claimed in claim 8, wherein the release agent is able to provide chloride ions to form the charged species.10. A method as claimed in claim 9, wherein the release agent is NaC1 or HC1.11. A method as claimed in any preceding claim, further comprising the step of 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.12. A method as claimed in claim 11, further comprising the step of incubating the analyte with a further binding moiety capable of binding to the analyte and which is secured to a solid support.13. A method as claimed in claim 12, wherein the solid support is mobile or fixed. I... * . . * ** W.* 14. A method as claimed in claim 13, wherein the mobile solid support is magnetised * *..: 15. A method as claimed in claim 13 or 14 wherein the metal label is a metal S...nanoparticle and wherein the solid support is a particle that is larger or smaller than the * : metal nanoparticle so as to allow filtering of unbound from bound metal nanoparticles.* h.J * 5*.16. A method as claimed in any of claims 13, 14 or 15, wherein the solid support is a charged particle in order to enable electrical separation.17. A method as claimed in claim 13, wherein the fixed solid support is a three-dimensional surface or structure. 1.18. A method as claimed in claim 17, wherein the fixed solid support is a three-dimensional porous structure.19. A method as claimed in claim 10, wherein the fixed solid support is a two-dimensional surface or structure.20. A method as claimed in any preceding claim wherein the metal label is a particulate label or a nanoparticle.21. A method as claimed in claim 19, where the metal label is a silver nanoparticle or a gold nanoparticle.22. A method as claimed in claim 21, wherein the metal label is a silver nanoparticle.23. A method as claimed in any preceding claim, wherein the quantitative determination procedure is a voltammetric method.24. A method as claimed in claim 23, wherein the voltammetric method is anodic S..* : stripping voltamrnetry. I... * S25. A method substantially as bereinbefore described with reference to, and as illustrated S...*, in, the accompanying drawings. *.. 5.5 * . SIS S *S.. * S *..S
GB0812845A 2007-11-26 2008-07-14 Electrochemical assay Withdrawn GB2462062A (en)

Priority Applications (10)

Application Number Priority Date Filing Date Title
GB0812845A GB2462062A (en) 2008-07-14 2008-07-14 Electrochemical assay
AU2008328588A AU2008328588B2 (en) 2007-11-26 2008-11-25 Electrochemical detection of a metal - labelled analyte
CN200880125056.2A CN101971029B (en) 2007-11-26 2008-11-25 Electrochemical detection of a metal-labelled analyte
EP08854118A EP2220494B1 (en) 2007-11-26 2008-11-25 Electrochemical detection of a metal labelled analyte
US12/744,645 US8337692B2 (en) 2007-11-26 2008-11-25 Electrochemical detection of a metal—labelled analyte
GB0912670A GB2458420B (en) 2007-11-26 2008-11-25 Electrochemical detection of a metal-labelled analyte
AT08854118T ATE533054T1 (en) 2007-11-26 2008-11-25 ELECTROCHEMICAL DETECTION WITH METAL MARKED DETECTION AGENT
PCT/GB2008/003931 WO2009068862A1 (en) 2007-11-26 2008-11-25 Electrochemical detection using silver nanoparticle labelled antibodies
NZ585851A NZ585851A (en) 2007-11-26 2008-11-25 Electrochemical detection of a metal - labelled analyte
JP2010534545A JP5055433B2 (en) 2007-11-26 2008-11-25 Electrochemical detection of metal-labeled specimens

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
GB0812845A GB2462062A (en) 2008-07-14 2008-07-14 Electrochemical assay

Publications (2)

Publication Number Publication Date
GB0812845D0 GB0812845D0 (en) 2008-08-20
GB2462062A true GB2462062A (en) 2010-01-27

Family

ID=39722243

Family Applications (1)

Application Number Title Priority Date Filing Date
GB0812845A Withdrawn GB2462062A (en) 2007-11-26 2008-07-14 Electrochemical assay

Country Status (1)

Country Link
GB (1) GB2462062A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120080324A1 (en) * 2010-09-30 2012-04-05 Sysmex Corporation Method of electrochemically detecting target substance, method of electrochemically detecting analyte, test chip, and detection set
EP2458383A1 (en) * 2010-11-30 2012-05-30 Sysmex Corporation Method of electrochemically detecting a sample substance

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103207160B (en) * 2013-04-11 2014-12-10 福建医科大学 Rapid determination method for thiocyanate with nanogold as coloring probe

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004016160A2 (en) * 2002-08-19 2004-02-26 Iowa State University Research Foundation, Inc. Redox polymer nanoparticles
US20060228814A1 (en) * 2000-06-26 2006-10-12 Centre National De La Recherche Scientifique (Cnrs) Electrochemical immunoassays using colloidal metal markers
US20060270049A1 (en) * 2005-05-25 2006-11-30 Board Of Supervisors Of Louisiana State University & A & M College Enhanced detection of analytes on surfaces using gold nanoparticles
EP1756574A1 (en) * 2004-06-07 2007-02-28 Inverness Medical Switzerland GmbH Method
WO2009068862A1 (en) * 2007-11-26 2009-06-04 The Secretary Of State For Innovation, Universities And Skills Of Her Majesty's Britannic Government Electrochemical detection using silver nanoparticle labelled antibodies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060228814A1 (en) * 2000-06-26 2006-10-12 Centre National De La Recherche Scientifique (Cnrs) Electrochemical immunoassays using colloidal metal markers
WO2004016160A2 (en) * 2002-08-19 2004-02-26 Iowa State University Research Foundation, Inc. Redox polymer nanoparticles
EP1756574A1 (en) * 2004-06-07 2007-02-28 Inverness Medical Switzerland GmbH Method
US20060270049A1 (en) * 2005-05-25 2006-11-30 Board Of Supervisors Of Louisiana State University & A & M College Enhanced detection of analytes on surfaces using gold nanoparticles
WO2009068862A1 (en) * 2007-11-26 2009-06-04 The Secretary Of State For Innovation, Universities And Skills Of Her Majesty's Britannic Government Electrochemical detection using silver nanoparticle labelled antibodies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Nanotechnology, vol 14, 2003, R63-R73, Institute of Physics Publishing, "Metal nanoparticles as labels for heterogeneous, chip based DNA detection", Fritzsche et al. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120080324A1 (en) * 2010-09-30 2012-04-05 Sysmex Corporation Method of electrochemically detecting target substance, method of electrochemically detecting analyte, test chip, and detection set
US9157885B2 (en) * 2010-09-30 2015-10-13 Sysmex Corporation Method of electrochemically detecting target substance, method of electrochemically detecting analyte, test chip, and detection set
EP2458383A1 (en) * 2010-11-30 2012-05-30 Sysmex Corporation Method of electrochemically detecting a sample substance
CN102539506A (en) * 2010-11-30 2012-07-04 希森美康株式会社 Method of electrochemically detecting a sample substance
CN102539506B (en) * 2010-11-30 2014-11-05 希森美康株式会社 Method of electrochemically detecting a sample substance
US8920626B2 (en) 2010-11-30 2014-12-30 Sysmex Corporation Method of electrochemically detecting a sample substance

Also Published As

Publication number Publication date
GB0812845D0 (en) 2008-08-20

Similar Documents

Publication Publication Date Title
EP2220494B1 (en) Electrochemical detection of a metal labelled analyte
de La Escosura-Muñiz et al. Size-dependent direct electrochemical detection of gold nanoparticles: application in magnetoimmunoassays
Wang Nanoparticle‐based electrochemical bioassays of proteins
US7045364B2 (en) Electrochemical immunoassays using colloidal metal markers
US8617907B2 (en) Determining the presence or amount of a metal-labelled species
Szymanski et al. Preparation and quality control of silver nanoparticle–antibody conjugate for use in electrochemical immunoassays
Camilo et al. Improving direct immunoassay response by layer-by-layer films of gold nanoparticles–Antibody conjugate towards label-free detection
Hao et al. An electrochemical immunosensing method based on silver nanoparticles
Chen et al. Preparation of protein-like silver–cysteine hybrid nanowires and application in ultrasensitive immunoassay of cancer biomarker
Du et al. Plasmonic gold nanoparticles stain hydrogels for the portable and high-throughput monitoring of mercury ions
Xie et al. Polyacrylamide gel-contained zinc finger peptide as the “lock” and zinc ions as the “key” for construction of ultrasensitive prostate-specific antigen SERS immunosensor
Fan et al. Ultrasensitive photoelectrochemical microcystin-LR immunosensor using carboxyl-functionalized graphene oxide enhanced gold nanoclusters for signal amplification
Paleček et al. Ionic strength-dependent structural transition of proteins at electrode surfaces
Feng et al. Gold microstructures/polyaniline/reduced graphene oxide/prussian blue composite as stable redox matrix for label-free electrochemical immunoassay of α-fetoprotein
Liu et al. Covalent anchoring of multifunctionized gold nanoparticles on electrodes towards an electrochemical sensor for the detection of cadmium ions
GB2462062A (en) Electrochemical assay
Pollok et al. Dual-shaped silver nanoparticle labels for electrochemical detection of bioassays
JP3897285B2 (en) Biopolymer detection reagent and biopolymer detection method
Ting et al. The solid-state Ag/AgCl process as a highly sensitive detection mechanism for an electrochemical immunosensor
Szymanski et al. Electrochemical dissolution of silver nanoparticles and its application in metalloimmunoassay
Ostatná et al. Simple protein structure-sensitive chronopotentiometric analysis with dithiothreitol-modified Hg electrodes
Silveri et al. Impedimetric immunosensor for microalbuminuria based on a WS2/Au water-phase assembled nanocomposite
KR101809905B1 (en) Electrochemical biosensor for detecting silver ions using single cytosine, and method for preparing the same
Mehta et al. Advances and Challenges in Nanomaterial-Based Electrochemical Immunosensors for Small Cell Lung Cancer Biomarker Neuron-Specific Enolase
Pollok The Development of a Metalloimmunoassay for the Detection of NT-proBNP

Legal Events

Date Code Title Description
WAP Application withdrawn, taken to be withdrawn or refused ** after publication under section 16(1)