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WO2004068141A1 - Detecteurs spectroscopique de resonance a guide-d'onde et a plasmons couples pour des tests pharmaceutiques et chimiques - Google Patents

Detecteurs spectroscopique de resonance a guide-d'onde et a plasmons couples pour des tests pharmaceutiques et chimiques Download PDF

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Publication number
WO2004068141A1
WO2004068141A1 PCT/US2003/002273 US0302273W WO2004068141A1 WO 2004068141 A1 WO2004068141 A1 WO 2004068141A1 US 0302273 W US0302273 W US 0302273W WO 2004068141 A1 WO2004068141 A1 WO 2004068141A1
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Prior art keywords
sensor
molecules
sensing area
group
dielectric
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PCT/US2003/002273
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English (en)
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Zdzislaw Salamon
Gordon Tollin
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The Arizona Board Of Regents On Behalf Of The University Of Arizona
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Priority to PCT/US2003/002273 priority Critical patent/WO2004068141A1/fr
Priority to AU2003217249A priority patent/AU2003217249A1/en
Publication of WO2004068141A1 publication Critical patent/WO2004068141A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides

Definitions

  • This invention pertains in general to the field of Coupled Plasmon Waveguide Resonance (CPWR) spectroscopy.
  • the invention relates to novel CPWR spectroscopic devices having a dielectric layer with surface modifications and sensor applications thereof.
  • SPR Surface plasmon resonance is a phenomenon used in many analytical applications in metallurgy, microscopy, and chemical and biochemical sensing. With optical techniques such as ellipsometry, multiple internal reflection spectroscopy, and differential reflectivity, SPR is one of the most sensitive techniques to surface and interface effects. This inherent property makes SPR well suited for nondestructive studies of surfaces, interfaces, and very thin layers.
  • a surface plasmon is an oscillation of free electrons that propagates along the surface of a conductor.
  • the phenomenon of surface plasmon resonance occurs under total internal reflection conditions at the boundary between substances of different refractive indices, such as glass and water solutions.
  • surface plasmon resonance occurs when the free electron clouds in the metal layer (the plasmons) absorb energy from the evanescent wave and cause a measurable drop in the intensity of the reflected light at a particular angle of incidence that depends on the refractive index of the second medium.
  • the conductor used for SPR spectrometry is a thin film of metal such as silver or gold; however, surface plasmons have also been excited on semiconductors.
  • the conventional method of exciting surface plasmons is to couple the transverse-magnetic (TM) polarized energy contained in an evanescent field to the plasmon mode on a metal film.
  • the amount of coupling, and thus the intensity of the plasmon is determined by the incident angle of the light beam and is directly affected by the refractive indices of the materials on both sides of the metal film.
  • changes in the refractive index of the sample material can be monitored by measuring changes in the surface plasmon coupling efficiency in the evanescent field.
  • total reflection can only occur above a particular critical incidence angle if the refractive index of the incident medium (typically a prism or grating) is greater than that of the emerging medium.
  • the refractive index of the incident medium typically a prism or grating
  • total reflection is observed only for incidence angles within a range narrower than from the critical angle to 90 degrees because of the physical limitations inherent with the testing apparatus.
  • total reflection is also observed only for a corresponding range of wavelengths. This range of incidence angles (or wavelengths) is referred to as the "observable range" for the purpose of this disclosure.
  • a metal film with a very small refractive index (as small as possible) and a very large extinction coefficient (as large as possible) is required to support plasmon resonance.
  • gold and silver are appropriate materials for the thin metal films used in visible-light SPR; in addition, they are very desirable because of their mechanical and chemical resistance.
  • the reflection of a monochromatic incident beam becomes a function of its angle of incidence and of the metal's refractive index, extinction coefficient, and thickness.
  • the thickness of the film is therefore selected such that it produces observable plasmon resonance when the monochromatic light is incident at an angle within the observable range.
  • the classical embodiments of SPR devices are the Kretschmann and Otto prism or grating arrangements, which consist of a prism with a high refractive index n (in the 1.4-1.7 range) coated on one face with a thin film of metal.
  • the Otto device also includes a very thin air gap between the face of the prism and the metal film.
  • the gap between the prism (or grating) and the metal layer which is in the order of nanometers, could be of a material other than air, even metal, so long as compatible with the production of observable plasmon resonance in the metal film when the monochromatic light is incident at an angle within the observable range.
  • Similar prior-art SPR devices are based on the phenomenon of long-range surface plasmon resonance, which is also generated with p-polarized light using a dielectric medium sandwiched between the incident medium and a thinner metal layer (than in conventional SPR applications).
  • the metal film must be sufficiently thin and the dielectric and emergent media must be beyond the critical angle (i.e., having refractive indices smaller than the refractive index of the entrant medium) so that they support evanescent waves to permit the simultaneous coupling of surface plasmons at the top and bottom interfaces of the thin metal layer (i.e., to permit excitation of surface waves on both sides of the thin metal film). This condition is necessary in order for the phenomenon of long-range surface plasmon resonance to occur.
  • the distinguishing structural characteristic between conventional surface plasmon resonance and long-range surface plasmon resonance is the tliickness of the metal film and of the inner dielectric film (the latter not being necessary for conventional SPR).
  • the metal film must be sufficiently thick and must be placed either directly on the entrant medium (i.e., prism or grating), or on a dielectric film which is too thin, to allow excitation of the surface bound waves on both metal surfaces to produce observable plasmon resonance when a monochromatic light is incident at an angle within the observable range.
  • LRSPR long-range surface plasmon resonance
  • the metal film In long-range surface plasmon resonance (LRSPR), in contrast, the metal film must be placed between two dielectric media that are beyond the critical angle so that they support evanescent waves, and must be thin enough to permit excitation of surface waves on both sides of the metal film.
  • the specific thickness depends on the optical parameters of the various components of the sensor in question, but film thicknesses in the order of 45-55nm for gold and silver are recognized as critical for conventional SPR, while no more than about half as much (15-28nm) can be used for LRSPR. It is noted that the thickness required to support either form of surface plasmon resonance for a specific system can be calculated by one skilled in the art on the basis of the system's optical parameters.
  • the main criterion for a material to support SP waves is that it have a negative real dielectric component, which results from the refractive and extinction properties mentioned above for the metal layer.
  • the surface of the metal film forms the transduction mechanism for the SPR device and is brought into contact with the sample material to be sensed at the interface between the metal film and the emerging medium contained in a cell assembly.
  • Monochromatic light is emitted by a laser or equivalent light source into the prism or grating and reflected off the metal film to an optical photodetector to create the sensor output.
  • the light launched into the prism and coupled into the SP mode on the film is p-polarized with respect to the metal surface where the reflection takes place.
  • TM transverse-magnetic
  • the surface plasmon is affected by changes in the dielectric value of the material in contact with the metal film. As this value changes, the conditions necessary to couple light into the plasmon mode also change.
  • SPR is used as a highly sensitive technique for investigating changes that occur at the surface of the metal film.
  • surface plasmon resonance spectroscopy to study the optical properties of molecules immobilized at the interface between solid and liquid phases.
  • the ability of the SPR phenomenon to provide information about the physical properties of dielectric thin films deposited on a metal layer, including lipid and protein molecules forming proteolipid membranes, is based upon two principal characteristics of the SPR effect.
  • the first is the fact that the evanescent electromagnetic field generated by the free electron oscillations decays exponentially with penetration distance into the emergent dielectric medium; i.e., the depth of penetration into the material in contact with a metal layer extends only to a fraction of the light wavelength used to generate the plasmons. This makes the phenomenon sensitive to the optical properties of the metal/dielectric interface without any interference from the properties of the bulk volume of the dielectric material or any medium that is in contact with it.
  • the second characteristic is the fact that the angular (or wavelength) position and shape of the resonance curve is very sensitive to the optical properties of both the metal film and the emergent dielectric medium adjacent to the metal surface.
  • SPR is ideally suited for studying both structural and mass changes of thin dielectric films, including lipid membranes, lipid-membrane/protein interactions, and interactions between integral membrane proteins and peripheral, water-soluble proteins. See Salamon, Z., H.A. Macleod and G. Tollin, "Surface Plasmon Resonance Spectroscopy as a Tool for Investigating the Biochemical and Biophysical Properties of Membrane Protein Systems. I: Theoretical Principles," Biochim. et Biophvs.
  • the additional layer of dielectric material functions as an optical amplifier that produces an increased sensitivity and enhanced spectroscopic capabilities in SPR.
  • the added dielectric layer makes it possible to produce resonance with either s- or p-polarized light.
  • the added dielectric protects the metal layer and could be used as a matrix for adsorbing and immobilizing the sensing materials in CPWR-based biosensor applications.
  • a biosensor is generally defined as a unique combination of a receptor for molecular recognition, for example a selective layer with immobilized antibodies, and a transducer for transmitting the values measured. Accordingly, a biosensor will detect the change which is caused in the optical properties of a surface layer due to the interaction of the receptor with the surrounding medium (such as could be detected by SPR or CPWR).
  • a biosensor will detect the change which is caused in the optical properties of a surface layer due to the interaction of the receptor with the surrounding medium (such as could be detected by SPR or CPWR).
  • the invention relates to a sensor unit for use in a system based on CPWR technology that includes a dielectric substrate having one or more sensing areas that contain at least one functional group supporting bi- or polyfunctional molecules capable of specific binding with molecules present in a sample analyte.
  • a sensor unit for use in a system based on CPWR technology that includes a dielectric substrate having one or more sensing areas that contain at least one functional group supporting bi- or polyfunctional molecules capable of specific binding with molecules present in a sample analyte.
  • a sensor unit for use in a system based on CPWR technology that includes a dielectric substrate having one or more sensing areas that contain at least one functional group supporting bi- or polyfunctional molecules capable of specific binding with molecules present in a sample analyte.
  • coupling between the bi- or polyfunctional molecules and the molecule(s) in the sample is selectively reversible such that the bi- or polyfunctional molecule-functionalized sensing areas can be regenerated, permitting repeated use of the functionalized sensor
  • the CPWR sensor of the present invention differs from SPR sensors in several key respects.
  • the invention features a surface-modified dielectric layer (not a modified metal layer).
  • the present sensor allows a user to benefit from the advantages (e.g., greater sensitivity) that CPWR-based systems offer over those utilizing SPR.
  • the present invention provides a surface- modified dielectric layer that is functionalized to support bi- or polyfunctional molecules capable of specifically binding a wide variety of chemical targets.
  • the invention may include an additional metal layer (i.e., in addition to the metal upon which the dielectric material is disposed in the CPWR device) that coats the dielectric material at the interface of the sensor and is functionalized to bind target molecules.
  • a goal of the invention is to provide the ability to perform CPWR for sensor applications.
  • Another object of the invention is to provide a sensor device that permits the practice of CPWR analysis on a wide variety of biological, chemical, and pharmaceutical molecules.
  • Another goal of the invention is to provide a method for functionalizing semi-conductor surfaces for use with CPWR.
  • Another important objective is to provide a CPWR-based technique that is suitable for testing more than lipid membranes that have either integral membrane proteins incorporated into them or peripheral membrane proteins bound to their surface.
  • Another goal of the invention is to provide a tool that is particularly suitable for obtaining information about more than one molecule in a given sample.
  • Yet another goal of the invention is to provide a technique that makes it possible to achieve high through-put testing applications with an efficient, practical and economically feasible implementation.
  • Another objective is to provide sensor devices that are suitable for direct incorporation with existing CPWR spectroscopic instruments.
  • the present invention pertains to novel and improved CPWR sensors featuring dielectric surfaces that have been functionalized for the binding of various molecules.
  • the sensors of the invention may be made in one piece, for example, from a glass slide (a dielectric material) that has been coated with a thin film of a metal suitable for CPWR procedures (e.g., silver or gold).
  • a layer of an organic polymer or a hydrogel forming a so-called basal surface that contains 5 functional groups for binding the desired ligands is applied to the surface of the glass slide.
  • the sensing surfaces have to be functionalized with different ligands capable of interaction with a target chemical or
  • the basal surface may be provided with a particular ligand during the manufacturing process or a user may provide her own.
  • the ligands employed are bi- or polyfunctional, meaning that the ligands contain a function that is utilized for immobilization on a corresponding sensing surface of the dielectric layer, plus one or
  • Fig. 1 is a schematic view of an embodiment of a coupled plasmon-waveguide resonance spectroscopic tool according to the prior art in an attenuated total reflection measuring system, wherein a glass prism coated with a 50nm-thick silver layer is protected by a 30 460nm-thick Si0 2 film; a lipid bilayer is deposited on the dielectric film and held in place by a TEFLON® spacer.
  • Fig. 2 is a schematic view of a preferred embodiment of the invention.
  • Fig. 3 is a schematic view of a second embodiment of the invention.
  • Fig. 4 is a schematic view of an embodiment of the invention featuring a very thin metal overcoating on the dielectric layer proximate to the sample interface.
  • Fig. 5 is a schematic view of fourth embodiment of the invention.
  • the invention is the result of further development of the CPWR devices described in our previous U.S. Patents (6,421,128; 6,330,387; 5,991,488).
  • the invention relates to sensor devices for use in CPWR-based systems. More specifically, the invention involves the surface modification and functionalization of a dielectric layer of a CPWR device with bi-and polyfunctional ligands capable of specifically binding one or more target molecules.
  • the invention also relates to methods for simultaneously measuring several properties of one type of molecule among a plurality of molecules in a given sample.
  • the invention relates to methods for simultaneously measuring the concentrations of different molecular species in a heterogeneous sample as well as measuring structural and electrical characteristics of such samples.
  • the invention is described herein with reference to the CPWR systems and techniques disclosed in the referenced patents. It is also understood that the dielectric layer of the invention is in addition to and separate from the sample material or analyte with which the invention is used.
  • the sample material at the interface with the emerging medium is often itself dielectric in nature, but its properties cannot be used to obtain the advantages of CPWR without the addition of an additional dielectric layer as disclosed in U.S. Patent No. 5,991,488. Therefore, all references to dielectric material pertain only to the additional layer contemplated by CPWR.
  • Fig. 1 illustrates in schematic form a typical prior art CPWR device 30.
  • the device 30 contains a metallic (or semiconductor) layer (or layers) 12, typically between 45 and 55nm thick, formed from either gold or silver deposited on either a glass prism or grating 16 for generating a surface plasmon wave. Note that the same elements could be used in an Otto configuration with a very thin air (or other material) gap between the glass and metal layer.
  • the silver film 12 is covered with a layer 32 of solid dielectric material characterized by an appropriate set of values of film thickness, t, refractive index, n, and extinction coefficient, k.
  • a lipid bilayer 34 (the material being tested) is deposited from the sample solution 20 on the dielectric film 32 and held in place by a TEFLON® spacer 36 according to the teachings of U.S. Patent No. 5,521,702 (Salamon et al.).
  • the dielectric material In the SiO 2 embodiment of Fig. 1, with a wavelength of about 633nm, the dielectric material must be at least 50nm thick to act as a waveguide. In addition, the resulting s- resonance will fall within the observable range for any thickness larger than 250nm; on the other hand, the p-resonance will be visible for any thickness greater than 400nm. In order to fulfill the conditions of the invention for both types of polarization, the dielectric layer must be at least about 420nm thick. Similarly, the same configuration embodied with a TiO 2 dielectric and a wavelength of about 633nm would require a thickness larger than 65nm for the s-resonance and larger than 140nm for the p- resonance to be observable. The conditions of the invention would be met for both types of polarization with a TiO 2 layer at least 750nm thick.
  • the dielectric film can be formed from any number of layers designed and optimized for different uses (see examples of dielectric materials, below). This feature is especially important in various sensor applications, where the dielectric overcoat can also be designed to adsorb and immobilize the sensing material either on its surface or within its interior.
  • any one layer of dielectric or combination of dielectric layers that satisfy the refractive index, extinction coefficient, and thickness requirements for producing resonance at incident angles (for a given wavelength) or at wavelengths (for a given incident angle) within the observable range is suitable for practicing the invention.
  • these materials include MgF 2 Al 2 O 3 , LaF 3 , Na 3 AlF 6 , ZnS, ZiO 2 , Y 2 O 3 , HfO 3 , Ta 2 ⁇ 5 , ITO, and nitrites or oxy-nitrites of silicon and aluminum, which are all normally used in optical applications.
  • Measurements using the CPWR sensor devices of the present invention are made in the same way as with conventional SPR techniques.
  • the attenuated total reflection method of coupling the light into the deposited thin multilayers is used, thereby exciting resonances that result in absorption of the incident radiation as a function of either the light incident angle ⁇ (with a monochromatic light source), or light wavelength ⁇ (at constant incident angle), with a consequent dip in the reflected light intensity.
  • Further details of experimental techniques employed to measure the resonance spectrum are given in Salamon and Tollin (1996), supra: Salamon et al.
  • chimeric molecules are employed for functionalizing the sensing surfaces of the dielectric layer.
  • the chimeric molecules comprise one portion binding to the basal surface, e.g., a dextran-coated sensing surface, and one portion having an affinity for the biomolecule to be detected.
  • Bi- or polyfunctional molecules for use according to the invention may be produced in a variety of ways, e.g., by means of chemical coupling of the appropriate molecules or molecule fragments, by means of hybridoma techniques for producing bifunctional antibodies, or by means of recombinant DNA techniques.
  • This last- mentioned technique involves the fusing of gene sequences coding for the structures which are wanted in the product, this product then being expressed in a suitable expression system such as, e.g., a culture of bacteria.
  • a suitable expression system such as, e.g., a culture of bacteria.
  • the covalent immobilization of nucleic acids to the sensing surface can then be accomplished as described in Swanson, M J. et al. "Reactive Polymer-Coated Surfaces for Covalent Immobilization of Nucleic Acids" (describing activated glass surfaces available from Amersham Biosciences as a “CodeLink Activated Slide” and formerly available from Surmodics, Inc. as a “3D-Link Microarray Slide.”).
  • Chemical coupling of biomolecules or fragments thereof can be performed in accordance with one of the coupling methods that have been developed for the immobilization of biomolecules (see, for example, Moser, I. et al., Sensors and Actuators B. 7, (1992) 356-362).
  • a suitable reagent is, e.g., SPDP N-succinimidyl 3-(2-pyridylthio)propionate, a heterobifunctional reagent (from Pharmacia AB, Sweden), and with a coupling technique as described by Carlsson et al. Biochem. J. 173:223 (1978).
  • the chimeric molecule may consist of an antibody against dextran conjugated with a biospecific ligand, e.g., an immunoglobulin.
  • a sensing surface is modified with a so-called hapten for binding chimeric molecules to the surface.
  • a reactive ester surface as described above may be derivatized with a theophylline analog which is then utilized for binding chimeric molecules.
  • the chimeric molecule consists of an antibody against theophylline conjugated with a biospecific ligand.
  • a glass prism 42 is coated with a layer of silver 44 and includes two solid dielectric layers 46, 48.
  • a sensing surface 50 (or basal layer) in the form of a dextran coating is created on dielectric layer 48.
  • the sensing surface 50 is then functionalized through the addition of bi-functional sensing molecules 52.
  • the sensing molecules 52 are ready to bind a sample analyte present at the interface of the CPWR sensor 40 with an emergent medium 54 (for example, target molecule 55 would be immobilized by sensing molecules 52).
  • various sensor devices for CPWR analysis can be created by modifying and functionalizing the surface of the dielectric member on the CPWR device.
  • suitable modification and functionalization of that surface may be accomplished by many different protocols depending on which target molecule(s) are desired to be analyzed by CPWR.
  • Functionalization techniques can be divided into two groups: one which is based on using self-assembled molecular entities which are created on the CPWR device surface and used to immobilize sample molecules, and the other that is based on chemical modification of the dielectric surface in order to attach the sensing molecules by chemical bonds.
  • self-assembled methods include, among others, Langmuir-Blodgett monolayers, self-assembled bilayer systems, and various polymeric and hydrogel films capable of immobilizing sensing molecules (e.g., organic polymers such as polyvinyl alcohol, polyacrylic acid, polyacrylamide and polyethylene glycol, or a hydrogel such as a polysaccharide).
  • organic polymers such as polyvinyl alcohol, polyacrylic acid, polyacrylamide and polyethylene glycol
  • hydrogel such as a polysaccharide
  • chimeric molecules i.e., bi- or poly-functional molecules such as protein antibodies
  • the chimeric molecules include one portion that binds to the basal surface, e.g., the dielectric layer of the CPWR device, and one portion having an affinity for the analyte (i.e., molecule(s) to be detected).
  • Bi- or poly-functional molecules for use in this invention may be produced in a variety of ways. For example, chemical coupling of appropriate molecules or molecular fragments, hybridoma techniques that produce bifunctional antibodies, or even recombinant DNA techniques may be utilized.
  • FIG. 3 illustrates schematically a sensor embodiment with multiple chimeric molecules as functionalizing agents.
  • the CPWR sensor device 58 includes a prism 60 coated with a layer of metal 62.
  • a dielectric layer 64 Disposed upon the metal 62 is a dielectric layer 64 that has been modified with a sensing area 66 and functionalized to harbor chimeric molecules 68 and 69.
  • Two different chimeric molecules are shown, A-Y and B-Z, which are attached to two different functional groups, a and b, respectively, on sensing area 66 by reversible bonds, a ⁇ A and b ⁇ B.
  • Examples of surface binding structures of the chimeric molecule (A and B) are auxin-binding protein or an antibody, while the analyte binding structure (Y and Z) may be exemplified by Ab/Fab, streptavidin or protein G, or cloned parts of these molecules, (see Lorrick J. W. et al. Biotechnolgy 7:934-938 (1989) and Ward E. S. et al. Nature 341544-546 (1989)).
  • the functional groups (a and b) are here preferably relatively small molecules which are covalently bound to the surface and which are stable, such that the biospecific bonds between these molecules and the respective chimeric molecules may be broken down by, e.g., HC1, NaOH or additives such as miscible organic solvents without the molecules a and b being destroyed, thereby permitting the surface to be regenerated.
  • HC1, NaOH or additives such as miscible organic solvents
  • Proteins as a base for chimeric molecules i.e., the surface binding structure thereof
  • the surface binding structure thereof must have strong bonds to their low-molecular chemically stable partners on the measuring basic surface.
  • no part thereof should, of course, be capable of interacting with the biosystems to be analyzed.
  • a biospecific pair derived from a plant for example, could be used in the measurement of analytes containing human proteins.
  • the analytical system is such that the binding of the chimeric molecule to the analyte may be reversed under conditions differing from those permitting the binding between the measuring surface and the chimeric molecule to be broken.
  • the sensing surface may be regenerated at two different levels, i.e., either for binding a new analyte, or for refunctionalizing the surface with the same or other chimeric molecules.
  • the effects on the dielectric layer of the invention are not diminished by the addition of a very thin (l-5nm) layer of gold (or other metal or a semiconducting material) at the interface with the emerging medium for the purpose of fixating the analyte to the sensing molecules.
  • a very thin layer of gold or other metal or a semiconducting material
  • Such a combination of properties in one interface permits the construction of a durable CPWR sensor device with very high sensitivity and an expanded dynamic range of measurements.
  • the addition of a very thin overcoating of the dielectric layer with a metal or semiconductor determines the type of chemistry required for surface modification and functionalization.
  • sulfur-bearing compounds can easily modify metal surfaces (including noble metal surfaces).
  • Long-chain thiols such as HS(CH 2 ) n X with n>10, adsorb from solution onto noble metals and form densely packed oriented monolayers (See, in general, S. Heyse et al. Biochimica et Biophysica Acta 85507 (1998) 319-338).
  • the terminal group, X, of the thiol can be chosen from a wide variety of functional groups to interact properly with sample molecules. The choice of functional group determines the properties of such modified surfaces and its interaction with sample molecules.
  • Monolayers of alkylthiols may serve as hydrophobic supports, allowing samples to be immobilized by hydrophobic interactions.
  • hydroxythiols make the surface hydrophilic.
  • More complex thiols can be used to serve special functions.
  • polyethylene-glycol-therminated thiols, biotyinylated thiols or thoalkanes bearing metal chelating groups allow the immobilization of histidine-tagged proteins and lipids to metal surfaces.
  • More complex surfaces can easily be created by co-adsorbing two or more thiols with different functional groups.
  • Fig. 4 depicts a sensor 70 that includes a prism 72 upon which a layer of metal 74 is disposed. Atop the metal layer 74 is a dielectric material 76, thus forming a basic CPWR device. However, disposed upon dielectric 76 is a very thin layer of gold 78. The layer of gold 78 has been chemically modified with molecules 80 and 81 (which are, in this case, molecules of tetradecanethiol and 11-mercatoundecanoic acid, respectively). Molecules 80 may act as sensing molecules (for example, to bind a hydrophobic sample molecule, such as a phospholipid). Molecule 81 may provide a basal layer for further functionalization of sensor 70 (e.g., by binding a bi- or polyfunctional hydrophillic sensing molecules).
  • an oxide as the dielectric layer conveys the advantage of intrinsic hydrophilicity.
  • silane chemistry which is well known in the chemical literature, is the most appropriate approach (see, for example, Bhatia, Suresh K. Analytical Biochemistry (1989) 178, 408-413 (1989).
  • antibodies can be immobilized on the dielectric SiO 2 , for example.
  • surface properties can be controlled using silane monolayers in the same way as with the thiol-metal chemistry described above.
  • the invention thus relates to a replaceable sensor unit which is to be used in systems based on the CPWR technique.
  • Each sensing surface contains at least one functional group, with sensor units for use in high-throughput screening having different functional groups or ligands for interaction with a plurality of molecules present in the sample to be analyzed.
  • the invention moreover relates to processes for further functionalization of these sensing surfaces by binding to them bi- or polyfunctional ligands which will interact with molecules in the sample when the measuring operation is taking place.
  • the invention also comprises a method for surface characterization of molecules, in that sample molecules are made to pass over a combination of sensing surfaces, each of which carries at least one unique ligand. The degree of interaction with each of the ligands will then provide information about the surface structure of the molecules.
  • Such combinations of sensing surfaces may, for example, contain ligands such as ion exchanger groups, hydrophobic/hydrophilic groups, metal ion chelating groups, groups showing different degrees of bioselectivity or biospecificity, etc.
  • ligands such as ion exchanger groups, hydrophobic/hydrophilic groups, metal ion chelating groups, groups showing different degrees of bioselectivity or biospecificity, etc.
  • the structure of a bound molecule can be studied by subjecting the sensing surface to flows of reagent solutions containing molecules that will bind to different structural elements on the target molecule which is being studied. Examples of such reagent molecules are monoclonal antibodies against various epitopes.
  • Silane chemistry is the most widely applied approach for functionalization of dielectric materials.
  • a variety of suitable mono-and multi-functional silanes i.e., with one or more reactive groups
  • a specific example of the application of silane functional immobilization of proteins on a dielectric sensor surface is described as follows.
  • This procedure is an extension of immobilized metal ion affinity chromatography, which allows the purification of proteins tagged with a poly-histidine sequence as described in Porath, J. Journal of Protein Purif. Expression 3, pp.263-281 (1992).
  • Histidine- tagged receptor molecules can be immobilized by the chelator nitrilotriacetic acid, which is covalently bound to the sensor surface and subsequently loaded with divalent cations, such as the metal ions Ni 2+ , Cu 2+ , or Zn 2+ .
  • the free coordination sites of the chelator- metal complex are then filled by additional electron donating groups such as histidine residues in the sequence of a natural or recombinant protein (in the latter case, these are usually added at the N-terminus).
  • the formation of the protein-chelator complex is highly specific and is fully reversible upon addition of a competitive ligand (e.g., histidine, imidazole), protonation of histidines, or removal of the metal ion by EDTA complexation.
  • the chelator is covalently bound to the sensor surface in two steps. First, the dielectric surface is modified by mercaptosilane, followed by the second step of crosslinking with nitrilotriacetic acid-maleimide in aqueous solution as described in detail in Schmid, E . et al., Anal. Chem. 69, pp. 1979-1985 (1997).
  • such a functionalized sensor surface 84 is able to immobilize any histidine-tagged receptor 86 (or membrane-associated protein) to the surface via its histidine residues 88 and Ni 2+ (or other divalent) metal ions 90 loaded via chelating groups 92 on the surface.
  • the CPWR aspect of the present invention provides significant advantages over alternative techniques for the detection and measurement of small optical changes based on optical waveguides.
  • the coupling arrangements are simple and convenient.
  • the geometric arrangement in CPWR spectroscopy is characterized by a complete isolation of the optical probe from the system under investigation, as is also the case in conventional SPR spectroscopy.
  • the CPWR sensors of the present invention may be utilized in the ultraviolet and infrared spectral ranges in order to enable the testing of materials sensitive to specific UN and IF wavelengths as described in our previous patents.
  • the dielectric layer of the sensor may be designed to serve both as a waveguide and as an electrode for monitoring simultaneously electrical characteristics and optical parameters as described in our U.S. Patent No. 6,330,387.

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Abstract

L'invention concerne des unités détecteurs à utiliser dans des systèmes de résonance à guide d'onde et à plasmons, permettant de mesurer simultanément plusieurs propriétés d'une molécule ainsi que la mesure simultanée des concentrations d'une pluralité de molécules dans un échantillon hétérogène. Dans un mode de réalisation préféré, une unité de détection (40) de l'invention comprend au moins une zone de surface de détection (50) disposée sur une couche diélectrique (48). Dans un autre mode de réalisation (70) préféré de l'invention, une couche (78) très fine de métal ou semi-conductrice est disposée sur la matière diélectrique (76). Le métal ou le semi-conducteur est alors modifié et fonctionnalisé de manière à lier un analyte d'échantillons. L'invention concerne également des procédés pour fonctionnaliser des unités de détection contenant lesdites surfaces et leur utilisation dans la caractérisation des molécules.
PCT/US2003/002273 2003-01-22 2003-01-22 Detecteurs spectroscopique de resonance a guide-d'onde et a plasmons couples pour des tests pharmaceutiques et chimiques WO2004068141A1 (fr)

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AU2003217249A AU2003217249A1 (en) 2003-01-22 2003-01-22 Coupled plasmon-waveguide resonance spectroscopic sensors for pharmaceutical and chemical testing

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007015556A1 (fr) * 2005-08-01 2007-02-08 Canon Kabushiki Kaisha Dispositif de detection d'une substance cible, procede de detection d'une substance cible l'utilisant, et appareil de detection et ensemble destine a cet effet
EP2019309A3 (fr) * 2007-07-20 2009-03-11 Hitachi High-Technologies Corporation Dispositif d'analyse d'acide nucléique et analyseur d'acide nucléique l'utilisant
KR20100075478A (ko) * 2007-09-06 2010-07-02 래크리사이언시즈, 엘엘씨. 눈물 샘플의 성분들의 농도를 측정하기 위한 장치
US7898667B2 (en) 2006-12-27 2011-03-01 Canon Kabushiki Kaisha Optical element and method for preparing the same, sensor apparatus and sensing method
EP1943500A4 (fr) * 2005-09-30 2013-01-16 Fujifilm Corp Systeme de détection

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US5242828A (en) * 1988-11-10 1993-09-07 Pharmacia Biosensor Ab Sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems

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US5242828A (en) * 1988-11-10 1993-09-07 Pharmacia Biosensor Ab Sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems
US5436161A (en) * 1988-11-10 1995-07-25 Pharmacia Biosensor Ab Matrix coating for sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007015556A1 (fr) * 2005-08-01 2007-02-08 Canon Kabushiki Kaisha Dispositif de detection d'une substance cible, procede de detection d'une substance cible l'utilisant, et appareil de detection et ensemble destine a cet effet
US8023114B2 (en) 2005-08-01 2011-09-20 Canon Kabushiki Kaisha Target substance detecting device, target substance detecting method using the same, and detecting apparatus and kit therefor
EP1943500A4 (fr) * 2005-09-30 2013-01-16 Fujifilm Corp Systeme de détection
US7898667B2 (en) 2006-12-27 2011-03-01 Canon Kabushiki Kaisha Optical element and method for preparing the same, sensor apparatus and sensing method
EP2019309A3 (fr) * 2007-07-20 2009-03-11 Hitachi High-Technologies Corporation Dispositif d'analyse d'acide nucléique et analyseur d'acide nucléique l'utilisant
US8865459B2 (en) 2007-07-20 2014-10-21 Hitachi High-Technologies Corporation Nucleic acid analysis device and nucleic acid analyzer using the same
KR20100075478A (ko) * 2007-09-06 2010-07-02 래크리사이언시즈, 엘엘씨. 눈물 샘플의 성분들의 농도를 측정하기 위한 장치
EP2188615A4 (fr) * 2007-09-06 2014-08-13 Osmopen Llc Dispositif permettant de mesurer les concentrations des constituants d'un échantillon de larme
KR101714752B1 (ko) * 2007-09-06 2017-03-09 래크리사이언시즈, 엘엘씨. 눈물 샘플의 성분들의 농도를 측정하기 위한 장치

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