WO2022115080A1 - Électrodes jetables à base de composites de nanoargile - Google Patents

Électrodes jetables à base de composites de nanoargile Download PDF

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Publication number
WO2022115080A1
WO2022115080A1 PCT/TR2021/051236 TR2021051236W WO2022115080A1 WO 2022115080 A1 WO2022115080 A1 WO 2022115080A1 TR 2021051236 W TR2021051236 W TR 2021051236W WO 2022115080 A1 WO2022115080 A1 WO 2022115080A1
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WO
WIPO (PCT)
Prior art keywords
electrode
nanoclay
determination
target analyte
electrochemical
Prior art date
Application number
PCT/TR2021/051236
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English (en)
Inventor
Kadriye Arzum ERDEM GÜRSAN
Esma YILDIZ
Original Assignee
Ege Üni̇versi̇tesi̇
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 Ege Üni̇versi̇tesi̇ filed Critical Ege Üni̇versi̇tesi̇
Priority to EP21898840.0A priority Critical patent/EP4248199A1/fr
Publication of WO2022115080A1 publication Critical patent/WO2022115080A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/20—Silicates
    • C01B33/36—Silicates having base-exchange properties but not having molecular sieve properties
    • C01B33/38—Layered base-exchange silicates, e.g. clays, micas or alkali metal silicates of kenyaite or magadiite type
    • C01B33/40—Clays
    • 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
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors
    • 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
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
    • 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
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

Definitions

  • the present invention is related to a disposable carbon-based electrode developed by modifying the surface with the nanocomposite formed by a nanoclay; halloysite nanoclay (HNT) with ionic liquid (IL).
  • a diagnostic kit that can be used by all laboratories, hospitals, and healthcare institutions, R&D units and personnel working in universities is presented in order to monitor the diagnosis and course of many diseases (cancer, neurodegenerative diseases-Alzheimer, metabolic diseases-Diabetes) by performing qualitative and quantitative determination of biomarkers (miRNA, DNA, protein, toxin, etc.) in various biological materials (serum analysis, analysis in artificial serum environment, total RNA analysis, blood protein analysis, etc.) with the electrode within the scope of the invention.
  • Electrodes subject to the invention also have the potential to be used in agricultural studies and for the determination of environmental contaminants. Therefore, the electrodes subject to the invention can be used by R&D units and university personnel for environmental analysis and in laboratories for the determination of environmental pollutants (pesticides, heavy metals, etc.) and toxins in groundwater and drinking water.
  • environmental pollutants pesticides, heavy metals, etc.
  • chromatographic based chemical analysis techniques such as a high-cost technique; HPLC/DAD (High Pressure Liquid Chromatography), LC/MS/MS (Liquid Chromatography -Mass Spectrometry/Mass Spectrometry) and GC/MS (Gas Chromatography-Mass Spectrometry) are used in pesticide analyses in environmental samples, which is another proposed application area, in the present art.
  • HPLC/DAD High Pressure Liquid Chromatography
  • LC/MS/MS Liquid Chromatography -Mass Spectrometry/Mass Spectrometry
  • GC/MS Gas Chromatography-Mass Spectrometry
  • Present invention is related to a disposable carbon-based electrode with modified surface with nanocomposite containing nanoclay and conductive material which meets the aforementioned needs, eliminates all the disadvantages and provides some additional advantages.
  • Halloysite nanoclay which is in the kaolin group nanoclay, was used to show the application of nanoclay-conductive material nanocomposite without any restrictive effect when developing the electrodes subject to the invention.
  • Conductive material can be ionic liquid, metallic nanoparticle, or conductive polymer.
  • Halloysite nanoclay (HNT) as the kaolin group nanoclay and ionic liquid as the conductive material were preferred in the invention.
  • An ionic liquid containing the imidazolium salt was used in the invention.
  • the primary object of the invention is to determine the target analytes (biomarkers and various environmental pollutants in various diseases, etc.) in a fast, sensitive, precise, highly selective, and cost-effective manner compared to fluorescence, spectroscopy that are used in analysis of various disease like the determination of cancer biomarkers and chemical analysis methods used in the determination of various pollutants.
  • the electrodes according to the invention are practical electrodes that can perform the analysis in a short time and can be easily made specific to the target analyte and enable the analysis resulting with low detection limit.
  • the differential pulse voltammetry (DPV) technique was used compared to other electrochemically based studies used for the determination of cancer biomarkers known in the art, and no chemical agent or indicator was required.
  • the applicability of the electrodes of the invention to the diagnostic kit in the medical field has been demonstrated in the presence of a cancer biomarker- specific sensing DNA probe.
  • the determination of the cancer biomarker is clearly shown depending on the electrochemical signal measured in the total RNA samples obtained from healthy and cancerous cell lines by using HNT/IL developed electrodes within the scope of the invention.
  • the nanoclay-ionic liquid nanocomposite modified electrodes prepared can be applied to the direct analysis of target analytes (cancer biomarkers, miRNA, DNA, pesticides, endocrine dismptors, heavy metals, proteins, toxins, environmental pollutants, drugs, etc.) in the sample matrix.
  • target analytes cancer biomarkers, miRNA, DNA, pesticides, endocrine dismptors, heavy metals, proteins, toxins, environmental pollutants, drugs, etc.
  • the invention has a fast and practical method, and includes electrodes modified with composites containing non-toxic biocompatible natural nanoclay compared to techniques such as fluorescence, spectroscopy, which are used in the determination of cancer biomarkers.
  • the invention is a basis for the diagnostic kit due to the clear demonstration of the electrochemical response measured for the determination of the cancer biomarker.
  • Halloysite nanoclay a natural type of nanoclay used in the modification of carbon-based electrodes, is a non-toxic and environmentally friendly material.
  • Ionic liquid which is the other material that forms the composite structure with halloysite nanoclay, has played a role in both increasing electrical conductivity and increasing the electrode surface area by providing a homogeneous distribution of nanoclay.
  • the application of a diagnostic kit based on halloysite nanoclay-ionic liquid nanocomposite modified carbon-based electrodes is shown for the first time within the scope of the invention to be used in the determination of biomarkers (miRNA, protein, toxin, DNA, etc.) in various diseases such as cancer, etc. and in the determination of environmental pollutants (pesticides, endocrine dismptors, heavy metals, etc.) with the invention.
  • HNT/IL-PGE halloysite nanoclay-ionic liquid composite modified electrodes
  • EIS impedance spectroscopy
  • CV cyclic voltammetry
  • the determination of cancer biomarker is carried out over a period of 60-100 minutes in total with the invention.
  • the invention includes a disposable nanoclay-ionic liquid nanocomposite modified electrode that can perform voltametric analysis for the target analyte in a very short time compared to the methods known in the art.
  • Figure 1 (A) Voltammograms, (B) Niquist diagrams obtained in redox probe solution, (a) pencil graphite electrode (PGE), (b) ionic liquid-pencil graphite electrode (IL-PGE), (c) halloysite nanoclay-ionic liquid composite modified pencil graphite electrode (HNT/IL-PGE)
  • Figure 2 Histogram presenting the average guanine signals measured after the hybridization step with DNA probe in total RNA samples isolated from (A) cancerous cell line and (B) the healthy cell line.
  • Inner figure presenting the voltammograms (a) signal received in ABS solution with HNT/IL modified electrode, (b) signal measured with DNA probe, guanine signals measured in the presence of hybridization with DNA probe in total RNA sample isolated from (c) 2 pg/mL healthy cell line, (d) 2 pg/mL cancerous cell line.
  • the invention is related to a carbon based electrode that its surface is modified with halloysite nanoclay (HNT) and ionic liquid (IL) nanocomposite and also related to the application of the inventive electrode in the medical field and environmental pollution.
  • HNT halloysite nanoclay
  • IL ionic liquid
  • Carbon-based electrode can be glassy carbon electrode, carbon paste electrode, screen printed carbon electrode and electrode whose surface is covered with carbon materials.
  • a disposable pencil graphite electrode was preferred as a carbon-based electrode in the invention.
  • electrodes were prepared by making them into nanocomposites containing ionic liquid, which provides an increase in electrical conductivity, a homogeneous distribution of nanoclay and an increase in surface area, with halloysite nanoclay, which is a non-toxic natural nanoclay, to provide rapid, sensitive, selective electrochemical method-based analysis of cancer biomarkers.
  • ionic liquid which provides an increase in electrical conductivity, a homogeneous distribution of nanoclay and an increase in surface area
  • halloysite nanoclay which is a non-toxic natural nanoclay
  • RNA samples prepared from healthy (human embryonic kidney cell-HEK-293) and cancerous (human breast cancer cell-MCF-7) cell lines in real samples with the developed electrodes.
  • the results of the analysis in the real sample revealed that the method based on the developed electrodes was selective and sensitive.
  • the electrochemical determination of the target analyte with a disposable carbon-based electrode modified with nanocomposite containing nanoclay and conductive material on the surface contains the following process steps.
  • the target analyte can be miRNA, protein, DNA, pesticides, endocrine disruptors, heavy metals, toxins, or drugs.
  • the recognizing molecule specific to the target analyte may be nucleic acid, aptamer, or antibody.
  • Pencil graphite electrodes PGE
  • halloysite nanotubes with a non-toxic nanotube-like structure belonging to the nanoclay family and ionic liquids were used in the design of nanoclay-ionic liquid modified electrodes developed for the determination of biomarkers in various diseases and various environmental pollutants.
  • target cancer biomarker microRNA-21 (miRNA-21) was carried out in cancerous and healthy cell lines in the application for the determination of cancer biomarkers with the developed HNT/IL modified pencil graphite electrodes.
  • the electrochemical determination of the target analyte with the electrode modified with nanocomposite containing halloysite nanoclay (HNT) and ionic liquid (IL) comprises the following process steps; i) Preparation of HNT/IL nanocomposites, ii) Modification and electrochemical characterization of HNT/IL nanocomposites on the electrode surface, iii) Electrochemical determination of the target analyte with HNT/IL modified electrode.
  • HNT/IL nanocomposites Preparation of HNT/IL nanocomposites: The distribution of halloysite nanotubes in the appropriate solvent is ensured and a nanoclay composite is formed in a concentration range of 100-1500 mg/mL HNT in the presence of an ionic liquid in the environment in a concentration range of 1% -25%. ii) Modification and electrochemical characterization of HNT/IL nanocomposites on the electrode surface:
  • the electrodes were kept in the prepared composite solution for a period of 15-90 minutes and the modification of electrode surface was occurred by the passive adsorption method.
  • the miRNA-21 target analyte which is defined as a biomarker in various cancers (such as breast cancer, lung cancer, liver cancer) was selected and the determination on the total RNA samples obtained from cancerous and healthy cell lines was carried out with the electrode subject to the invention.
  • the peak potential of the guanine signal observed between +0,85-1-1,05 V was measured in the voltametric analysis taken with this electrode subject to the invention in the sample prepared after hybridization between the miRNA-21 specific DNA probe and the existing miRNA-21 in the sample.
  • the miRNA-21 -specific DNA probe which is used as a catcher bioreceptor that detects target cancer biomarkers selected as cancer biomarkers, was studied in the concentration range of 0.01 - 2 pg/mL.
  • the miRNA-21 DNA probe with the target cancer biomarker miRNA-21 target was allowed to interact for the period deemed appropriate in the range of 1-30 minutes. Then, it was kept on the HNT/IL modified PGE surface for 5-90 minutes. Electrochemical measurements were performed using differential pulse voltammetry (DPV) technique.
  • the miRNA-21 DNA probe used is amino labelled at the 5’ end and contains inosine base, which is the synthetic analogue instead of guanine base.
  • the guanine signal observed in peak potential between + 0.85 - + 1.05 V was measured when hybridization occurred between the miRNA-21 target and the miRNA-21 DNA probe. However, no guanine signal was observed when the target analyte miRNA-21 was absent.
  • the miRNA-21 DNA probe hybridization times with the target cancer biomarker miRNA-21 were optimized between the time range of 1 minute-30 minutes.
  • the hybridization time between miRNA-21 DNA probe and the target cancer biomarker miRNA-21 was optimized in the range of 1 minute- 30 minutes.
  • the immobilization time on the surface of the inventive electrode was optimized between 5 minutes and 90 minutes after the hybridization between miRNA-21 DNA probe and the target cancer biomarker miRNA-21.
  • the detection limit of the miRNA-21 target sequence can be calculated in the linear concentration range of 0,25- 2 pg/mL.
  • RNA samples obtained from the selected cell lines were used to show their applicability to real patient samples.
  • the guanine signal (Figure 2) was measured in the total RNA sample obtained from cancerous cell line with the electrodes (HNT/IL-PGE) subject to the invention compared to healthy cell line.
  • An increase in the measured guanine signal was observed ( Figure 2-A) depending on the increasing concentration after hybridization of the total RNA samples obtained from the cancer cell line in the concentration range of 1-4 pg/mL with the miRNA-21- specific DNA probe, as can be seen in Figure 2.
  • guanine signal depending on the increasing concentration was not observed (Figure 2-B) after hybridization of total RNA samples obtained from the healthy cell line in the concentration range of 1-4 pg/mL with miRNA-21 -specific DNA probe.
  • the limit of detection for miRNA-21 can be calculated in the range of 1-4 pg/mL linear concentration of the total RNA samples obtained from the selected cancer cell line to show its applicability to real patient samples.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Dispersion Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

L'invention concerne une électrode à base de carbone jetable mise au point par modification de la surface avec un nanocomposite constitué de nanoargile halloysite (HNT) et de liquide ionique (IL).
PCT/TR2021/051236 2020-11-26 2021-11-19 Électrodes jetables à base de composites de nanoargile WO2022115080A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP21898840.0A EP4248199A1 (fr) 2020-11-26 2021-11-19 Électrodes jetables à base de composites de nanoargile

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
TR2020/19070 2020-11-26
TR202019070 2020-11-26
TR2021/009966 2021-06-18
TR202109966 2021-06-18

Publications (1)

Publication Number Publication Date
WO2022115080A1 true WO2022115080A1 (fr) 2022-06-02

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EP (1) EP4248199A1 (fr)
WO (1) WO2022115080A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016148665A1 (fr) * 2015-03-18 2016-09-22 ERDEM GURSAN, Arzum K. Biocapteur d'acide nucléique électrochimique à usage unique à base d'électrodes de graphite de crayon modifiées par des nanoparticules d'hydroxyapatite

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016148665A1 (fr) * 2015-03-18 2016-09-22 ERDEM GURSAN, Arzum K. Biocapteur d'acide nucléique électrochimique à usage unique à base d'électrodes de graphite de crayon modifiées par des nanoparticules d'hydroxyapatite

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRONDANI DANIELA, SCHEEREN CARLA WEBER, DUPONT JAIRTON, VIEIRA IOLANDA CRUZ: "Halloysite clay nanotubes and platinum nanoparticles dispersed in ionic liquid applied in the development of a catecholamine biosensor", ANALYST, ROYAL SOCIETY OF CHEMISTRY, UK, vol. 137, no. 16, 1 January 2012 (2012-01-01), UK , pages 3732, XP055940382, ISSN: 0003-2654, DOI: 10.1039/c2an35313j *
YAN YUNSONG, ET AL.: "Electroactive Ionic Soft Actuators with Monolithically Integrated Gold Nanocomposites Electrodes", ADV. MATER., vol. 29, 18 April 2017 (2017-04-18), XP055940383, DOI: 10.1002/adma.201606109 *
YARAH ECE, ET AL.: "Ionic Liquid Modified Single-use Electrode Developed for Voltammetric Detection of miRNA -34a and its Application to Real Samples", ELECTROANALYSIS, vol. 32, 20 September 2019 (2019-09-20), pages 384 - 393, XP055940381, DOI: 10.1002/elan.201900353 *

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