+

WO1997004313A1 - Electroimmunodosages analytiques et electrodosages de reconnaissance biologique - Google Patents

Electroimmunodosages analytiques et electrodosages de reconnaissance biologique Download PDF

Info

Publication number
WO1997004313A1
WO1997004313A1 PCT/ES1996/000151 ES9600151W WO9704313A1 WO 1997004313 A1 WO1997004313 A1 WO 1997004313A1 ES 9600151 W ES9600151 W ES 9600151W WO 9704313 A1 WO9704313 A1 WO 9704313A1
Authority
WO
WIPO (PCT)
Prior art keywords
electrode
analytical
electroassays
electroimmunoassays
biological
Prior art date
Application number
PCT/ES1996/000151
Other languages
English (en)
Spanish (es)
Inventor
Agustin Costa Garcia
Maria Begoña GONZALEZ GARCIA
Cesar Fernandez Sanchez
Original Assignee
Universidad De Oviedo
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 Universidad De Oviedo filed Critical Universidad De Oviedo
Priority to AU64195/96A priority Critical patent/AU6419596A/en
Publication of WO1997004313A1 publication Critical patent/WO1997004313A1/fr

Links

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
    • G01N33/54366—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes

Definitions

  • the invention which has application in the field of biomedical analyzes, relates to analytical electroimmunoassays and biological recognition electroassays that combine the selectivity of immunological reactions with the sensitivity of the electrode-adsorptive properties of a carbon paste electrode that It is used as support and transducer of the analytical signal. It also refers to a method to increase the sensitivity of determinations for low levels of concentration of the protein material that covers the electrode.
  • Enzyme-modified carbon pulp electrodes have recently been used as electrochemical biosensor transducers (Dom ⁇ nguez Sánchez, P., O'Suliivan, CLK., Miranda Ordieres, AJ, Tu ⁇ ón Blanco, P., Smyth, MR, 1 994, Chim. Actu. 291: 349-356).
  • Electrochemistry at solid electrodes 1 969, 280-283 at the beginning of the 1960s and widely used since then, has adsorption characteristics on different organic substances that make it possible to carry out coating studies of different materials reliably on its surface. These coatings result in low concentrations of the aforementioned organic substances can be easily quantified, if in their structure there were electroactive groups or electrochemical marks capable of being oxidized or reduced by applying a certain potential to the electrode on which they are located (Cortina Villar , JV, Costa Garc ⁇ a, A., Tu ⁇ ón Blanco, P., 1 993, Tatania
  • concentrations of the order of 1 0 21 M of alkaline phosphatase-labeled IgG can be detected amperometrically with a carbon electrode in an ELISA with amperometric detection (Thompson, RQ et al., 1 993, Anal. Chim. Minute 271: 223-229) and other ELISAs with similar characteristics have been developed with low quantification limits for different analytes.
  • a device and method could be designed both to quantify biotin and to quantify avidin, and since biotin is one of the most universal haptens and macromolecule markers, through it any carbon paste could be attached molecule or structure capable of being labeled with biotin.
  • objects of the invention are analytical electroimmunoassays and biological recognition electroassays, which use a carbon paste electrode as a transducer of the analytical signal, and which have a protein part adsorbed on the surface of the electrode for biochemical interactions. .
  • Said analytical electroimmunoassays and biological recognition electroassays use the same electrode surface in each test, which is recoverable after a convenient chemical-electrochemical treatment, with small gold particles and gold colloidal particles being provided as a necessary mark for monitoring the biological reaction , and the use of silver as a sensitizing element of the analytical signal, as well as alkaline phosphatase as an enzymatic brand for monitoring biological interaction.
  • Another object of the invention is a method of cleaning and activating the electrode surface comprising:
  • the method uses silver to sensitize the analytical signal by:
  • the method uses alkaline phosphatase by:
  • the analytical electroimmunoassays and biological recognition electroassays according to the invention could use other electrodes or ultramicroelectrodes as transducers of the analytical signal, as well as using other complexing or precipitating means, such as iodide, which are used as a sensitizing medium in the oxidation of the silver.
  • the analytical electroimmunoassays and biological recognition electroassays of the invention would be usable for the quantification of antigens, haptens and antibodies.
  • a first exemplary embodiment of the invention comprises a carbon pulp electrode that acts as a transducer of the analytical signal and a protein part adsorbed on the pulp that acts as the site of biochemical interactions.
  • the transducer consists of a carbon paste electrode, whose basic scheme can be seen in the examples illustrated in the attached Figures 1 and 2.
  • the carbon paste is prepared, for example, by mixing in a mortar 5 grams of graphite powder and 1.8 ml of paraffin, which is placed in a lower drain of a Teflon cylinder 1 which contains a rod of one conductive metal 2 which acts as an electrical contact between the carbon paste 3 and the measuring instrument (for example, a potentiostat) not shown.
  • the outer surface thereof is polished on a sheet of paper and then subjected to the following electrode pretreatment:
  • the electrode is introduced into a solution of NH 3 1.0 M, this solution is stirred and a potential of + 1 .00 V is imposed on the electrode for a period of 1 20 seconds.
  • This electrode pretreatment prior to any determination or test is absolutely essential. It is the one that always works with an electrode surface with similar characteristics and that reproducibilities higher than 96% are achieved.
  • the electrode surface of the carbon paste 3 is pretreated, it is washed in a phosphate buffer solution of pH 8.0 and then the electrode is introduced into another phosphate buffer solution of pH 8.0 which is at the same time 10 "4 M in Streptavidin is maintained for 5 minutes in this streptavidin solution, which is stirring slightly, thus fixing a layer of streptavidin 5 molecules on the carbon paste surface 3.
  • the electrode once its surface has been modified with streptavidin, is introduced 5 minutes in another buffer solution of pH 8.0 with 1% albumin.
  • albumin molecules 4 are also fixed on the electrode surface and block the centers of nonspecific adsorption.
  • the electrode thus modified becomes a biosensor that can be applied both to the quantification of biotin and to the quantification of avidin from an unknown sample.
  • the electrode thus prepared would be introduced into the unknown biotin sample of pH 8.0 during a 60 minute incubation time in which the biotin molecules would bind to a part of the adsorbed streptavidin molecules about him electrode (always in excess).
  • the electrode is removed from this first incubation and introduced into another pH 8.0 buffer solution containing biotinylated albumin adsorbed on colloidal gold particles or marked with small gold particles, and held in this solution for another 60 minutes in order to that the streptavidin molecules that had remained unreacted with the biotin molecules in the sample now react with the adsorbed biotin molecules on the colloidal gold particles.
  • the electrode is removed and taken to the quantization cell containing a 0.1 M HCI solution in addition to a platinum auxiliary electrode and a saturated Ag / AgCI reference electrode.
  • the electrode processes that will be described later for colloidal gold are caused, so that the recorded signal will depend directly on the number of biotiny colloidal gold particles 6 attached to the electrode surface through the streptavidin-biotin interactions. colloidal gold Therefore, the analytical signal will be inversely related to the concentration of biotin in the sample.
  • the streptavidin adsorbed on the electrode should be competed with the avidin of the sample in solution, for the same concentration of biotinylated albumin adsorbed on colloidal gold also in solution. As there was less concentration of avidin in the sample, more biotiny colloidal gold particles could bind to the streptavidin modified electrode surface. Therefore, also in this case the analytical signal would be inversely related to the concentration of anaite in the sample, in this case avidin.
  • the methodology would be similar to that described for the scheme in Figure 1, but now the coating protein that would be adsorbed on the previously activated carbon paste 3 would be biotinylated albumin 4 'and albumin 4.
  • the concentration used of biotinylated albumin would be 1 0 "4 M and, as in the scheme of Figure 1, could also be used to quantify both biotin and avidin, however, in this case streptavidin adsorbed onto 6 'colloidal gold would be used to obtain the analytical signal.
  • the electrochemical brand is colloidal gold. This brand is used for the first time as the basis of the voltammetric signal for monitoring this interaction. Biotin or avidin control will be carried out indirectly through the oxidation-reduction process of colloidal gold, which consists of the following:
  • colloidal gold adsorbed through the interaction is perfectly adhered to the carbon paste surface, since a mechanical stirring of the 2000 rpm solution is unable to release any colloidal gold particles from the electrode.
  • the first process is caused by subjecting the electrode to a potential of + 1 .25 V in relation to an Ag / AgCI reference electrode saturated with KCI, in a 0.1 M HCI medium.
  • the second process is the one that gives rise to the analytical signal. Once the first process has been verified, the same electrode is subjected to a sweep of potentials from + 1 .25 V towards less positive potentials in which a voltammetric peak around + 0.43 V appears as a result of reduction of adsorbed AuCI 4 .
  • It consists of oxidizing the deposited silver in the presence of a complexing or precipitating agent of the silver.
  • the methodology consists of the following phases:
  • Gold particle fixation which takes place through the biological reaction between the protein part fixed in the electrode and the analyte fixed or marked with the gold particles.
  • sandwich-type enzyme electro-immunosensor designs work with the same reproducibility benefits as biotin / avidin biosensors.
  • the electrode is used for the first time, its surface is subjected to a potential of + 1 .6 V for 1 0 minutes in a pH 9 phosphate buffer. This step is done only once. And then as a pretreatment that is repeated for each test, the following is done: 1 o ) Wash for 2 minutes in 0.1 M NaOH with stirring. 2 o ) Imposition of a potential of + 1.5 V for 5 minutes in a pH 9 phosphate buffer.
  • This electrode pretreatment makes the same surface of carbon paste can be used in successive tests with similar electrode-adsorptive properties, and these tests comprise the following stages: A) Controlled coating of the protein part on the electrode surface, which consists of adsorbing on said surface the protein necessary for biological interaction.
  • the basic instrumentation for practicing the present invention would consist, for example, of an electrolytic cell for three electrodes (working, auxiliary and reference) and a potentiostat plus a wave generator or a polargraph.
  • An electroimmunotechnology according to the invention would compete with known immunotechnologies, such as RIA, EMIT, ELISA, etc., of which the most representative and most frequently used today would be ELISA, a technique against which electroimmunotechnology of the invention has, among others, the following advantages:
  • the electrode material is a relatively very material cheap, and the same electrode can be used for more than 20 analyzes approximately.
  • the concentration range may range from 10 '9 M and 1 0 "6 M.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Manufacture Of Metal Powder And Suspensions Thereof (AREA)

Abstract

Electroimmunodosages analytiques et électrodosages de reconnaissance biologique, lesquels combinent la sélectivité propre aux réactions immunologiques avec la sensibilité des propriétés électrodico-adsorptives d'une électrode en pâte carbonique qu'on utilise comme support et transducteur du signal analytique. Application aux analyses biomédicals.
PCT/ES1996/000151 1995-07-18 1996-07-18 Electroimmunodosages analytiques et electrodosages de reconnaissance biologique WO1997004313A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU64195/96A AU6419596A (en) 1995-07-18 1996-07-18 Analytical electroimmunoassays and electroassays for biological recognition

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES9501475A ES2102970B1 (es) 1995-07-18 1995-07-18 Electroinmunoensayos analiticos y electroensayos de reconocimiento biologico.
ESP9501475 1995-07-18

Publications (1)

Publication Number Publication Date
WO1997004313A1 true WO1997004313A1 (fr) 1997-02-06

Family

ID=8291128

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES1996/000151 WO1997004313A1 (fr) 1995-07-18 1996-07-18 Electroimmunodosages analytiques et electrodosages de reconnaissance biologique

Country Status (3)

Country Link
AU (1) AU6419596A (fr)
ES (2) ES2102970B1 (fr)
WO (1) WO1997004313A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2810739A1 (fr) * 2000-06-26 2001-12-28 Centre Nat Rech Scient Immunodosage electrochimiques a l'aide de marqueurs metalliques colloidaux
US7655261B2 (en) 2001-05-25 2010-02-02 Gorm Danscher Medicament and method of treatment of patients with heavy metals

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990005300A1 (fr) * 1988-11-10 1990-05-17 Midwest Research Technologies, Inc. Methode de detection electrique d'une reaction de liaison
RU1441991C (ru) * 1986-07-18 1995-01-20 Казанский государственный технический университет им. А.Н.Туполева Способ очистки поверхности изделия

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU1441991C (ru) * 1986-07-18 1995-01-20 Казанский государственный технический университет им. А.Н.Туполева Способ очистки поверхности изделия
WO1990005300A1 (fr) * 1988-11-10 1990-05-17 Midwest Research Technologies, Inc. Methode de detection electrique d'une reaction de liaison

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 9529, Derwent World Patents Index; AN 95-222453, XP002018788 *
E. LORENZO ET AL.: "Immobilized enzyme carbon paste electrodes as amperometric sensors.", ELECTROANALYSIS, vol. 3, 1991, pages 319 - 323, XP000609698 *
JIAN-XING FENG ET AL.: "Electrochemical pretreatment of carbon fibers for in vivo electrochemistry: effects on sensitivity and response time.", ANAL. CHEM., vol. 59, 15 July 1987 (1987-07-15), pages 1863 - 1867, XP000611032 *
M. B. GONZALEZ ET AL.: "adortive stripping voltammetric behaviour of colloidal gold and immunogold on carbon paste electrode.", BIOELECTROCHEMISTRY AND BIOENERGETICS, vol. 38, October 1995 (1995-10-01), pages 389 - 395, XP000609801 *
MI-SOOK WON ET AL.: "Determination of copper(I) Ion with a chemically modified carbon paste electrode based on di(2-imino-cyclopentylidine mercaptomethyl) disulfide.", ELECTROANALYSIS, vol. 5, June 1993 (1993-06-01), pages 421 - 426, XP000609699 *
S. H. JENKINS ET AL.: "Extending the detection limit of solid-phase electrochemical enzyme immunoassay to the attomole level", ANALYTICAL BIOCHEMISTRY, vol. 168, 1988, pages 292 - 299, XP000609815 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2810739A1 (fr) * 2000-06-26 2001-12-28 Centre Nat Rech Scient Immunodosage electrochimiques a l'aide de marqueurs metalliques colloidaux
WO2002001178A3 (fr) * 2000-06-26 2003-02-13 Centre Nat Rech Scient Immunodosages electrochimiques a l'aide de marqueurs metalliques colloïdaux
US7045364B2 (en) 2000-06-26 2006-05-16 Centre National De La Recherche Scientifique (Cnrs) Electrochemical immunoassays using colloidal metal markers
US7655261B2 (en) 2001-05-25 2010-02-02 Gorm Danscher Medicament and method of treatment of patients with heavy metals

Also Published As

Publication number Publication date
ES2102970A1 (es) 1997-08-01
ES2149104B1 (es) 2001-05-16
ES2149104A1 (es) 2000-10-16
AU6419596A (en) 1997-02-18
ES2102970B1 (es) 1998-04-01

Similar Documents

Publication Publication Date Title
Tang et al. Electrochemical immuno-bioanalysis for carcinoma antigen 125 based on thionine and gold nanoparticles-modified carbon paste interface
Bagal-Kestwal et al. Invertase inhibition based electrochemical sensor for the detection of heavy metal ions in aqueous system: Application of ultra-microelectrode to enhance sucrose biosensor's sensitivity
EP1105736B1 (fr) Analyse d'affinite electrochimique
AU705417B2 (en) Determination of an analyte in a liquid medium
Liu et al. Square wave voltammetry versus electrochemical impedance spectroscopy as a rapid detection technique at electrochemical immunosensors
Turyan et al. Patterning and characterization of surfaces with organic and biological molecules by the scanning electrochemical microscope
Viet et al. Gold-linked electrochemical immunoassay on single-walled carbon nanotube for highly sensitive detection of human chorionic gonadotropinhormone
de la Escosura-Muñiz et al. Controlling the electrochemical deposition of silver onto gold nanoparticles: Reducing interferences and increasing the sensitivity of magnetoimmuno assays
US20060160100A1 (en) Enzymatic electrochemical detection assay using protective monolayer and device therefor
Fernández-Sánchez et al. AC voltammetric carbon paste-based enzyme immunosensors
US6063259A (en) Microfabricated thick-film electrochemical sensor for nucleic acid determination
Pedano et al. Immobilization of DNA on glassy carbon electrodes for the development of affinity biosensors
Balkenhohl et al. Screen-printed electrodes as impedimetric immunosensors for the detection of anti-transglutaminase antibodies in human sera
Zhang et al. Silica-nanoparticle-based interface for the enhanced immobilization and sequence-specific detection of DNA
CA2585183A1 (fr) Procede ameliore d'analyse electrochimique d'un analyte
Hernando et al. Immobilization of horseradish peroxidase via an amino silane on oxidized ultrananocrystalline diamond
KR101991563B1 (ko) 도파민 검출용 센서 및 이의 제조 방법
US8617907B2 (en) Determining the presence or amount of a metal-labelled species
Zacco et al. Electrochemical biosensing based on universal affinity biocomposite platforms
Mohd Said Electrochemical biosensor based on microfabricated electrode arrays for life sciences applications
JP3393361B2 (ja) バイオセンサ
US6458600B1 (en) Method for producing laterally organized structures on supporting surfaces
Wilhelm et al. Scanning electrochemical microscopy of enzymes immobilized on structured glass-gold substrates
de la Escosura-Muñiz et al. DNA hybridization sensor based on aurothiomalate electroactive label on glassy carbon electrodes
US20160281147A1 (en) Ultrasensitive diagnostic device using electrocatalytic fluid displacement (efd) for visual readout

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR CA HU JP KR NO PL RO RU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载