WO2004068141A1

WO2004068141A1 - Coupled plasmon-waveguide resonance spectroscopic sensors for pharmaceutical and chemical testing

Info

Publication number: WO2004068141A1
Application number: PCT/US2003/002273
Authority: WO
Inventors: Zdzislaw Salamon; Gordon Tollin
Original assignee: The Arizona Board Of Regents On Behalf Of The University Of Arizona
Priority date: 2003-01-22
Filing date: 2003-01-22
Publication date: 2004-08-12
Also published as: AU2003217249A1

Abstract

Sensor units to be used in coupled plasmon waveguide resonance systems for measuring simultaneously several properties of one molecule as well as for simultaneously measuring the concentrations of a plurality of molecules in a heterogenous sample. In a preferred embodiment, a sensor unit (40) of the invention features at least one sensing surface area (50) disposed upon a dielectric layer (48). In another preferred embodiment (70), a very thin metal or semiconductor layer (78) is disposed over the dielectrical material (76). The metal or semiconductor is then modified and functionalized for binding of a sample analyte. The invention also relates to methods for functionalizing sensor units containing such surfaces and to the use thereof for characterization of molecules.

Description

COUPLED PLASMON-WANEGUIDE RESONANCE SPECTROSCOPIC SENSORS FOR PHARMACEUTICAL AND CHEMICAL TESTING

U.S. GOVERNMENT RIGHTS This invention was made with Federal Government support under Contract Number MCB-9904753 awarded by the National Science Foundation and Contract Number GM59630 awarded by the National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention pertains in general to the field of Coupled Plasmon Waveguide Resonance (CPWR) spectroscopy. In particular, the invention relates to novel CPWR spectroscopic devices having a dielectric layer with surface modifications and sensor applications thereof.

Description of the Related Art

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.

The SPR phenomenon has been known for decades and the theory is fairly well developed. Simply stated, 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. When an incident light beam is reflected internally within the first medium, its electromagnetic field produces an evanescent wave that crosses a short distance (in the order of nanometers) beyond the interface with the second medium. If a thin metal film is inserted at the interface between the two media, 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.

Typically, 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. By including the sample material to be measured as a layer on one side of the metallic 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. When changes occur in the refractive index of the sample material, the propagation of the evanescent wave and the angle of incidence producing resonance are affected. Therefore, by monitoring the angle of incidence at a given wavelength and identifying changes in the angle that causes resonance, corresponding changes in the refractive index and related properties of the material can be readily detected.

As those skilled in the field readily understand, 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. In practice, 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. Similarly, for systems operating with variable wavelengths and a given incidence angle, 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. Moreover, 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. Accordingly, 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.

Thus, once materials are selected for the prism, metal film and emerging medium that satisfy the described conditions for total reflection and plasmon resonance, 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. In fact, 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. For a given set of parameters, 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). In the conventional technique, 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.

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.

As well understood by those skilled in the art, 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. In all these prior-art devices and techniques, only p-polarized light is coupled into the plasmon mode because this particular polarization has the electric field vector oscillating normal to the plane that contains the metal film. This is sometimes referred to as transverse-magnetic (TM) polarization.

As mentioned, 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. Thus, SPR is used as a highly sensitive technique for investigating changes that occur at the surface of the metal film. In particular, over the last several years there has been a keen interest in the application of 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. As a consequence of these characteristics, 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. Acta 1331: 117-129 (1997); and 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. II: Applications to Biological Systems," Biochim. et Biophvs. Ada. 1331: 131-152 (1997).

In U.S. Patent No. 5,991,488, herein incorporated by reference, we disclosed new thin- film interface designs that couple surface plasmon and waveguide excitation modes. The new technique, defined as coupled plasmon-waveguide resonance (CPWR), is based on the totally new concept of coupling plasmon resonances in a thin metal film with the waveguide modes in a dielectric overcoating. Accordingly, a metallic layer, typically either gold or silver, is used with a prism so as to provide a surface plasmon wave by conventional SPR (or waves by long-range SPR) and is further covered with a solid dielectric layer characterized by predetermined optical parameters. The dielectric member inserted between the metal film and the emergent medium is selected such that coupled plasmon-waveguide resonance effects are produced within an observable range.

The emergent dielectric medium is then placed in contact with this solid dielectric layer. As disclosed in the patent, we found that the additional layer of dielectric material functions as an optical amplifier that produces an increased sensitivity and enhanced spectroscopic capabilities in SPR. In particular, the added dielectric layer makes it possible to produce resonance with either s- or p-polarized light. In addition, 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). Thus far, however, work on the development of biosensors has focused almost exclusively on various methods for binding a particular biomolecule to the metal surface of an SPR device. For example, in U.S. Patent 5,492,840, Malmqvist et al. discloses a sensor unit for use in SPR-based systems that features a metal film having several sensing surfaces that have been disposed thereon and functionalized with antibodies and various other chemical elements.

Thus, it would be desirable to create a biosensor for use specifically with CPWR-based systems. Moreover, there continues to be a need in the art for sensors adapted to immobilize a wide variety of biological, chemical, and pharmaceutical molecules for testing applications in CPWR-based systems.

BRIEF SUMMARY OF THE INVENTION

The main goal of this invention is the development of coupled plasmon-waveguide resonance devices that are useful in a wider range of sensor applications than has previously been known. To that end, 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. Preferably, 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 unit.

The CPWR sensor of the present invention differs from SPR sensors in several key respects. First, the invention features a surface-modified dielectric layer (not a modified metal layer). Second, the present sensor allows a user to benefit from the advantages (e.g., greater sensitivity) that CPWR-based systems offer over those utilizing SPR. Moreover, while previously disclosed CPWR devices have limited CPWR analysis to certain membrane or lipid bound biomolecules, 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. Optionally, 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.

In particular, 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.

Finally, another objective is to provide sensor devices that are suitable for direct incorporation with existing CPWR spectroscopic instruments.

Therefore, according to these and other objectives, 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). Accordingly, in one embodiment of the invention, 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. These organic polymer or hydrogel layers thus become sensing surfaces.

For CPWR measurements to be carried out, the sensing surfaces have to be functionalized with different ligands capable of interaction with a target chemical or

10 molecule. Several routes are available in achieving such functionalization. For example, 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

15 more bioselective functions for interaction with molecules in a sample.

Various other purposes and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, to the accomplishment of the objectives 20 described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiment and particularly pointed out in the claims. However, such drawings and description disclose but one of the various ways in which the invention may be practiced.

25 BRIEF DESCRIPTION OF THE DRAWINGS

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 Si0₂ 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.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

This 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). In general 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. Furthermore, 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.

Up until now, only the use of a biomembranes, such as lipid bilayers, have been described and used to modify the dielectric member of a CPWR device. While this type of surface modification allows the use of CPWR in biosensor applications, a great number of chemical or pharmaceutical samples will not be immobilized. Hence, functionalization of a modified dielectric surface provides the ability to perform CPWR with wide variety of biological and non-biological molecules.

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.

Referring to the drawings, wherein like reference numerals and symbols are used for like parts, 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.

Suitable dielectric materials must have a refraction index n_d greater than the refractive index n_e of the emerging medium; they must have an extinction coefficient k_ά as small as possible for a given wavelength (for example, <0.1, preferably between 0 and 0.01, for λ = 633nm); and they must be selected with a thickness that will support a guided wave and result in resonance effects occurring at an angle of incidence within the observable range, as explained above. For example, a glass prism coated with a 50nm-thick silver layer protected by a 460nm-thick Si0₂ film (n_d = 1.4571, k_ά = 0.0030) is suitable to practice the invention with an aqueous analyte (n_e = 1.33). 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.).

In the SiO₂ 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 TiO₂ 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 TiO₂ layer at least 750nm thick.

There is no limitation on the dielectric materials that can be used in the dielectric coatings 32 of the invention, as long as the optical characteristics are favorable, as explained above. Therefore, 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.

Furthermore, a large variety of dielectric overcoat film combinations exists that can be used in particular applications. In essence, 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. For example, these materials include MgF₂ Al₂O₃, LaF₃, Na₃AlF₆, ZnS, ZiO₂, Y₂O₃, HfO₃, Ta₂θ₅, 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. As well understood in the art, 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 α (with a monochromatic light source), or light wavelength λ (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. (1996), supra: Salamon et al., "Plasmon Resonance Spectroscopy: Probing Molecular Interactions within Membranes," Trends in Biochemical Sciences, 24, 213-219 (1999); Salamon et al., "Surface Plasmon Resonance, Theory," Encyclopedia of Spectroscopy & Spectrometry, Academic Press, Vol. 3, 2311-2319; and Salamon et al., "Surface Plasmon Resonance: Applications," Encyclopedia of Spectroscopy & Spectrometry, Academic Press, Vol. 3, 2294-2302.

In one embodiment of the invention, so-called chimeric molecules (bi- or polyfunctional 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. 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). In the case of dextran, the chimeric molecule may consist of an antibody against dextran conjugated with a biospecific ligand, e.g., an immunoglobulin.

According to an alternative procedure, a sensing surface is modified with a so-called hapten for binding chimeric molecules to the surface. Thus, for example, a reactive ester surface as described above may be derivatized with a theophylline analog which is then utilized for binding chimeric molecules. In this case, the chimeric molecule consists of an antibody against theophylline conjugated with a biospecific ligand. This alternative embodiment very clearly reveals the great versatility attainable with the use of surfaces according to the present invention, inasmuch as it is very easy for the user to provide the same single basal surface with the desired ligand (e.g., receptor).

Thus, in a preferred embodiment 40 of the invention, illustrated in Fig. 2, a glass prism 42 is coated with a layer of silver 44 and includes two solid dielectric layers 46, 48. One 50nm layer 46 of TiO₂ (n_d = 2.2789, k_d = 0.000151) protects the silver film 44; a second 750nm layer 48 of a lower density, lower refractive index (n = 1.35) dielectric material (Na₃AlF₆) is applied over the first layer. In this embodiment, 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. Thus, 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).

According to the invention, various sensor devices for CPWR analysis can be created by modifying and functionalizing the surface of the dielectric member on the CPWR device. Thus, 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.

Examples of 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).

Chemical functionalization requires a covalent attachment of sensing molecules to the dielectric surface. The dielectric surface therefore has to be functionalized to allow selective interaction with the desired molecules. Therefore, so-called chimeric molecules (i.e., bi- or poly-functional molecules such as protein antibodies) are employed for functionalizing the sensing surface. 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.

With a series of chimeric molecules that contain a dextran antibody and a group of a different specificity, a plurality of identical sensing surfaces on one CPWR sensor can easily be subjected to parallel activations for the purpose of detecting a plurality of biomolecules in a single sample. Thus, the invention also relates to methods which allow one to measure the surface concentration of different molecules in a heterogeneous analyte sample. 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. 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. As examples of such small molecules may be mentioned theophylline, steroid hormones and thyroxine, which may be regenerated by sodium hydroxide and additives. Many proteins can also withstand extreme conditions, but normally proteins are less stable. Some antibodies may also be reversed in spite of high binding strength.

Proteins as a base for chimeric molecules (i.e., the surface binding structure thereof) must have strong bonds to their low-molecular chemically stable partners on the measuring basic surface. Also, no part thereof should, of course, be capable of interacting with the biosystems to be analyzed. Thus, in order to control non-specific binding, a biospecific pair derived from a plant, for example, could be used in the measurement of analytes containing human proteins.

Preferably, 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. In such manner, depending on the conditions, 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.

It is noted that 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. 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. In other words, 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.

As is known to those skilled in the art, sulfur-bearing compounds can easily modify metal surfaces (including noble metal surfaces). Long-chain thiols, such as HS(CH ₂)_nX 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 (X=CH₃) may serve as hydrophobic supports, allowing samples to be immobilized by hydrophobic interactions. Alternatively, hydroxythiols (X=OH) make the surface hydrophilic. Titratable terminal groups (e.g., X=COO or X=NH₃ ⁺) confer hydrophilicity and charge the surface, which varies as a function of pH. Some other examples are: amino, aldehyde, hydrazide, carbonyl or vinyl groups.

The reaction occurring when these or other groups are used for coupling various types of sample molecules are well known from the literature. More complex thiols can be used to serve special functions. For example, 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.

Turning to an example of chemical surface modification, 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).

Employing an oxide as the dielectric layer conveys the advantage of intrinsic hydrophilicity. Thus, for certain applications (e.g., for self-assembled bilayer formation, see above) it need not be chemically modified. If functionalization is desired, 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). Thus, antibodies can be immobilized on the dielectric SiO₂, for example. In principle, 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. Also, by real time measurement, information on kinetic parameters can be obtained. 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. Moreover 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.

The following example further illustrates the invention and is not meant to limit this disclosure in any way.

Example

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) are available for this purpose. 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). Such 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 Zn²⁺. 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).

Therefore, as shown in Fig. 5, 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.

Thus, it has been shown that, by functionalizing dielectric (with or without very thin overcoatings) materials with appropriate optical parameters and physical properties, it is possible to create a CPWR sensor assembly that can be utilized in a wide variety of applications. In effect, CPWR devices can now be designed for use with a wide range of chemical, biological, and pharmaceutical molecules. Thus, this disclosure expands the application of plasmon resonance techniques beyond its use with membrane or lipid bound biomolecules.

Because of its characteristics, 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. Moreover, 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. Various changes in the details, steps and components that have been described may be made by those skilled in the art within the principles and scope of the invention herein illustrated and defined in the appended claims. For example, 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. Also, 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.

Therefore, while the present invention has been shown and described herein in what is believed to be the most practical and preferred embodiments, it is recognized that departures can be made therefrom within the scope of the invention, which is not to be limited to the details disclosed herein but is to be accorded the full scope of the claims so as to embrace any and all equivalent processes and devices.

Claims

What is claimed is:

1. In a coupled plasmon waveguide resonance spectroscopic system, wherein a surface plasmon is excited by a light beam and propagated along a metallic or semiconductor film to measure a property of a sample material placed at an interface of an emergent medium and a dielectric member is inserted between said film and said emergent medium such that resonance effects are produced within an observable range, a sensor comprising at least one sensing area formed upon said dielectric member, wherein said at least one sensing area has undergone functionalization such that a target chemical within said sample material is immobilized.

2. The sensor of claim 1, wherein said at least one sensing area comprises a layer of an organic polymer and at least one functional group supporting bi- or polyfunctional sensing molecules.

3. The sensor of claim 2, wherein said bi - or polyfunctional sensing molecules are selected from a group of chimeric molecules having an identical sensing area binding structure, but having varying analyte binding structures.

4. The sensor of claim 2, wherein said at least one functional group is selected from the group consisting of hydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, and vinyl.

5. The sensor of claim 2, wherein at least one of said organic polymer comprises a polysaccharide.

6. The sensor of claim 2, wherein said functional group is selected from the group consisting of ion exchanger groups, metal chelating groups, and receptors for biological molecules, or combinations thereof.

7. The sensor of claim 5, wherein said polysaccharide is selected from the group consisting of agarose, dextran, carrageenan, alginic acid, starch, cellulose, or a derivative of any one of the foregoing.

5 8. The sensor of claim 7, wherein said derivative is a carboxymefhyl derivative.

9. The sensor unit of claim 5, wherein said polysaccharide is dextran.

10. The sensor unit of claim 9, wherein said dextran is activated for binding a ligand or 10 ligands by the generation of at least one member selected from the group consisting of carboxymethyl, carboxyl, hydrazide, ester, and disulfide groups thereon.

11. The sensor of claim 10, wherein said ester groups are derivatized with a hapten.

15 12. The sensor of claim 11 , wherein said hapten is theophylline.

13. In a coupled plasmon waveguide resonance spectroscopic system, wherein a surface plasmon is excited by a light beam and propagated along a metallic or semiconductor film to measure a property of a sample material placed at an interface of an emergent 20 medium and a dielectric member is inserted between said film and said emergent medium such that resonance effects are produced within an observable range, a sensor comprising: a very thin film of metal or semiconductive material disposed upon said dielectric member, and 25 at least one sensing area formed upon said metal or semiconductive material, wherein said at least one sensing area has undergone functionalization such that a target chemical within said sample material is immobilized.

30

14. The sensor of claim 13, wherein said at least one sensing area comprises sulfur- bearing compounds.

15. The sensor of claim 14, wherein said sulfur-bearing compounds are selected from 5 the group consisting of HS(CH ₂)_nX, wherein n>10 and X is CH₃, OH, COO, or NH₃ ⁺.

16. The sensor of claim 13, wherein said at least one sensing area comprises a layer of an organic polymer and at least one functional group supporting bi- or polyfunctional molecules.

10

17. The sensor of claim 16, wherein said bi - or polyfunctional molecules are selected from a group of chimeric molecules having an identical sensing area binding structure, but having varying analyte binding structures.

15 18. The sensor of claim 16, wherein said at least one functional group is selected from the group consisting of hydroxyl, carboxyl, amino, aldehyde, hydrazide, carbonyl, and vinyl.

19. A method for detecting binding sites on a molecule, comprising: 0 contacting a sample with said at least one sensing area of claim 1 and detecting any binding interaction between said molecule and said at least one sensing area through standard CPWR procedures.

20. A method for detecting binding sites on a molecule, comprising:

25 contacting a sample with said at least one sensing area of claim 13 and detecting any binding interaction between said molecule and said at least one sensing area through standard CPWR procedures.