Patent Application
20070222995
Surface plasmon resonance (SPR) is an optical method for measuring the refractive index of very thin layers of material adsorbed on a metal film. SPR is used to measure biomolecular interactions in real-time in a label-free environment. Label-free detection refers to a method for determining the identity of a biomolecule without fluorescence tagging. For example, in the case of protein adsorption, the refractive index of a thin metal film in a buffer solution (e.g., an aqueous solution) differs depending on whether an adsorbate is bound on its surface and can be easily quantitatively measured using SPR.
The specific binding of biomolecules changes the refractive index of the metal film. The change in refractive index is measured as a change in resonance angle or resonance wavelength. The change in refractive index on the surface is linear with respect to the quantity of molecules specifically bound.
The SPR technique exploits the fact that, under certain conditions, surface plasmons on metallic surfaces can be excited by photons, thereby transforming a photon into a surface plasmon. The conditions depend on the properties of the metal film, the wavelength of the incident light, temperature, and the refractive index of the media on both sides of the metal film. Since the metal film, the wavelength of the incident light and temperature are kept constant, the SPR signal is directly dependent on the change of the refractive index of the medium on the side of the SPR surface having the metal film due to the adsorption.
At an interface between two transparent media of different refractive indices (e.g., glass and water), light coming from the side of higher refractive index is partly reflected and partly refracted. Above a certain critical angle of incidence, no light is refracted across the interface, and total internal reflection is observed. While incident light is totally reflected, the electromagnetic field component penetrates a short distance (tens of nanometers) into the medium of the lower refractive index creating an exponentially-decaying evanescent wave. If the interface between the media is coated with a thin film of metal (such as gold, silver, platinum, or another chemically-stable metal), and the light is monochromatic and polarized, the intensity of the reflected light is reduced at a specific angle of incidence, producing a sharp shadow. The sharp shadow is due to surface plasmon resonance and appears as a narrow line due to the resonant energy transfer between the photons of the evanescent wave and surface plasmons.
The resonance conditions are influenced by material adsorbed onto the thin metal film. For example, the velocity of the plasmons changes when the composition of the medium changes. Because of the change in velocity, in other words, the change in momentum, the angle of incidence at which the resonance occurs changes accordingly. Therefore, a monolayer of antigen molecules on the surface of the metal film has a characteristic surface plasmon resonance angle. The angle shifts when the corresponding antibody binds to the antigen molecules on the surface. Therefore, measuring surface plasmon resonance via the resonance angle can be used to determine whether a binding event takes place. Moreover, reaction rate constants as well as equilibrium constants can be determined. This type of SPR-based biosensing is referred to as angular SPR. Alternatively, the angle of incidence can remain fixed and the wavelength of the incident light can be varied until resonance occurs. This means that the analyte and ligand association and dissociation can be observed and rate constants and equilibrium constants can be calculated.
SPR is useful for probing the interactions of various biomolecules with various ligands, biomolecules, and membranes, including, for example, protein:ligand; protein:protein; protein:DNA; and protein:membrane binding. It provides not only a method for identifying these interactions and quantifying their equilibrium constants, kinetic constants and underlying energenitics, but also for performing label-free biomolecule detection.
In a typical SPR biosensing application, one interactant in the interactant pair (i.e., a ligand or biomolecule) is immobilized on a glass slide coated with a thin film of metal, such as gold. The immobilized interactant forms a thin layer. The other interactant is located in an aqueous buffer solution and is induced to flow across the surface of the glass slide. When light of a given wavelength is directed through the glass slide and onto the surface of the metal film at an angle near the so-called “surface plasmon resonance” condition for the wavelength, the optical reflectivity of the metal film changes very sensitively with the concentration of biomolecules on the surface of the metal film or in a thin coating on the metal film. The extent of binding between the solution-phase interactant and the immobilized interactant is easily observed and quantified by measuring the resonance angle or the resonance frequency of the reflected light. The SPR-detected concentration measurement is highly sensitive without the need for any fluorescent or other labeling of the interactants.
The use of a textured surface having nanometer-scale metallic features instead of a smooth metal surface enhances the ability to detect biomolecules using SPR. Measurement of SPR at a flat metal surface is difficult due to angular dependence and temperature sensitivity. A metal surface (e.g., gold, silver, platinum) with nanometer-scale features relaxes the stringent angular and temperature requirements and exhibits strong absorption in the ultraviolet (UV)-visible frequency range. This absorption is referred to as localized surface plasmon resonance (LSPR). LSPR can be thought of as an optical enhancement of the electromagnetic field facilitated by the presence of the nanometer scale features. Moreover, a nanotextured surface provides larger surface area than a smooth surface. Therefore, a nanotextured surface can have a higher population of the immobilized interactant and has a better chance to capture interactants of interest.
Similarly, the sensitivity of electrochemical impedance spectroscopy (EIS) can be enhanced by the use of a metal substrate having a nanometer-scale textured surface. EIS is an effective tool for screening anti-cancer drugs by determining the interaction between immobilized DNA and potential drugs. In one example, the sensitivity of SPR detection of nogalamycin, an anti-tumor drug, was 40 times greater using SPR on a textured metal surface than on a smooth metal surface. The increased sensitivity results from the greater ability of biomolecules to attach to targets arranged in a concentric fashion on a surface textured with nanometer-scale particles.
Unfortunately, current methods for preparing nanometer-scale textured surfaces suffer from surface defects and from inferior process robustness. For example, electrochemical deposition has been used to deposit nanometer-scale gold particles on a surface, but accurate control of the particle size and density is difficult. In another process, textured nanometer-scale gold surfaces have been prepared by depositing a thin layer of gold onto a surface coated with a monolayer of polystyrene spheres. Unfortunately, it is difficult to obtain a consistent monolayer of the spheres. This causes irregularities in the layer of gold. Further, the chemical suspensions that are used to deposit the polystyrene spheres have a limited shelf life.
In an embodiment, an artifact having a textured metal surface with nanometer-scale features comprises a substrate, a substructure over the substrate, the substructure comprising a periodic array of nanometer-scale structural elements comprising an inorganic oxide, and a metal film over the substructure. In an example application, the artifact can be used to enhance the sensitivity of an apparatus used to perform surface plasmon resonance analysis.
The invention can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present invention. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.
An artifact having a textured metal surface with nanometer-scale features will be described below in the context of an artifact whose textured metal surface is used in a surface plasmon resonance (SPR) biosensing application. However, the artifact having a textured metal surface with nanometer-scale features can be used in other applications in which a nanometer-scale textured metal surface is needed.
Prior to describing embodiments of the invention, a description of a block copolymer is provided to aid in the understanding of the embodiments to be described below. The term “polymer” refers to a chemical compound formed by polymerization and consisting essentially of repeating structural units. The basic chemical “units” that are used in building a polymer are referred to as “repeat units.” A polymer may have a large number of repeat units or a polymer may have relatively few repeat units, in which case the polymer is often referred to as an “oligomer.”
When a polymer is made by linking only one type of repeat unit together, it is referred to as a “homopolymer.” When two (or more) different types of repeat units are joined in the same polymer chain, the polymer is called a “copolymer.” In copolymers, the different types of repeat units can be joined together in different arrangements. For instance, two repeat units may be arranged in an alternating fashion, in which case the polymer is referred to as an “alternating copolymer.” As another example, in a “random copolymer,” the two repeat units may follow in any order. Further, in a “block copolymer,” all of one type of repeat unit are grouped together, and all of the other type of repeat unit are grouped together. Thus, a block copolymer can generally be thought of as two homopolymers joined in tandem. A block copolymer can include two or more units of a polymer chain joined together by covalent bonds. A “diblock copolymer” is a block copolymer that contains only two units joined together by a covalent bond. A “triblock copolymer” is a block copolymer that contains only three units joined together by covalent bonds.
A polymer that may be processed to deliver an inorganic payload on the surface of a substrate is referred to herein as a “vector polymer.” As described further below, such a vector polymer self-assembles into a desired structure for controlling the size and/or distribution of nanoparticles produced by the inorganic payload carried by such vector polymer. Thus, the vector polymer self-assembles into a desired structure of inorganic material-containing domains. The non-payload (e.g., organic) components of the vector polymer can then be removed, resulting in the inorganic nanoparticles remaining on the substrate with their size and/or distribution controlled by the vector polymer's self-assembly. While in certain exemplary embodiments described herein a diblock copolymer (A-B) is used as a vector polymer for carrying an inorganic payload, the scope of the present invention is not so limited. Rather, any polymer (e.g., triblock polymer, etc.) that is capable of self-assembly and in which at least one repeat unit thereof includes an inorganic payload may be utilized in accordance with the concepts presented herein. For instance, in certain embodiments a block copolymer A-B-A may be used. Further, in certain embodiments, a mixture of block copolymers (e.g., diblock copolymers) and homopolymers or a miscible blend of two homopolymers (A) and (B) is used to form a film containing self-assembling polymers. As an example, a diblock polymer and two homopolymers are used for forming the film containing self-assembling polymers.
Amphiphilic block copolymers are known self-assembly systems in which chemically distinct blocks microphase-separate into the periodic domains. The domains adopt a variety of nanoscale morphologies, such as lamellar, double gyroid, cylindrical, or spherical, depending on the polymer chemistry and molecular weight. Embodiments are described herein in which such amphiphilic block copolymers are used as carriers of inorganic payloads, wherein the self-assembly of the block copolymers into a desired nanoscale morphology results in a controlled arrangement of the inorganic nanoparticles formed from the carried inorganic payloads.
The block that contains the inorganic payload is referred to as a payload-containing block. One or more instances of such a payload-containing block is present in each block polymer. For instance, in certain embodiments, a diblock copolymer has one block that is a payload-containing block and another block that contains no inorganic payload. The block that contains no inorganic payload is referred to as the matrix. As described further below, a block copolymer deposited on the surface of a substrate and subject to annealing will self-assemble into a predetermined structure (i.e., a desired nanoscale morphology). The structure into which the block copolymer self-assembles controls the size and relative spacing of the inorganic nanoparticles formed from the inorganic payload carried by the block copolymer.
Various techniques can be used for forming block copolymers containing an inorganic payload. One exemplary technique involves complexation of an inorganic payload (e.g., atoms of an inorganic species) with a block of a diblock copolymer. For instance, incorporation of an inorganic species, which may be a metal, such as iron, cobalt, and molybdenum, into one block of a diblock copolymer is accomplished by complexation of the atoms of the inorganic species with the pyridine units of polystyrene-b-poly(vinyl pyridine) (PS-b-PVP). Another exemplary technique involves direct synthesis of a payload-containing diblock copolymer. For instance, sequential living polymerization of the nonmetal-containing styrene monomer followed by the inorganic species-containing monomer of ferrocenylethylmethylsilane to form polystyrene-b-poly(ferrocenylethylmethylsilane) (PS-b-PFEMS) is an exemplary technique for direct synthesis of an inorganic species-containing diblock copolymer.
By controlling the volume of each of the blocks (A and B) of the diblock copolymer, the structures into which the diblock copolymer self-assembles during annealing can be controlled. The volume ratio between the blocks of the diblock copolymer determines the morphology, such as lamellar, double gyroid, cylindrical, or spherical, of the microdomains into which the diblock copolymer self-assembles. Additionally, the volumes of the blocks determine the size of the microdomains and the spacing between the microdomains in the matrix after the self-assembly process. Accordingly, a volume ratio between the blocks of a diblock copolymer is first determined based on the desired morphology of the microdomains that are to be formed by the self-assembly process, and the volumes of the blocks are next determined based on the desired size and spacing of the microdomains. The blocks are then deposited onto the surface of a substrate as a thin film. The blocks have the volume and volume ratio that provide the desired morphology, size and spacing.
An annealing process is then performed to cause the diblock copolymers to self-assemble. The microdomains and matrix into which the diblock copolymers self-assemble dictate the size and distribution (e.g., relative spacing) of the inorganic structural elements that will later be formed from the carried inorganic payloads. Further, this self-assembly technique provides a high yield as substantially all of the inorganic structural elements formed by the self-assembled diblock copolymers remain on the substrate after an oxidation process (e.g., UV-ozone or oxygen plasma) treatment is performed to remove the organic component of the diblock copolymer, as will be described further below. The oxidation process additionally oxidizes the inorganic species to form a non-volatile inorganic oxide. The inorganic oxide forms structural elements that collectively constitute a substructure having nanometer-scale features.
In accordance with an embodiment of the invention, a layer of metal is deposited over the substructure. The surface of the metal layer is a textured metal surface having nanometer-scale features. The substructure underlying the metal layer defines the topology of the textured metal surface.
In the example shown in
When the artifact 100 is used as part of a surface plasmon resonance analysis device, functionalizing biomolecules can easily be attached to the textured metal surface 145 by, for example, a thiol linkage. The functionalizing biomolecules are biomolecules that bind to a specific analyte of interest. In an example, the functionalizing biomolecules are antigens that bind to a specific antibody of interest. The surface area of the textured metal surface 145 is much larger than the area of the surface 105 of the substrate 101 covered by the metal layer 140. Accordingly, for a given area of the surface 105, textured metal surface 145 allows many more functionalizing biomolecules to be available to bind with the analyte of interest than would be available if the functionalizing biomolecules were directly attached to surface 105. The larger surface area of the nanometer-scale textured metal surface 145 increases the probability of the analyte of interest binding to one of the functionalizing biomolecules. This increases the sensitivity of the SPR analysis device. Further, localized surface plasmon resonance occurs when plasmons produced at textured metal surface with nanometer-scale features match the energy and momentum of the incident photons. As a result, the detection sensitivity is increased.
In the example shown in
A prism 622 is located on a surface 626 of the surface of the substrate 101 of the artifact 100 opposite the textured metal surface 145. A light source 614 is located to direct polarized light 616 into the prism 622 as shown. The polarized light passes through the prism 622 and the substrate 101, reflects off the textured metal surface 145 and passes back through the substrate 101 and the prism 622 as reflected light 624. The angle at which the SPR absorption line occurs depends on whether the analyte of interest has bound to the functionalizing biomolecules 604. The difference in the angle at which the SPR absorption line occurs with and without an analyte of interest can be represented by a signal whose amplitude is proportional to the volume of biomolecules bound near the textured metal surface 145 of the artifact 100.
This disclosure describes the invention in detail using illustrative embodiments. However, it is to be understood that the invention defined by the appended claims is not limited to the precise embodiments described.
