ITRM20090450A1 - Chip nanoforato di nitruro di silicio per l'analisi di profili di espressione genica e relativi biosensori. - Google Patents
Chip nanoforato di nitruro di silicio per l'analisi di profili di espressione genica e relativi biosensori. Download PDFInfo
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- ITRM20090450A1 ITRM20090450A1 IT000450A ITRM20090450A ITRM20090450A1 IT RM20090450 A1 ITRM20090450 A1 IT RM20090450A1 IT 000450 A IT000450 A IT 000450A IT RM20090450 A ITRM20090450 A IT RM20090450A IT RM20090450 A1 ITRM20090450 A1 IT RM20090450A1
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- Italy
- Prior art keywords
- biosensor
- solution
- nanophores
- chip
- silicon
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- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 title description 2
- 238000004458 analytical method Methods 0.000 title description 2
- 230000014509 gene expression Effects 0.000 title description 2
- 229910052710 silicon Inorganic materials 0.000 title description 2
- 239000010703 silicon Substances 0.000 title description 2
- 108091034117 Oligonucleotide Proteins 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 230000009849 deactivation Effects 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonium chloride Substances [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 2
- 230000008021 deposition Effects 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- 239000012153 distilled water Substances 0.000 claims description 2
- 239000012154 double-distilled water Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 239000001509 sodium citrate Substances 0.000 claims description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims 1
- 238000010884 ion-beam technique Methods 0.000 claims 1
- 239000012472 biological sample Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 230000001173 tumoral effect Effects 0.000 description 2
- 208000032236 Predisposition to disease Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000003869 genetically modified organism Nutrition 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 229910052814 silicon oxide Inorganic materials 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
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
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
- G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Dispersion Chemistry (AREA)
- Nanotechnology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
secondo l'invenzione permette di analizzare, in diverse patologie (tumorali e non), i profili di espressione genica a partire da campioni biologici di varia natura (materiale bioptico, sangue periferico o midollare ecc.). Tale applicazione consente di valutare marcatori molecolari con significato prognostico e/o consentire un migliore inquadramento del paziente nelle categorie di rischio e la conseguente personalizzazione della terapia. In particolare, il dispositivo permette di identificare SNP in campioni biologici di diverse patologie (tumorali e non) . Essendo noto che gli SNP sono associati alla suscettibilità di alcune malattie e alla diversa risposta alla terapia farmacologica, tale analisi consentirà di effettuare un fingerprinting dei pazienti al fine di valutare la predisposizione a patologie e la risposta ai farmaci. Inoltre, il dispositivo secondo l'invenzione può essere utilizzato per applicazioni in campo botanico, zootecnico e zooprofilattico, ad esempio per l'identificazione di OGM in alimenti o in coltivazione o per la caratterizzazione di virus.
membrana di di silicio nanoforata
nano fori diametro efficace
da 20 a 50 nm spessore
da 10 nm a 50 nm preferibilmente da 10 a 20 nm.
Ί nanofori possono essere formati mediante
ioni focalizzati, ad esempio mediante l'impiego di un Costituisce ulteriore oggetto della presente invenzione un procedimento di preparazione del biosensore descritto sopra comprendente o consistente nelle seguenti fasi: deposizione di una soluzione di oligonucleotide di lunghezza da 40 a 55 basi, preferibilmente 45 basi, detta soluzione avendo una concentrazione che varia da 10 a 200 nM, preferibilmente 100 nM in acqua bidistillata oppure in SSC 2X ossia una soluzione di 0,3 M cloruro di sodio, 0,03 M citrato di sodio e pH 7,1; b) disattivazione della superficie del biosensore. La disattivazione della superficie del biosensore può avvenire mediante lavaggi con una soluzione di ammoniaca 28% e successivi lavaggi con acqua bi-distillata.
ulteriormente due serbatoi contenenti una soluzione ionica comunicanti attraverso i nanofori e due elettrodi per l'applicazione di una tensione e rilevazione di corrente.
Costituisce ulteriore oggetto della presente invenzione l'uso biosensore o del dispositivo come definiti sopra per rilevazioni di molecole in campo medico, botanico, zootecnico o zooprofilattico.
La presente invenzione verrà ora descritta a titolo illustrativo, ma non limitativo, secondo sue forme preferite di realizzazione, con particolare riferimento alle figure dei disegni allegati in cui:
Figura,1 mostra uno schema di cella tratto da A J Storm, J H Chen, H W Zandbergen, and C Dekker, Translocation of doublé- strand dna through a Silicon oxide nanopore . Physical Review, 71:051903, 2005 [8], Figura 2 mostra la cella della figura 1 posizionata su un tavolo antivibrante all'interno di una doppia gabbia di Faraday.
Figura 3 mostra una rampa di corrente rilevata applicando diverse tensioni alla cella di misura contenente il chip nanoforato (figura 3A) e la curva tensione -corrente ricostruita a partire dalla rampa di corrente con evidenziato in linea tratteggiata il fit lineare realizzato per ricavare una stima della resistenza R del nanoporo (figura 3B).
Figura 4 mostra uno schema di nanoporo in cui r à ̈ il raggio e 1 la lunghezza.
Claims (1)
- Rivendicazioi chip secondo la rivendicazione 1, in cui i nanofori sono formati mediante fascio di ioni Biosensore comprendente o consistente in chip, in cui almeno un nanoforo à ̈ funzionalizzato con un oligo nucleotide lunghezza da 13 ,6 nm a 18,7 nm, preferibilmente 15,3 nmy IX 1 . Biosensore secondo la rivendicazione 44,/ in cui i nanofori sono funzionali zzati con oligonucleotidi uguali o diversi tra loro. 4, il diametro effice var ia da 3 a 20 nm, preferibilmente da 4 a 10 nm, ancora più preferibilmente da 4 a 6 mn - --Procedimento di preparazione del biosensore secondo ognuna delle rivendicazioni 1-2 comprendente o consistente nelle seguenti fasi deposizione di una soluzione di oligonucleotide di lunghezza da 40 a 55 basi, preferibilmente 45 basi, detta soluzione avendo una concentrazione che varia da 10 a 200 nM, preferibilmente 100 nM in acqua bidistillata oppure in una soluzione di 0,3 M cloruro di sodio, 0,03 M citrato i sodio, pH 7,1; b) disattivazione della superficie del biosensore. 4 Procedimento secondo la rivendicazione 3 in cui la disattivazione della superficie del biosensore avviene mediante lavaggi con una soluzione di ammoniaca 28% e successivi lavaggi con acqua bi-distillata. 5 Dispositivo per la rilevazione di comprendente il come definito in ognuna delle rivendicazioni 1-2 6 Dispositivo secondo la rivendicazione comprendente ulteriormente due serbatoi contenenti una soluzione ionica comunicanti attraverso i nanofori e due elettrodi per l'applicazione di una tensione e rilevazione di corrente. Uso rivendicazioni iosensore come definito in ognuna delle rivendicazioni o del dispositivo come definito in ognuna delle rivendicazioni per rilevazioni di molecole in campo medico, botanico, zootecnico o zooprofilattico.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITRM2009A000450A IT1398771B1 (it) | 2009-09-04 | 2009-09-04 | Chip nanoforato di nitruro di silicio per l'analisi di profili di espressione genica e relativi biosensori. |
| EP10774286.8A EP2473849B1 (en) | 2009-09-04 | 2010-09-02 | Nanopored silicon nitride chip for the analysis of gene expression profiles |
| PCT/IT2010/000382 WO2011027379A1 (en) | 2009-09-04 | 2010-09-02 | Nanopored silicon nitride chip for the analysis of gene expression profiles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ITRM2009A000450A IT1398771B1 (it) | 2009-09-04 | 2009-09-04 | Chip nanoforato di nitruro di silicio per l'analisi di profili di espressione genica e relativi biosensori. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| ITRM20090450A1 true ITRM20090450A1 (it) | 2011-03-05 |
| IT1398771B1 IT1398771B1 (it) | 2013-03-18 |
Family
ID=41510001
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| ITRM2009A000450A IT1398771B1 (it) | 2009-09-04 | 2009-09-04 | Chip nanoforato di nitruro di silicio per l'analisi di profili di espressione genica e relativi biosensori. |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP2473849B1 (it) |
| IT (1) | IT1398771B1 (it) |
| WO (1) | WO2011027379A1 (it) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051578B (zh) * | 2011-04-04 | 2020-07-24 | 哈佛大学校长及研究员协会 | 通过局部电位测量进行的纳米孔感测 |
| FR2975986A1 (fr) * | 2011-06-01 | 2012-12-07 | Commissariat Energie Atomique | Procede de fonctionnalisation de la paroi d'un pore. |
| EP3158330B1 (en) * | 2014-06-19 | 2019-03-06 | The Board of Trustees of The Leland Stanford Junior University | Apparatuses and methods for determining analyte charge |
| CA2976043A1 (en) | 2015-02-10 | 2016-10-06 | Multerra Bio, Inc. | Apparatuses and methods for detecting molecules and binding energy |
| CN105039539A (zh) * | 2015-07-13 | 2015-11-11 | 中国矿业大学 | 一种利用原位纳米力学测试系统识别dna碱基的方法 |
| US10444179B2 (en) | 2016-08-10 | 2019-10-15 | Multerra Bio, Inc. | Apparatuses and methods for detecting molecules and binding energy |
| US20200326325A1 (en) * | 2019-04-12 | 2020-10-15 | Lisa Diamond | Nanosensor chip with compound nanopores |
| JP2024081540A (ja) * | 2022-12-06 | 2024-06-18 | 株式会社アドバンテスト | ポアチップおよび微粒子測定システム |
-
2009
- 2009-09-04 IT ITRM2009A000450A patent/IT1398771B1/it active
-
2010
- 2010-09-02 EP EP10774286.8A patent/EP2473849B1/en not_active Not-in-force
- 2010-09-02 WO PCT/IT2010/000382 patent/WO2011027379A1/en not_active Ceased
Non-Patent Citations (6)
| Title |
|---|
| HAN A ET AL: "Label-free detection of single protein molecules and protein-protein interactions using synthetic nanopores", ANALYTICAL CHEMISTRY 20080615 AMERICAN CHEMICAL SOCIETY US, vol. 80, no. 12, 15 June 2008 (2008-06-15), pages 4651 - 4658, XP007911227 * |
| IQBAL S M ET AL: "Solid-state nanopore channels with DNA selectivity", NATURE NANOTECHNOLOGY NATURE PUBLISHING GROUP UK, vol. 2, no. 4, April 2007 (2007-04-01), pages 243 - 248, XP007911218, ISSN: 1748-3387 * |
| MUSSI V ET AL: "Solid state nanopores for gene expression profiling", SUPERLATTICES AND MICROSTRUCTURES, ACADEMIC PRESS, LONDON, GB, vol. 46, no. 1-2, 10 October 2008 (2008-10-10), pages 59 - 63, XP026152421, ISSN: 0749-6036, [retrieved on 20081010] * |
| SMEETS R M M ET AL: "Noise in solid-state nanopores.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 15 JAN 2008, vol. 105, no. 2, 15 January 2008 (2008-01-15), pages 417 - 421, XP007911228, ISSN: 1091-6490 * |
| SMEETS R M M ET AL: "Translocation of RecA-coated double-stranded DNA through solid-state nanopores.", NANO LETTERS SEP 2009, vol. 9, no. 9, 3 December 2008 (2008-12-03), pages 3089 - 3096, XP007911221, ISSN: 1530-6992 * |
| WANUNU M ET AL: "Chemically modified solid-state nanopores", NANO LETTERS AMERICAN CHEMICAL SOCIETY USA, vol. 7, no. 6, June 2007 (2007-06-01), pages 1580 - 1585, XP007911217, ISSN: 1530-6984 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2011027379A1 (en) | 2011-03-10 |
| EP2473849A1 (en) | 2012-07-11 |
| EP2473849B1 (en) | 2014-10-29 |
| IT1398771B1 (it) | 2013-03-18 |
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