EP1678488A1 - Nouvelle configuration de capteur - Google Patents

Nouvelle configuration de capteur

Info

Publication number
EP1678488A1
EP1678488A1 EP04789600A EP04789600A EP1678488A1 EP 1678488 A1 EP1678488 A1 EP 1678488A1 EP 04789600 A EP04789600 A EP 04789600A EP 04789600 A EP04789600 A EP 04789600A EP 1678488 A1 EP1678488 A1 EP 1678488A1
Authority
EP
European Patent Office
Prior art keywords
layer
membrane
hydrophobic
well
silicon
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Withdrawn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
EP04789600A
Other languages
German (de)
English (en)
Inventor
Bruce Andrew Cornell
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.)
Ambri Ltd
Original Assignee
Ambri Ltd
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
Priority claimed from AU2003905808A external-priority patent/AU2003905808A0/en
Application filed by Ambri Ltd filed Critical Ambri Ltd
Publication of EP1678488A1 publication Critical patent/EP1678488A1/fr
Withdrawn legal-status Critical Current

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
    • G01N33/5436—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand physically entrapped within the solid phase
    • 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

Definitions

  • the present invention relates to a novel device for use in a lipid membrane biosensor, methods of preparing the device and applications thereof.
  • Biosensors based on ion channels or ionophores contained within lipid membranes that arc deposited onto metal electrodes and where the ion channels are switched in the presence of analyte molecules have been described in International Patent Application Numbers WO92/17788, WO 93/21528, WO 94/07593 and US.
  • ionophores such as gramicidin ion channels ma be co-dispersed with amphiphilic molecules, thereby forming lipid membranes with altered properties in relation to the permeability of ions.
  • the present invention provides a device comprising a well defined within a substrate, said substrate comprising, in sec ⁇ ence, a first base layer, a second hydrophobic layer, and a third hydrophilic layer; said well extending from the upper surface of the base layer through the second and third layers to provide an opening in the upper surface of the third layer wherein a lipid membrane comprising a closely packed array of self-assembling amphiphilic molecules extends across the well within the region defined between the first base layer and the third hydrophilic layer.
  • the device further comprises a fourth hydrophobic layer wherein said well urther extends to the upper surface of the f urth layer.
  • the method further comprises the step of depositing a fourth hydrophobic layer on the third hydrophilic layer wherein said well is f rmed so as to extend to the upper sur ace of the fourth layer.
  • a membrane-based biosensor comprising the steps of:
  • lipid membrane of said device comprises one or more biotinylated gramicidin ion channels and/or one or more biotinylated membrane spanning lipids;
  • FIGURES Figure 1 shows in schematic form a well of a device according to one embodiment of the present invention.
  • Figure 2 shows in schematic form a well of a device according to another embodiment of the present invention in whkih the opening of the well is overlaid with a hydrophilic mesh.
  • Figure 3 shows in schematic form a device according to an embodiment of the present invention.
  • the present invention provides a device comprising a well defined within a substrate, said substrate comprising, in sequence, EX first base layer, a second hydrophobic layer, and a third hydrophilic layer; said well extending from the upper surface of the base layer through the second and third layers to provide an cjpening in the upper surface of the third layer wherein a lipid membrane comprising a. closely packed array of self-assembling amphiphilic molecules extends across the ell wit-hin the region defined between the first base layer and the third hydrophilic layer.
  • the device further comprises a fourth Hydrophobic layer wherein said well further extends to the upper surface of the fourth layer.
  • the lipid membrane is composed such that the impedance of the membrane is dependent on the presence or absence of an analyte to be detected.
  • the composition of such membranes are described in detail in the publications referred to herein.
  • the device cluding die features of the first aspect can retain a protective bead of a polar liquid, such as water or an aqueous solution, on the membrane surface thereby stabilising the membrane to the introduction of air.
  • a polar liquid such as water or an aqueous solution
  • the dimensions of the well are preferably selected such that the bead of retained polar liquid is of sufficient size to prevent contact of the memb-cane with air, but still be capable of rapid exchange with analyte solutions or other solutions-
  • suitable dimensions are: for the first layer, 50 to 150 nm, preferably 100 nm; for the second layer, 100 to 300 ⁇ m, preferably 250 run; for the third layer, 400 to 600 nm, preferably 500 run; and for the optional fourth layer, 100 to 300 nm, preferably 200 ran.
  • the opening of the well is substantially circular with a diameter of from abomt " 10 to 200 microns, more preferably 20 to 150 microns and most preferably 100 microns.
  • the first base layer is a conductive layer.
  • the conductive layer may be formed from any conductive material capable of acting as electrode including gold, silver, copper, conducting polymers and the like. Gold is particularly preferred.
  • the third hydrophilic layer comprises a hydrophilic material.
  • the hydrophilic material may be of any suitable type which has some affinity for the polar liquid and is preferably metallic, ceramic or polymeric.
  • the hydrophilic material is selected from, the group consisting of silicon carbide, silicon oxide, silicon dioxide, titanium dibromidc, titanium oxide, titanium nitride, zinc oxide, zirconium dioxide, magnesium oxide, iron oxides, graphite, boron nitride, chromium nitride, and poly vinylidene fluoride. Titanium oxide is particularly preferred.
  • the second and optional fourth hydrophobic layers maybe formed from any suitable hydrophobic material that resist wetting by the polar liquid.
  • the materials to form d e second and optional fourth layers may be the same or different.
  • the hydrophobic material is an organic polymer and/or is selected from the group consisting of pol amides, PVC, polystyrenes, polyesters, polycarbonates, polyurethanes, nylons Glass fibre, Plastics, Silicon rubbers, Latex, glass, vinyl, phenolic, resins, brass, Te rafluoroethylene Octadccyltrichlorosilane, Teflon, Silicon, nitride, Silicon carbide, alurni um nitride, oxidised silicon carbide, Butadiene Styrene, Ethylene vinyl acetate, and PTFE (polytetrafluoroethylerie) polymer. It is preferred that the hydrophobic material is oxidised silicon carbide or aluminium nitride.
  • the third hydrophilic layer comprises titanium oxide and the second hydrophobic layer comprises oxidised silicon carbide.
  • the internal circumfexence of the well is varied such that it is greater in the region defined by the fourth hydrophobic layer than in the region deployed by the second hyc ophilic layer.
  • a mesh covers at least a portion of the well opening. This allows the well to retain the polar liquid more tenaciously by increasing the capillary force and therefore allows for the size of the well and hence the size of the electrode to be maintained.
  • the mesh is formed as an extension of the optional fourth hydrophobic layer.
  • the mesh may form any tesselating pattern, such as rectangular, square, triangular, hexagonal and the like. A hexagonal pattern is particrularly preferred.
  • the wall partitions of the mesh are preferably from 1 to 10 nm in thickness and the individual cells are preferably from 20 to 100 nm wide at their widest point
  • sensor according to the present invention may include a plurality of wells in a single substrate thus allowing for an array of the wells to be formed.
  • the sensor comprises a laminar substrate comprising a silicon support 1, a titanium layer 2 of about 5 nm thickness, a first conductive base layer 3 of gold of about 100 nm in thickness, a second hydrophobic layer 4 of silicon nitride of about 200 m in thickness; a third hydrophilic layer 5 of silicon oxide of abut 500 nm in thickness, and a fourth hydrophobic layer 6 of about 200 nm thickness.
  • a well 9 is defined within the substrate such that it extends from the upper surface of the base layer through the second, third and fourth layers to provide a well opening 10 in the upper surface of the fourth layer.
  • a membrane is located within the region of the weE defined by the second hydrophobic layer, the membrane comprising a lower first layer 7 and an upper second layer 8 of closely packed amphiphilic molecules and a plurality of ionophores with at least a proportion of the molecules and ionophores of the lower first layer 7 being connected to the upper surface of the first conductive layer 3 by means of linker groups.
  • the internal circumference of the well is varied such that it is greater in the region defined by the fourth hydrophobic layer 6 than in the region defined by the second hydrophobic layer 4.
  • FIG 2 a top view of four wells ⁇ in a sttbstrate is displayed in which the openings of the wells are covered with a hexagonal mesh 12.
  • the mesh in this case is silicon dioxide.
  • a further layer of silicon nitride 13 coats the remainder of the substrate surface.
  • Figure 3 displays the membrane-based biosensor of Figure 1 in which a bead of liquid 14 is trapped in the well above the surface of the membrane by the arrangement of hydrophilic and hydrophobic layers.
  • the method further comprises the step of depositing a fourth hydrophobic layer on the third hydrophilic layer wherein said well is formed so as to extend to the upper surface of the fourth layer.
  • the first base layer is deposited on a layer of titanium of a support material comprising a silicon support and the layer of titani-um. More preferably, the support material is formed by depositing a layer of titanium on the silicon support. Even more preferably, the silicon support is a single crystal silicon wafer,
  • the first base layer is from 50 nm to 150 ran thick, more preferably 100 nm thick.
  • the second hydrophobic layer is from 1 . O0 ran to 300 nm thick, more preferably 200 n thick.
  • the third hydrophilic layer is from 400 nm to 600 nm thick, more preferably 500 nm thick.
  • d e optional fourth hydrophobic layer Ls from 100 nm to 300 nm thick, more preferably about 200 nm thick.
  • the opening of the well is substantially circular with a diameter of from about 10 to 200 microns, more preferably 20 to 150 microns and most preferably 100 microns.
  • the first base layer is a conductive layer, more preferably the conductive layer is formed from gold.
  • the third hydrophilic layer comprises a hydrophilic material.
  • the hydrophilic material may be of any suitable type which has some affinity for the polar liquid and is preferably metallic, ceramic or polymeric.
  • the hydrophilic material is selected from the group consisting of silicon carbide, silicon oxide, silicon dioxide, titaruum dibromide, titanium oxide, titanium nitride, zinc oxide, zirconium dioxide, magnesium oxide, iron oxides, graphite, boron nitride, chromium nitride, and poly vinylidene fluoride. Titanium oxide is particularly preferred.
  • the second and optional fourth hydrophobic layers may be formed from any suitable hydrophobic material that resist wetting by the polar liquid.
  • the materials to form the second and optional fourth layers may be the same or different.
  • the hydrophobic material is an organic polymer and/ r is selected from the group consisting of polyamides, PVC, polystyrenes, polyesters, polycarbonates, polyurcthanes, nylons Glass fibre, Plastics, Silicon rubbers, Latex, glass, vinyl, phenolic, resins, brass, Tetrafluoroethylene Octadecyltrichlorosilane, Teflon, Silicon nitride, Silicon carbide, aluminium nitride, oxidised silicon carbide, Butadiene Styrene, Ethylene vinyl acetate, and PTFE (polytetrafluoroethylene) polymer. It is preferred that the hydrophobic material is oxidised silicon carbide or alurniniurn nitride.
  • the well is formed by etching.
  • the lipid membrane comprises a lower first membrane layer and an upper second membrane layer and wherein the lipid membrane further comprises a plurality of ionophores with at least a proportion of the molecules and ionophores of the lower first layer being connected to the upper surface of the first base layer by means of linker groups,
  • the step of forming the lipid membrane comprises: forming a first solution containing one or more amphiphilic molecules, one or more linker groups and one or more ionophores in a first organic solvent (preferably ethanol); contacting the first base layer of the well ith the first solution to form the lower first membrane layer comprising a closely packed array of said amphiphilic molecules and said ionophores wherein said lower first membrane layer is connected to the first base layer " by means of said linker groups; rinsing the device with a suitable second organic solvent (preferably ethanoi); removing the excess second organic solvent; forming a second solution of one or more amphiphilic molecules and one or more ionophores in a suitable third organic solvent (preferably ethanoi); contacting the second solution with the device corr_ ⁇ rising said first lower membrane layer to form the second layer membrane layer; rinsing the device with an aqueous solution; and removing the device from the aqueous solution and allowing to drain.
  • said second organic solvent is removed " by rapid air drying.
  • the device is immersed in the third solution.
  • the one or more ionophores comprise gramicidin A or an analogue thereof. More preferably, the one or more ionophores are biotinylated.
  • one or more receptors are attached to the surface of the membrane. More preferably, the one or more receptors are attached to the membrane by using streptavidin, avidin or one of the related biotin binding-proteins. Even more preferably, one or more receptors are coupled to one or more biotinylated gramicidin ion channeLs and /or to one or more biotinylated mernbrane-spanning lipid.
  • Tn a third aspect there is provided a method of preparing a membrane-based biosensor comprising the steps of;
  • the electrode is stored at between minus 20°C and plus 5"C
  • the preparation of a device in accordance w th an embodiment of the present invention will now be described.
  • the embodiment is concerned with device that can be used as a component in an electrode sensor that detects the presence of an analyte by an adjustment in the conductivity of the membrane, it would be clear to a person skilled in the art, however, that devices and methods of the present invention also extends to other techniques for detecting the presence of an analyte using a device such as by using fluoresence techniques and the like.
  • a layer of titanium typically 5nm in thickness
  • a layer of gold typically 100 nm in thickness which forms an electrode of die membrane-based biosensor
  • a hydrophobic layer typically 200 nm in thickness
  • a hydrophilic layer typically 500 nm in thickness
  • optionally a hydrophobic layer typically 200 nm in thickness to form a laminar substrate
  • the support material is a single crystal silicon wafer.
  • the electrode area is wet etched using a photolithographic patterning approach.
  • the gold electrode consists of a freshly evaporated or sputtered gold electrode.
  • a membrane can then be formed in the well of the device by:
  • the membrane so formed extends across the well within the region defined between the first base layer and e third, hydrophilic layer.
  • the solvent for the adsorbing solutions in steps (4) and (8) and for the rinsing step (6) is ethanoi. It is further preferred that in step (6) the solvent is removed by rapid air drying,
  • step (7) it is preferred that immediately upon removal of excess solvent in step (7) the electrode is immersed in the solution described in step (8).
  • Tt is preferred that the ionophorc in step (8) is gra ⁇ ucic ⁇ n A or an analogue thereof. It is further preferred that this molecule is biotinylated to enable subsequent binding of streptavidin or analogues thereof. It is preferred that rinsing step (10) is carried out before the solvent drains from the bilayer membrane formed in step (9) .
  • the membrane can be further functionalised in order to provide for the detection of the presence of analyte by the membrane-based biosensor.
  • One convenient method to attach appropriate receptors to the urface of a membrane is by using streptavidin, avidin or one of the related biotin binding-proteins as a means of coupling a wide range of receptors onto a biotinylated gramicidin ion channel or membrane- spanning lipid.
  • An example of such a process comprises the steps of:
  • biotinylated receptor molecules are introduced into the well of the device using an ink jet robot.
  • This simple sequential dipping technology allows for a desired sensor configuration to be rapidly assembled by combining a stored membrane with a an appropriate receptor solution.
  • This process, combining previously prepared and stored components, allows for simplified fabrication which can be carried out remotely from the point of manufacture.
  • the membrane of a device of the present invention may be dried in a relatively controlled fashion such that the lipid membrane retains its function, structure and activity when resolvatcd. This will assist in the storage and handling of devices according to the present invention.
  • the amount of liquid retained above the membrane be reproducibly and precisely controlled, hence methods of drying such as lyophilisation, evaporation, or evaporation over controlled humidity, are preferred.
  • the devices of the present invention provide further advantages in simplifying the analyte detection process in that it allows for the use of air bubbles to separate different components of a liquid flow stream.
  • the use of air to separate different components of a liquid flow stream and prevent their mixing is well known in the art, but has not previously been applicable to lipid membrane sensors due to the possibility of permanent disruption of the membrane.
  • the devices of the invention provide a membrane which is protected from the effects of the uncontrolled introduction of air or gas by the presence of a trapped bead of polar liquid or solvent. By retaining liquid on the lipid membrane surface even in the presence of air, the present invention allows, for example, the sequential passage of rinse, calibration and analyte solutions over the membrane with each solution being separated from the next by interposition of an air bubble.

Abstract

L'invention porte sur un dispositif comprenant un puits formé dans un substrat, ce substrat comprenant, successivement, une première couche de base, une deuxième couche hydrophobe et une troisième couche hydrophobe, le puits s'étendant depuis la surface supérieure de la couche de base dans les deuxième et troisième couches de façon à former une ouverture dans la surface supérieure, et une membrane lipidique comprenant une matrice de molécules amphiphiles à auto-assemblage, très concentrées, s'étendant dans le puits dans la région formée entre la première couche de base et la troisième couche hydrophile. L'invention porte également sur un procédé de fabrication de ce dispositif.
EP04789600A 2003-10-22 2004-10-22 Nouvelle configuration de capteur Withdrawn EP1678488A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2003905808A AU2003905808A0 (en) 2003-10-22 Novel sensor configuration
PCT/AU2004/001453 WO2005040783A1 (fr) 2003-10-22 2004-10-22 Nouvelle configuration de capteur

Publications (1)

Publication Number Publication Date
EP1678488A1 true EP1678488A1 (fr) 2006-07-12

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EP04789600A Withdrawn EP1678488A1 (fr) 2003-10-22 2004-10-22 Nouvelle configuration de capteur

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EP (1) EP1678488A1 (fr)
WO (1) WO2005040783A1 (fr)

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DK2122344T3 (da) 2007-02-20 2019-07-15 Oxford Nanopore Tech Ltd Lipiddobbeltlags-sensorsystem
GB0724736D0 (en) 2007-12-19 2008-01-30 Oxford Nanolabs Ltd Formation of layers of amphiphilic molecules
CN102203618B (zh) * 2008-10-30 2014-10-15 郭培宣 用于dna测序和其他用途的膜集成病毒dna包装马达蛋白连接器生物传感器
WO2013007570A1 (fr) * 2011-07-12 2013-01-17 Centre National De La Recherche Scientifique Procédé de criblage grande vitesse de l'activité lipase et/ou d'inhibiteurs de lipase dans des échantillons biologiques et des milieux de culture
GB201202519D0 (en) 2012-02-13 2012-03-28 Oxford Nanopore Tech Ltd Apparatus for supporting an array of layers of amphiphilic molecules and method of forming an array of layers of amphiphilic molecules
CN102608103B (zh) * 2012-03-30 2014-04-30 中国科学院长春应用化学研究所 表面增强拉曼散射基底及其制备方法
GB201313121D0 (en) 2013-07-23 2013-09-04 Oxford Nanopore Tech Ltd Array of volumes of polar medium
GB201418512D0 (en) 2014-10-17 2014-12-03 Oxford Nanopore Tech Ltd Electrical device with detachable components
CN105154879B (zh) * 2015-09-23 2018-08-31 北京机械工业自动化研究所 风速管复合涂层、其制备方法及具有该复合涂层的风速管
GB201611770D0 (en) 2016-07-06 2016-08-17 Oxford Nanopore Tech Microfluidic device
CN114456524B (zh) * 2017-10-18 2023-08-11 大金工业株式会社 交联性弹性体组合物和氟橡胶成型品
AU2020239385A1 (en) 2019-03-12 2021-08-26 Oxford Nanopore Technologies Plc Nanopore sensing device and methods of operation and of forming it
CN110567935A (zh) * 2019-09-04 2019-12-13 宁波工程学院 一种亲疏水组装表面增强拉曼散射基底及其制备方法
CN112705279B (zh) * 2019-10-25 2022-09-23 成都今是科技有限公司 微流控芯片及其制备方法
CN114471755B (zh) * 2021-12-30 2023-10-20 上海天马微电子有限公司 微流控芯片及其制作方法

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CA2145996A1 (fr) * 1992-10-01 1994-04-14 Burkhard Raguse Membranes de detection ameliorees
WO1994025862A1 (fr) * 1993-05-04 1994-11-10 Washington State University Research Foundation Substrat de biocacteur concu pour supporter une membrane lipidique bicouche contenant un recepteur
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