WO2010010211A2 - Biocapteur ampérométrique jetable, procédé de production de ce dernier et procédé de détermination de la présence d'analytes dans des aliments - Google Patents

Biocapteur ampérométrique jetable, procédé de production de ce dernier et procédé de détermination de la présence d'analytes dans des aliments Download PDF

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Publication number
WO2010010211A2
WO2010010211A2 PCT/ES2009/000381 ES2009000381W WO2010010211A2 WO 2010010211 A2 WO2010010211 A2 WO 2010010211A2 ES 2009000381 W ES2009000381 W ES 2009000381W WO 2010010211 A2 WO2010010211 A2 WO 2010010211A2
Authority
WO
WIPO (PCT)
Prior art keywords
biosensor
stage
gold
enzyme
amperometric biosensor
Prior art date
Application number
PCT/ES2009/000381
Other languages
English (en)
Spanish (es)
Other versions
WO2010010211A3 (fr
Inventor
Angel Julio Reviejo Garcia
José Manuel PINGARRON CARRAZON
Susana Campuzano Ruiz
María GAMELLA CARBALLO
Valentín VINCENTE GARCIA-ECHAVE
Javier Manso Lorenzo
Ana Vazquez De Prada
Francisco Javier Ferero Martin
Juan Campo Rodriguez
Marta Valledor Llopis
Original Assignee
Inbea Biosensores, S.L.
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 Inbea Biosensores, S.L. filed Critical Inbea Biosensores, S.L.
Publication of WO2010010211A2 publication Critical patent/WO2010010211A2/fr
Publication of WO2010010211A3 publication Critical patent/WO2010010211A3/fr

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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
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • 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
    • 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
    • G01N33/02—Food
    • 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
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/403—Cells and electrode assemblies
    • G01N27/404—Cells with anode, cathode and cell electrolyte on the same side of a permeable membrane which separates them from the sample fluid, e.g. Clark-type oxygen sensors

Definitions

  • the purpose of the present specification refers to a disposable amperometric biosensor for the determination of, for example, fructose in all types of foods containing the analyte to be detected.
  • Biosensors of fructose have been constructed using different immobilization strategies of the FDH: adsorption on colloidal gold particles that are subsequently deposited on the vitrified carbon electrode [Yabuki, S .; Mizutani, F. Electroanalysis 9 (1997) 23-25]; incorporation of the enzyme in carbon paste electrodes [Parellada, J .; Dominguez, E .; Fernández, V.M. Anal.Chim. Minutes 330 (1996) 71-77.], [Paredes, P.A .; Parellada, J .; Fernández, V.M .; Katakis, l .; Dominguez, E. Biosens. & Bioelectron.
  • the biosensor in which the enzyme has been immobilized in phospholipid bilayers deposited on the electrode has greater stability [Kinnear, K.T .; Monbouquette, H. G. Anal. Chem. 69 (1997) 1771-1775], but the immobilization stage is usually excessively long, requiring dialysis processes lasting several days (2-6 days).
  • the best results have been achieved with biosensors manufactured by modifying carbon paste with the enzyme [Ikeda, T .; Matsushita, F .; Senda, M. Biosens. & Bioelectron.
  • a fructose dehydrogenase biosensor has also been developed that improves the previous ones in which the co-mobilization of the FDH enzyme and the TTF redox mediator is produced by cross-linking with glutaraldehyde on a conventional gold electrode modified with a mercaptopropionic acid (MPA) monolayer [Campuzano , OA Loaiza, M. Pedrero, FJ Manuel de Villena, JM Pingarrón, Bioelectrochemistry 63 (2004) 199-206], this monolayer is necessary to separate the surface of the gold electrode from the enzyme so that the deactivation of Ia is not produced FDH Operational times of more than 30 days are achieved with this biosensor and more than 150 analyzes can be performed with the same biosensor. However, the time required for its manufacture is more than one day, because this time is necessary to deposit the MPA monolayer necessary so that the enzyme deactivation does not occur.
  • MPA mercaptopropionic acid
  • the gold deposited on the stainless steel surface by sputtering has a structure different from that of the conventional gold electrode that does not produce the deactivation of the enzyme and therefore the modification with MPA monolayers of the gold deposited on the surface of the stainless steel electrode is not necessary.
  • the gold-plated stainless steel electrode maintains all other characteristics of the conventional gold electrode in electrochemical applications.
  • the coating of quartz crystals with gold by sputtering is a usual technique in the construction of piezoelectric biosensors. Recently it has also been used in the construction of electrochemical biosensors mainly in the form of tracks on polymeric non-conductive materials or on other metals previously deposited on polymeric surfaces such as Cr as is done in the construction of piezoelectric crystals.
  • Biosensors have been constructed with gold deposited by sputtering for the construction of enzymatic biosensors [Masatoshi Hashimoto, Sanjay Upadhyay, Hiroaki Suzuki, Biosensors and Bioelectronics 21 (2006) 2224-2231; Huaqing Li, Zonghui Guoa, Hui Wang, Dafu Cui, Xinxia Cai; Sensors and Actuators B 119 (2006) 419-424] and immunosensors [Eva M. Abad-Villar, M. Maria Fernández-Abedul, Agusti ⁇ n Costa-Garc ⁇ a, Biosensors and Bioelectronics 17 (2002) 797-780; Eva M. Abad-Villar, M. Maria Fernández-Abedul, Agusti ⁇ n Costa-Garc ⁇ a, Analytica Chimica Acta 453 (2002) 63-69] who need to modify the gold surface to achieve good biosensor stability.
  • biosensors constructed by sputtering deposition have never been deposited directly on metal surfaces such as stainless steel.
  • the deposition of gold on stainless steel by sputtering is simple and economical to construct gold electrodes (biosensors) in which the enzyme is immobilized directly on the surface of the electrode without there being a deactivation of the enzyme as mentioned above.
  • the present invention relates to a disposable enzymatic electrode for the determination, preferably of fructose, in all types of foods containing this analyte.
  • the biosensor consists of an electronic matrix of stainless steel or any other conductive material embedded in an insulating material in which a gold film has been deposited by means of the sputtering technique on one of the surfaces of the matrix. electrode, the other surface being in contact with the potentiostat.
  • the corresponding enzyme is deposited on the gold film with the appropriate mediator and encapsulated with a dialysis membrane.
  • This method is based on the use of a device comprising the biosensor object of the invention, together with an Ag / AgCI reference electrode and an auxiliary electrode of a conductive material.
  • This device is immersed in a working solution to which an aliquot of the food sample is added.
  • the biosensor is connected to an instrument that in addition to being used as a potentiostat allows, by means of a previous calibration, to relate the current generated in the biosensor with the content of the analyte (in the appropriate units) in the analyzed food sample.
  • This measuring instrument has been specifically designed for the biosensor, solving communication and data logging problems to provide a reliable result.
  • the determination of the analyte content in the sample can be done in two different ways: a) by external calibration and b) using the standard addition method.
  • the present invention relates to a process for preparing disposable amperometric enzyme electrodes.
  • the procedure is characterized by preparing the bioelectrodes according to the methodology described below:
  • a thin layer of gold is placed by spputering, then, without performing any cleaning or conditioning treatment of the gold, the amount of enzyme is placed suitable in solution, the solution is allowed to dry, then an aliquot with the appropriate amount of mediator is added, the solution is allowed to dry. Finally, a dialysis membrane (or any other analyte permeable membrane) is placed on the gold surface containing the enzyme and the mediator.
  • a device that allows individual recognition by the measuring instrument is implemented to the biosensor in order to count the number of measurements.
  • the enzymatic biosensor is manufactured by simple physical entrapment between the surface of the gold and the membrane, without the need to separate by any type of procedure (normally self-assembled mono-layers of thiols) from the enzyme, since The enzyme in contact with the surface Conventional gold electrode is deactivated.
  • the non-deactivation of the enzyme by the gold particles deposited on the stainless steel surface is due to the different microscopic structure that it has with respect to conventional gold surfaces.
  • the stability of the disposable biosensor is good as evidenced when different aspects are considered: a) Repeatability of the amperometric response for a biosen ⁇ or, b) Reproducibility in the manufacture of different electrodes manufactured in the same way and c) Lifetime of the biosensor that is 1 month or 100 measures, once these requirements have been met the biosensor is discarded
  • the biosensor thus constructed is placed in the sensor unit and immersed in the working solution obtaining the analyte measurements in the working solution by amperometry, measuring the current when the steady state is reached at a constant potential of 150 mV versus Ag / AgCI.
  • the electronics of the device allows the currents obtained in the biosensor to be correlated with the analyte concentration in the sample, by means of a previously established calibration and taking into account the dilution factor for each sample in particular.
  • FIGURE 1 View of the disposable amperometric biosensor, object of the present invention, in an exploded view (figure 1a) and overall view (figure 1b).
  • FIGURE 2 View of the use device of the disposable amperometric biosensor, object of the present invention. Preferred embodiment of the invention.
  • the disposable amperometric biosensor (100) comprises, at least: a first conductive body (1), preferably cylindrical and steel
  • a second insulating body (2) preferably cylindrical, located on the first body (1) such that said first body (1) is embedded in the second body (2); and where, on the lower surface (12) of both bodies (1, 2) at least: a third layer of gold (3) deposited by sputtering is placed, on which the corresponding fourth layer of enzyme (4) is deposited with the right mediator; and where, in addition, said lower face (12) of the set of bodies (1, 2) is encapsulated with a dialysis membrane (5) closed with a ring (6).
  • the disposable amperometric biosensor (100), object of the present invention is used by a device shown in Figure 2.
  • Said device comprises, at least: a first body (200) container of the connections, including the connecting element (202) with a potentiostat, not shown in the figures, and configured for device calibration; a second body (201) located in the lower part of the first body (200), which in its lower part comprises, in turn: a first disposable amperometric biosensor (100), object of the present invention; a second reference electrode (203) preferably of Ag / AgCI; and a third auxiliary electrode of a conductive material (204); where said device is configured to be immersed in a working solution to which an aliquot sample of the food is added.
  • the manufacturing process of the biosensor, second aspect of the present invention comprises, at least, the following steps: (i) a first stage of placing a layer of gold by sputtering on a surface of a conductive material embedded in an insulating material;
  • the method of determining the presence of analytes in food comprises, at least, the following steps:

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Food Science & Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Inorganic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)

Abstract

La présente invention concerne un biocapteur ampérométrique jetable (100), un procédé de production de ce dernier et un procédé de détermination de la présence d'analytes dans des aliments. Cette invention repose principalement sur l'utilisation d'une mince couche d'or (3) déposée par pulvérisation cathodique sur une électrode.
PCT/ES2009/000381 2008-07-24 2009-07-20 Biocapteur ampérométrique jetable, procédé de production de ce dernier et procédé de détermination de la présence d'analytes dans des aliments WO2010010211A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200802211A ES2337328B1 (es) 2008-07-24 2008-07-24 Biosensor amperometrico desechable, metodo de fabricacion del mismo ymetodo de determinacion de la presencia de analitos en alimentos.
ESP200802211 2008-07-24

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WO2010010211A2 true WO2010010211A2 (fr) 2010-01-28
WO2010010211A3 WO2010010211A3 (fr) 2010-03-18

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3321670A4 (fr) * 2015-07-09 2019-01-02 Biolan Microbiosensores, S.L. Système et méthode pour mesurer les sulfites dans des échantillons d'aliments au moyen d'un biocapteur ampérométrique et utilisation dudit biocapteur
EP3512958B1 (fr) * 2016-09-16 2022-04-06 Eastman Chemical Company Électrodes de biocapteur préparées par dépôt physique en phase vapeur
US11624723B2 (en) 2016-09-16 2023-04-11 Eastman Chemical Company Biosensor electrodes prepared by physical vapor deposition
US11835481B2 (en) 2016-06-15 2023-12-05 Eastman Chemical Company Physical vapor deposited biosensor components
US11881549B2 (en) 2017-06-22 2024-01-23 Eastman Chemical Company Physical vapor deposited electrode for electrochemical sensors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1496354A1 (fr) * 2002-03-08 2005-01-12 Matsushita Electric Industrial Co., Ltd. Procede de determination de substrat
ES2255861B1 (es) * 2004-12-23 2007-11-01 Intecsa-Inarsa S.A. Dispositivos y procedimientos para la medida del contenido de etanol en sangre.

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1496354A1 (fr) * 2002-03-08 2005-01-12 Matsushita Electric Industrial Co., Ltd. Procede de determination de substrat
ES2255861B1 (es) * 2004-12-23 2007-11-01 Intecsa-Inarsa S.A. Dispositivos y procedimientos para la medida del contenido de etanol en sangre.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PALMISANO, F. ET AL.: 'Flow injection analysis of L-lactate in milk and yoghurt by on-line microdialysis and amperometric detection at a disposable biosensor.' THE ANALYST. vol. 126, no. 6, June 2001, ISSN 0003-2654 pages 866 - 870 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3321670A4 (fr) * 2015-07-09 2019-01-02 Biolan Microbiosensores, S.L. Système et méthode pour mesurer les sulfites dans des échantillons d'aliments au moyen d'un biocapteur ampérométrique et utilisation dudit biocapteur
US11835481B2 (en) 2016-06-15 2023-12-05 Eastman Chemical Company Physical vapor deposited biosensor components
EP3512958B1 (fr) * 2016-09-16 2022-04-06 Eastman Chemical Company Électrodes de biocapteur préparées par dépôt physique en phase vapeur
US11624723B2 (en) 2016-09-16 2023-04-11 Eastman Chemical Company Biosensor electrodes prepared by physical vapor deposition
US11630075B2 (en) 2016-09-16 2023-04-18 Eastman Chemical Company Biosensor electrodes prepared by physical vapor deposition
US11881549B2 (en) 2017-06-22 2024-01-23 Eastman Chemical Company Physical vapor deposited electrode for electrochemical sensors

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Publication number Publication date
ES2337328B1 (es) 2011-02-14
WO2010010211A3 (fr) 2010-03-18
ES2337328A1 (es) 2010-04-22

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