WO2010120293A1

WO2010120293A1 - Non-planar biosensing surfaces

Info

Publication number: WO2010120293A1
Application number: PCT/US2009/040662
Authority: WO
Inventors: John Gerard Quinn; Aaron David Martin; Jeffery Michael Havard
Original assignee: Nomadics, Inc.
Priority date: 2009-04-15
Filing date: 2009-04-15
Publication date: 2010-10-21

Abstract

An improved sensing surface suitable for use in a biosensor. The sensing surface provides a substrate supporting a metal layer which in turn supports a self-assembled ligand binding monolayer of amphiphilic compounds. The preferred amphiphilic compounds have the general structure of X_n-R_m-Y_w, wherein X is selected from the group consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from the group consisting of oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands; and, n, m and w are integer numbers from one to 10. Additionally, a method is provided for preparing the sensing surface. In the method of the current invention a solution or dispersion of the amphiphilic compound is prepared in a polar organic solvent. The metal coated substrate is treated with the dispersion or solution for a period of time sufficient to deposit a self-assembled ligand binding monolayer of the amphiphilic compound on the surface of the metal. The resulting monolayer of amphiphilic compound has a non-planar topography.

Description

Non-Planar Biosensing Surfaces

Background of the Invention

[0001] Biosensors suitable for detecting drugs and biomolecules commonly use hydrogel based matrix coatings as a capture mechanism, i.e. a sensing surface, suitable for binding and retaining the target ligands. As depicted in Figure 1, hydrogel films 10 carried by a monolayer 12 of X-R-Y compounds provide three dimensional environments for immobilizing ligands 14 and subsequently capture injected analyte in the three dimensional space. Thus, three dimensional hydrogels 10 carried by a barrier layer 12 of a compound having the configuration of X-R-Y provide increased ligand 14 immobilization capacity compared to two dimensional coatings. The X-R-Y compound has the following make-up: X is selected from the group consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms; Y is selected from the group consisting of active groups suitable to covalently bind ligands.

[0002] Sensing surfaces using this configuration are commonly used to identify low molecular weight analytes. Low molecular weight analytes give rise to low mass loading onto the sensing surface; therefore, an increase in ligand loading capacity will advantageously maximize analyte loading capacity. Unfortunately, as shown in Fig. 2, three dimensional hydrogels 10 frequently undergo excessive cross-linking of ligand 14 to hydrogel 10. Excessive cross-linking results from the high degree of mobility of the soluble hydrogel polymer chains. The resulting cross-linked matrix obstructs binding of the analyte and gives rise to a population of differentially inaccessible ligands producing a condition commonly known as ligand heterogeneity.

[0003] A two dimensional sensing surface, as depicted in Fig. 3, will preclude ligand heterogeneity. However, the alternative planar configuration limits the surface area available for binding of the target analytes such as the aforementioned drugs and ligands. The limited binding area offered by a planar layer lowers the analyte detection sensitivity of surface plasmon resonance based systems commonly used to detect the presence of the target analytes. These systems measure the interaction of the layer with the target analytes by determining the changes in an evanescent field which extends about 200-300 nanometers from the surface of the layer. Thus, use of the planar hydrogel for the biosensing surface severely limits the binding capacity of the biosensor and in turn the sensitivity of the sensor.

[0004] In order to overcome the limitations of the prior art, the current invention provides a sensing surface in the form of a self-assembled ligand binding layer having an increased surface area when compared to the prior art two-dimensional layers. However, in contrast to prior art three dimensional hydrogels this aggregate-coated sensing surface is not a hydrogel. Further, the current invention does not promote excessive cross-linking of the ligand. Thus, the current invention substantially avoids ligand heterogeneity. In particular, the current invention provides a sensing surface in the form of a three-dimensional (3-D) ligand binding layer comprising amphophilic compounds having micelle like structures embedded or attached to a planar barrier layer. As described below, this self-assembled ligand binding layer of amphiphilic compounds has a three dimensional configuration. When compared to a planar binding layer, the added presence of micelles significantly increases the surface area available for interaction with the target analyte without introducing the problem of ligand heterogeneity.

Summary of the Invention

[0005] In one embodiment, the current invention provides a sensing surface comprising a substrate having a metal layer with a self-assembled ligand binding layer comprising amphiphilic compounds bound to the metal. The ligand binding layer has a non-planar topography. The typical metal layer is gold; however, other corrosion/oxidation resistant metals will also perform satisfactorily. The amphiphilic compounds have the general structure of X_n-R_n1-Yw, wherein X is selected from the group consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y provides active groups suitable to covalently bind ligands; and, n, m and w are integers from one to 10. Y is selected from the group consisting of hydrophilic chains with active groups located at the terminal end. In particular, Y is a chain structure selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatom chains carrying an active group at the terminal end. Suitable active groups include, but are not limited to carboxylic acids, hydroxyl groups, amine groups, carboxyl, aldehyde, hydrazide, carbonyl, epoxy, alkyne, thiol, vinyl groups, cyano, azide, maleimide. Following self-assembly onto the metal layer, the selected amphiphilic compound(s) produces a hydrophilic film wherein the Y component does not extend more than 3 nm from the R component. Thus, the selected compounds limit the range of motion of the Y component in the biosensing film. As such, the resulting biosensing film is not a hydrogel, [0006] Preferably, the generic structure of X_n-Rm-Y_w includes an Aryl group positioned either between the X and the R constituents or between the R and the Y constituents. The Aryl group suitable for use in the amphiphilic compounds is any aromatic group. Thus, the preferred amphiphilic compound has a structure of X_n-Aryl-R_m-Y_w or X_n-R_m-Aryl-Y_w. [0007] The current invention further provides a method for preparing a sensing surface. The method comprises the preparation of a dispersion in an organic solvent of an amphiphilic compound having the general structure of X_π-R_m-Y_w wherein X, R and Y are as defined above. The metal coated substrate is treated with the dispersion for a period of time sufficient to deposit a self-assembled ligand binding layer of the amphiphilic compound on the surface of the metal. The preferred solvents for preparing the dispersion of amphiphilic compound. The resulting dispersion is preferably agitated for a period of time to uniformly disperse the organic compound. The resulting self-assembled ligand binding layer of amphiphilic compounds carried by the metal coated substrate has a non-planar topography. Finally, the preferred amphiphilic compound includes an Aryl constituent located either between the X and the R constituents or between the R and the Y constituents. Typically the resulting non-planar layer of amphiphilic compound is bound by chemisorption bonds to the metal surface.

[0008] Still further, the current invention provides a method for preparing a sensing surface comprising the steps of preparing a first dispersion comprising a polar solvent and an amphiphilic compound having a general structure selected from the group consisting of X_n-R_m- Y_w, X_n-Aryl-R_m-Y_w and X_n-R_m-Aryl-Y_w. The amphiphilic compound is present in the dispersion as uniformly dispersed, well ordered aggregates, such as micelles. A metal coated substrate is immersed in the first dispersion and subsequently sealed in a container for an incubation period. Following incubation, the metal coated substrate is washed with water and the resulting amphiphilic layer is allowed to dry. The dried substrate is subsequently immersed in an alcohol/acid solution to remove any unbound amphiphilic compound. Following the alcohol/acid solution bath, the substrate is rinsed with water and allowed to dry thereby yielding a substrate carrying a non-planar layer of the amphiphilic compound. Optionally, the foregoing method may include the step of plasma oxidizing the barrier layer prior to the incubation period. The oxidizing step is preferably carried out using an oxygen plasma activator/cleaner. [0009] Still further, the current invention provides a method for preparing a sensing surface comprising the steps of preparing a stock dispersion of X₂-R₂- Aryl- Y by dispersing 100 mg of purified X₂-R₂- Aryl- Y in ethanol. This stock dispersion is subsequently diluted further with ethanol to produce a dispersion having a concentration of 50 μg/mL. Agitation of the dispersion for at least 60 seconds will uniformly disperse the amphiphilic compound. Subsequently, a gold coated glass wafer is exposed to the freshly prepared dispersion of amphiphilic compound and incubated for 20 minutes in an oven that has been pre-heated to 50°C. Following incubation, the wafer is washed with copious amounts of water and allowed to dry. Optionally following drying, the wafer is immersed in a 50/50 solution of 2-propanol and 50 mM hydrochloric acid for 10 minutes to remove any unbound amphiphilic compound. After rinsing with water and allowing to dry, the wafer now carrying a non-planar topography of amphiphilic compound is ready for use in a biosensor.

Brief Description of the Drawings

[0010] Figure 1 depicts a prior art representation of a 3-D hydrogel layer on a sensing surface.

[0011] Figure 2 depicts ligand heterogeneity resulting from excessive cross-linking in a prior art 3-D hydrogel.

[0012] Figure 3 depicts a prior art sensing surface carrying a 2-D planar coating with attached ligands,

[0013] Figures 4-a through 4-y depict amphiphilic compounds suitable for use in the current invention.

[0014] Figure 5 depicts a self-assembled monolayer prepared from a X₂-R₂-Aryl-Y amphiphilic compound.

[0015] Figure 6 depicts a self-assembled monolayer prepared from a X₂-R₂-Aryl-Y amphiphilic compound with an embedded micelle formed during self-assembly of the three dimensional interaction layer.

[0016] Figure 7 depicts a self-assembled monolayer prepared from an X-R-Y amphiphilic compound with an attached micelle formed from an amphiphilic compound having the structure of R-Y.

[0017] Figure 8 is a graph demonstrating the increased capacity of a sensing surface using the

3-D self-assembled monolayer of the present invention.

Detailed Disclosure of the Current Invention

Bio sensing Surfaces having Non-Planar Topography

[0018] The current invention provides improved biosensing surfaces and methods for preparing the same. The biosensing surface of the current invention will improve the sensitivity of a variety of optical biosensor systems. For example, surface plasmon resonance systems will benefit from the use of the biosensing surface of the current invention. In contrast to prior art biosensing surfaces, the present invention does not utilize a hydrogel layer. As known to those skilled in the art, a hydrogel films extends more than 3 nm from the surface of the substrate carrying the hydrogel. In contrast to the prior art hydrogels, the sensing surfaces provided by the current invention comprise a planar barrier layer having a three dimensional surface due to the presence of micelles or micelle like structures adhered to or embedded within the planar barrier film. The planar barrier layer and the micelles or micelle like structures are prepared from amphiphilic compounds. In the preferred embodiments, the amphophilic compounds will self- assemble into the desired biosensing surface.

[0019] In the simplest of terms, the biosensing surface 20 of the current invention comprises a substrate 22 carrying a metal layer 24 which in turn supports a self-assembled non-planar three dimensional interaction layer 26 also referred to as a biosensing film 26 of amphiphilic compounds 28. Self-assembled non-planar three dimensional interaction layer 26 is a high capacity surface suitable for binding ligands. As depicted in Figures 5-7, ligand binding three dimensional interaction layer 26 includes a barrier layer portion 32 and micelle or micelle like extensions 34, 44, 46. In Figure 5, a micelle 44 is attached to barrier layer 32 while in Figure 6 micelle like extension 34 is embedded in barrier layer 32. Finally, Figure 7 depicts an alternative embodiment with an attached micelle 46. The embedded micelle like extensions 34 and micelles 44 may be formed simultaneously with the formation of barrier layer 32 or may be subsequently added to barrier layer 32 to form biosensing film 26.

[0020] The preferred metal is a noble metal such as but not limited to gold. The self- assembled ligand binding layer of amphiphilic compounds is bound either covalently or through chemisorption bonds with the metal. The nature of the bond is not critical so long as the layer adheres to the metal surface. The resulting three dimensional biosensing surface 20 provides a non-planar topography with increased surface area having enhanced ligand immobilization capacity; and, therefore, enhanced sensitivity to binding of the target analyte. The resulting three dimensional interaction layer is substantially free of ligand heterogeneity. Preferably, the non- planar layer provided by the following described embodiments of the present invention is free of ligand heterogeneity. Biosensors using the surfaces of the current invention will have improved sensitivity to target analytes such as but not limited to: proteins, immunoglobulins, nucleic acids, peptides, and other biologically active organic molecules.

[0021] With regard to the improved biosensing surface, the compositions of the current invention encompass several different forms of self-assembled ligand binding layers having non- planar topography. Each of the compositions utilizes a substrate 22 carrying a metal layer 24. Substrates 22 include glass and other materials suitable for use in optical biosensors and other biosensors. The preferred metal is a noble metal such as gold; however, other corrosion resistant metals suitable for use in the current invention include but are not limited to copper, silver, and platinum. Preferably, the metal of choice will be capable of forming covalent or chemisorption bonds with thiol compounds. The preferred metal coated substrate carries a metal layer 24 on only one side of the substrate 22. [0022] The amphiphilic compound 28 has the general structure of X₁₁-R_1n-Yw More preferably, the amphiphilic molecule includes an Aryl group located either between the X and R constituents or between the R and Y constituents. Presently, the most preferred compound for use as the non-planar self-assembled ligand binding layer has the general formula OfX₂-R₂-ArVl- Y. As used herein with regard to the amphiphilic compound, X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with hetero atoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from the group consisting of active groups for covalently binding ligands and preferably resisting non-specific binding; and, n, m and w are integers from one to ten,

[0023] More precisely Y includes hydrophilic heteroatom chains with at least one active group located at the terminal end. Suitable active groups include but are not limited to carboxylic acids, hydroxyl groups, amine groups, carboxyl, aldehyde, hydrazide, carbonyl, epoxy, alkyne, thiol, vinyl groups, cyano, azide, maleimide. For example the amphiphilic compound of Figure 4x consists of a Y component that contains a hexaethylene oxide chain with a terminal carboxylic acid bound via an ester bond. Other chain type hydrophilic molecules such as those composed of oligoethylene oxides, glycerol, oligosaccharides and even branched heteroatom chains are suitable Y components.

[0024] Following self-assembly onto the metal layer, the selected compound(s) produces a hydrophilic film wherein the Y component does not extend more than 3nm from the R component. Thus, the selected compounds limit the range of motion of the Y component biosensing film thereby limiting the range of motion of subsequently attached ligand molecules. As such, the resulting biosensing film 26 is not a hydrogel. Thus, the current invention reduces and preferably precludes occurrences of ligand heterogeneity. Therefore, hydrophilic molecules that are composed of long chain polysaccharides, i.e. longer than 10 monomeric units, such as dextran are not suitable Y components.

[0025] In one preferred embodiment, biosensing film 26 comprises cross-linked amphiphilic compounds 28. A fraction of the cross-linked compounds extend beyond the primary surface of the binding layer. This fraction is not in direct contact with the surface of the metal. As shown in Figure 6, when the cross-linking occurs during self-assembly, biosensing film 26 includes an embedded micelle 34 with only a small fraction of the constituent amphiphilic compounds 28 in contact with the metal layer 24 of substrate 22. In this embodiment, micelle 34 extends upward and away from the metal layer 24 with the extended structure stabilized by cross-linking of adjacent amphophilic compounds 28 via the X components 36. In this instance the X components 36 are sulfides that that enable the formation of disulfide bonds between adjacent amphiphilic molecules.

[0026] Alternatively, cross-liking of the adjacent amphiphilic compounds within the micelle- like structure may also be accomplished via the R or Y groups. In each of these embodiments, embedded micelles 34 increase the surface area available for ligand and analyte binding, Ligands immobilized onto biosensing film 26 possess similar spatial restriction as compared to a true planar monolayer-based surface. Thus, the ligand is considered to be bound to biosensing film 26 as opposed to being distributed within it.

[0027] The embedded micelle configurations contrast with currently used high capacity surfaces employing bound hydrogel films as depicted in Fig. 3. In contrast to the embodiments of the current invention, bound ligand distributed within the hydrogel of prior art high capacity surfaces employing bound hydrogel films have a high range of motion. The high range of ligand motion in prior art hydrogel films allowed excessive cross-linking between the hydrogel polymer and the ligand leading to ligand heterogeneity as depicted in Fig, 2. Ligand heterogeneity is to be avoided as this condition hinders analyte binding due to the hydrogel polymer wrapping around the ligand and blocking analyte access. Thus, ligand heterogeneity also hinders kinetic analysis.

[0028] As discussed above, the current invention utilizes embedded or adhered micelles or micelle like structures to produce a three dimensional surface. A micelle is a specific type of aggregate that forms dxie to the behavior of amphiphiles in a solvent. A micelle has a well organized structure as opposed to a disorganized aggregate. The micelle retains its organized structure when embedded in the surface. As depicted in Fig. 7, the ordered structure allows the Y groups 40 to be highly packed thereby greatly improving surface quality. The R component is identified as element 52.

[0029] In an alternative embodiment depicted in Figure 5, biosensing film 26 comprises attached micelles 44 in the form of separately cross-linked amphiphilic compounds, subsequently bound to barrier layer 32. In this embodiment, a covalent bond joins micelles 44 to barrier layer 32. However, other bonds are also suitable in the present embodiment. As depicted in Figure 5, the Y component 40 of amphiphilic compounds 28 forming the barrier layer 32 and the Y component 48 of micelles 44 are selected for their ability to form the necessary bonds to yield biosensing film 26 including attached micelles 44. This embodiment provides for a wider range of options in the biosensor. For example, micelle 44 may be prepared from the same amphiphilic compound used to form the barrier layer 32. However, alternatives include micelles prepared from other X_n-R_m-Y_w, X_n-R_πτAryl-Y_w and X_n-Aryl-R_m-Y_w compounds.

[0030] In another embodiment, a second amphiphilic compound having the general structure of R-Y provides the non-planar topography of biosensing film 26, wherein R and Y are selected from the same components as discussed above. In this embodiment, the R-Y amphiphilic compound forms an independent micelle 46. A covalent bond at the Y components secures resulting micelle 46 to barrier layer 32 thereby providing additional binding sites for a variety of ligands. Figure 7 depicts the resulting modified biosensing film 26. Additionally, Figure 7 depicts an alternative embodiment of the present invention wherein the barrier layer 32 is a linear amphiphilic compound having the structure of X-R-Y. While the preferred compounds discussed above yield a better surface, linear compounds will perform satisfactorily. Although micelle 46 having linear structure R-Y is depicted as attached to a linear amphiphilic compound 28a, micelles 46 may be attached to barrier layers 32 formed from amphiphilic compounds having complex structures such as X_n-R_m-Aryl-Y_w and X_n-Aryl-R_m-Y_w. In another embodiment of the current invention, barrier layer 32 may be composed of non-amphiphilic compounds (i.e. X-Y) that lack the non-polar R component. For example a chemisorption bond between an X group such as an alkyne is sufficiently strong to allow elimination of the R segment without compromising the stability of barrier film 32.

[0031] One sMlled in the art will recognize that the Y-constituent of the amphiphilic compound will determine the biosensor's ability to bind certain ligands. Therefore, the current invention further provides a self-assembled ligand binding layer comprising two or more types of amphiphilic compounds having the general structures described above but with different Y- constituents. Thus, the biosensor of the present invention provides a great deal of flexibility for designing a surface suitable for determining the presence of wide variety of analytes. [0032] In this embodiment of the current invention, the self-assembled ligand binding layer may take several forms. In one embodiment, the self-assembled ligand binding layer comprises a heterogeneous blend of the two or more amphiphilic compounds. Amphiphilic compounds suitable for use in the heterogeneous monolayer include but are not limited to X_n-R_m-Y_w, Xn-Rm- Aryl-Y_w and X_n-Aryl-R_m-Y_w or a blend of X_n-R_n,- Y_w, X_n-R_m-Aryl-Y_w and X_n-Aryl-R_m-Yw, further including an additional compound of R-Y structure. In either case the non-planar topography includes extensions of the various Y-constituents associated with each amphiphilic compound. Alternatively, biosensing film 26 is a heterogeneous blend of one amphiphilic compound with a second amphiphilic compound where the second compound forms micelles which are embedded into the layer of the first amphiphilic compound in the same manner as depicted in Figure 6 or linked to the Y component of the planar layer formed from the first amphophilic compound as in Figure 5.

[0033] Figures 4a through 4x depict preferred micelle forming compounds. The non-limiting list of compounds include: (4a) 16-Phosphonohexadecanoic acid; (4b) 11-Mercaptoundecanoic acid; (4c) 15-Mercaptopentadecanoic acid; (4d) 16-Mercaptohexadecanoic acid; (4e) Biphenyl- 4,4'dithiol; (4f) Bis(10-carboxydecyl)disulfide; (4g) Bis(16-Hydroxyhexadecyl)disulfide; (4h) Bis(l 1-Hydroxyundecyl)disulfide; (41) Oxy-(3,5-(6-mercaptohexoxy))benzyloxy)-Oxy'-(heptoxy hydrazide)-2,2'-p-diphenol propane; O-(l-mercaptoundecyl)tri(ethylene glycol); (4j) l,2-( 12- mercaptododecoxy carbonyl)-3-( 1 -Phospho-2-trimethylammonium ethane) propane; (4k) Bis(l 1 -azidoundecyl) disulfide; (41) O-(l-mercaptoundecyl)tetra(ethylene glycol); (4m) 1,16-hexadec anedithiol; (4n) 11-aminoundecanethiol; (4o) O-(l-mercaptoundecyl)hexa(ethyleneglycol)grycolic acid; (4p) 11-mercaptoundecylphosphoric acid; (4q) O-(l-mercaptoundecyl)hexa(ethylene glycol)-O'-acetohydrazide; (4r) O-(l-mercaptoundecyl)hexa(ethylene glycol)glycolic acid NHS ester; (4s) 3,5-(6-mercaptohexoxy)-Oxy-((hexa(ethylene glycol)acetohydrazide)oxy)benzyl alcohol; (4t) N-(3-(0-(biotmarnidopropyl)tri(ethylene glycoppropy^-l l-mercaptoundecaneamide; (4u) 3,5-(6-mercaptohexoxy)-O-((hexa(ethylene glycol)glycolic acid)oxy) benzyl alcohol NHS ester; (4v) 3,5-(6-mercaptohexoxy)-O-(tri(ethylene glycol)oxy)benzyl alcohol; (4w) 3,5-(6- mercaptohexoxy)-O-((hexa(ethylene glycol)glycolic acid)oxy)benzyl alcohol; and (4x) ((3,5- bis(6-mercaptohexoxy)benzyloxy)hexaethylene glycol)glycolic acid. The preferred compound is ((3,5-bis(6-mercaptohexoxy)benzyloxy)hexaethylene glycol)glycolic acid (4x). [0034] The following portion of this disclosure will discuss particularly preferred compositions suitable for use as the self-assembled ligand binding layer, In one embodiment, the amphiphilic compound has the structure of X₂-R₂-ATyI-Y, for example, ((3,5-bis(6- mercaptohexoxy)ben2yloxy)hexaethylene glycol) glycolic acid. In this embodiment, the preferred X-constituent is selected from the group consisting of thiol, alkyne or sulfide; the R is selected from the group consisting of hydrocarbon chains, hydrocarbon chains with heteroatoms and branched hydrocarbon chains, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; the active terminal portion of the Y structure is selected from the group consisting of functional groups such as carboxylic acids, hydroxyl groups or amine groups, and the Aryl constituent is a conjugated aromatic ring. This compound is capable of forming at least two chemisorption or covalent bonds with the metal via the X-constituents. As previously noted, the type of bond is not critical so long as the non-planar self-assembled ligand binding layer adheres to the metal surface carried by the substrate. The Aryl group provides rigidity while the R constituent acts as the back bone of the amphiphilic compound providing increased stability of the film. Finally, the Y-constituent is selected for its ability to bind target analytes or support attachment of tether molecules such as but not limited to natural biological polymers such as proteins, nucleic acids, polysaccharides, lipoproteins. As noted above, a blend of amphiphilic compounds having differing Y-constituents may used such that the ligand binding layer presents more than one Y-component. In this case the differing Y- components may serve a secondary purpose such as preventing non-specific binding or controlling the orientation, or surface density, of the immobilized ligand.

[0035] The currently preferred structure of the compound for forming non-planar topography biosensing surfaces is X₂-R₂-Aryl-Y. This structure has a tendency to form cross-links between adjacent molecules when solubilized in polar organic solvents. The resulting three dimensional interaction layer includes bubbles or loops, i.e. micelles, of the amphiphilic compound protruding from the surface of the layer as depicted in Figure 6. Thus, while the amphiphilic molecule is capable of at least two covalent or chemisorption bonds with the metal, a significant fraction of the molecules do not bind to the gold surface, but rather provide the non-planar structure.

[0036] In the preferred embodiment, the self-assembled ligand binding layer, i.e. the Y component 40 has a thickness of about 0.5 nanometers to about 2.5 nanometers and the non- planar portions of the self-assembled ligand binding layer, i.e. embedded and attached micelles 34, 44, 46, extend outward from the layer a further distance of about 0.5 nanometers to about 100 nanometers.

Methods for Preparing a Biosensing Surface

[0037] Having described the compositions suitable for use in optical biosensors, the following discussion relates to the preferred methods for preparing the above-discussed biosensing surfaces. The current invention provides methods suitable for preparing a wide variety of biosensing surfaces. These methods include:

• Method 1 - Preparing a Non-Planar Amphiphilic Layer by Self Assembly; and,

• Method 2 - Preparing a Non-Planar Amphiphilic Layer by Self Assembly of Barrier Layer followed by Active Linkage of Aggregates.

[0038] Prior to discussing the specifics of each particular method, the current invention relates in general to a method for preparing a biosensing surface. According to the method of the current invention, the preparation of a substrate carrying a metal layer or coating uses methods known to those skilled in the art. Common substrates include a glass cover slide, wafer or any other optical variant suitable for use in commonly available biosensing systems. As discussed above, the metal is preferably gold; however, metals such as but not limited to copper, silver, and platinum will also perform satisfactorily in the current invention. Preferably, the metal of choice will be capable of forming covalent or chemisorption bonds with thiol compounds. The metal layer typically has a thickness of about 10 nm to about 70 nm thick and is found only on one side of the substrate. The prepared substrate is preferably stored in an air tight container until treated with the amphiphilic compound.

[0039] In general terms, the method for applying the amphiphilic compound as a self- assembled ligand binding layer to the prepared substrate entails the following steps:

A) preparing a stock dispersion of amphiphilic compound having the general formula of X_n- R_m-Y_w or X_n-R_m- Aryl-Y_w or X_n-Aryl-R_m-Y_w in a solvent;

B) diluting the stock dispersion with solvent to a concentration of about 30μg/ml to about 70μg/mL;

C) treating the prepared substrate with the diluted dispersion of B) for a period of one minute to 100 minutes in an oven or other conventional device which has been pre-heated to a temperature of about 40° C to about 100° C to deposit a self-assembled ligand binding layer of amphiphilic compound on the metal layer of the prepared substrate. More preferably, the incubation step occurs within 20 minutes when the oven has been pre-heated to a temperature of about 50° C;

D) washing the deposited layer of amphiphilic compound and allowing the amphiphilic compound to dry;

E) treating the dry layer of amphiphilic compound with an alcohol/acid solution to remove non-bound organic compounds;

F) rinsing the layer of amphiphilic compound with water; and,

G) allowing the layer to dry.

As an alternative, step C may be carried at room temperature over a period of about 3 hours to about 18 hours to deposit a self-assembled ligand binding layer of amphiphilic compound on the metal layer of the prepared substrate,

[0040] The above process steps, preferable use a polar solvent suitable for solvating the hydrophilic portion of the amphiphilic compound. The majority of preferred amphiphilic compounds will not dissolve completely in the solvents of choice because of the insolubility of the hydrophobic portion of the amphiphilic compound. Accordingly, the resulting mixture is a dispersion. However, inasmuch as some amphiphilic compounds may dissolve in the solvents, the current invention contemplates both solutions and dispersions for preparation of the biosensing surfaces. Therefore, as used herein, the term dispersion does not exclude the use of a solution of amphiphilic compound in a suitable solvent. For example, in the preferred embodiment the solvent must enable the simultaneous formation of a stable biosensing film and the formation of micelles that are embedded in the biosensing film as it forms. In an alternative method the micelles may be prepared in advance of exposure to the gold surface, and once stabilized by any mechanism known to those skilled in the art, may be reconstituted in a solvent of choice that may include solvents that do not promote micelle formation. Once micelles are formed and stabilized then micelle forming solvents are not essential to the procedure. [0041] The preferred concentration of the diluted dispersion is about 50μg/mL. Although the initial stock dispersion could be prepared at this concentration, the preferred method begins with the preparation of a concentrated stock dispersion containing lOOmg/mL of amphipbilic compound in ethanol. In the preferred embodiment, the substrate incubates in the diluted dispersion for about 14 hours at room temperature or alternatively for 20 minutes at 50°C. The preferred alcohol/acid bath is an alcohol selected from 2-propanol, ethanol, methanol, or any aromatic alcohol and an acid selected from hydrochloric acid, phosphoric acid, or acetic acid. The preferred alcohol/acid bath is 2-propanol and 5OmM HCl in equal parts by volume. [0042] The described basic procedure will provide a biosensing surface carrying a non-planar layer of amphiphilic compound suitable for use in conventional biosensing systems. Further improvements to this basic procedure and a description of the preferred embodiments are provided below.

Method 1 - Preparing a Non-Planar Amphiphilic Layer by Self Assembly [ΘΘ43] The following discussion details the preferred method for preparing a biosensing surface having a non-planar amphiphilic layer with cross-linked, embedded micelles 34 as depicted in Figure 6. In this method, a dispersion of amphiphilic compound is prepared in a polar organic solvent. The amphiphilic compound has a general structure of X_n-R_m-Y_w or X_n- R_m-Aryl-Y_w or X_n-Aryl-R_m-Y_w as described above. The preferred amphiphilic compound will include an Aryl constituent. More preferably, the amphiphilic compound has the X_n-R_m-Aryl-Y_w structure.

[0044] Figs. 4-a - 4-x depict a non-limiting list of suitable amphiphilic compounds. The chemical properties of the final three dimensional interaction surface may be tuned to meet the demands of a diverse range of applications by using a mixture of two or more amphiphilic compounds. The mole ratio of chemisorbed amphiphiles approximates the mole ratio of the amphiphiles present in the dispersion used during deposition. Preferably the structure of each amphiphile in such a heterogeneous non-planar amphiphilic layer will be analogous with the exception of the Y component. For example, the amphiphile shown in figure 4x possesses the same structure as the amphiphile in figure 4w other than a difference in the structure of the Y component. The carboxylic group of the amphiphile of figure 4x provides a means of covalent attachment of ligand while the hydroxyl groups of the amphiphile in Figure 4w are ideal for lowering non-specific binding to the surface. The relative density of each amphiphile in such a heterogeneous layer is chosen to meet the requirements of the application. For example, if a low ligand density is required then a surface composition of 5% carboxylic acid to 95% hydroxyl is suitable. In cases where high ligand density is required the substitution ratio may be the inverse. [0045] Polar solvents suitable for preparing the initial stock dispersion of the amph philic compound included, but are not limited to: ethanol, water, dimethyl sulfoxide, 2-propanol, and dimethyl formamide. Not all amphiphiles behave the same in these solvents, for example, the amphiphiles of Figure 4x and Figure 4w will readily form a dispersion in ethanol but will form a solution in DMSO. Therefore the selection of suitable solvents for generating a dispersion must be empirically optimized. For generation of a dispersion, the solvent will solvate the Y constituent or portion of the amphiphilic compound. Solvating the Y constituent of the amphiphilic compound but not the non-polar hydrophobic component causes the non-polar regions to aggregate leading to the formation of micelles in the bulk liquid. These colloidal particles will then integrate into the barrier layer 32 (Figure 6) or attach to the surface of barrier layer 32 (Figure 5). As shown in Fig. 6, a portion of biosensing film 26 is bound to the metal surface while other portions are cross-linked to one another and extend outwards from the surface of barrier layer 32. These micelle like extensions 34 increase the surface area of biosensing film 26 thereby improving the sensitivity of the resulting biosensing surface. Typically, micelle like extensions 34 range from about 3 to 10 nanometers beyond the surface of the amphiphilic layer.

[0046] Following preparation of the stock dispersion of amphiphilic compound(s) in polar organic solvent, the dispersion is further diluted to yield a final concentration of amphiphilic compound or mixture of amphiphilic compounds of about 30μg/mL to about 70μg/mL, with a preferred concentration of about 50 μg/mL. The resulting working dispersion contains uniformly distributed amphiphilic compound typically in the form of a dispersion.

[0047] Prior to applying the layer of amphiphilic compound to the metal substrate, the metal is optionally cleaned. The cleaning enhances the binding of the amphiphilic compound to the metal by limiting defect sites that occur where debris is located on the surface. The preferred cleaning method utilizes an oxygen plasma cleaner. According to this optional step, a vacuum is applied to the chamber of the plasma cleaner and a plasma is struck to clean the metal surface. The vacuum lowers the gas pressure allowing a plasma to be struck. Dry air, nitrogen, oxygen and many other gases may be used to generate a cleaning plasma. Alternatively, the surface may be cleaned by exposing the metal surface to piranha solution for 1-30 minutes at room temperature and then rinsing the surface extensively with ultrapure water. Optionally the surface of the metal may be deposited such that the average surface roughness (root mean square) is greater than 3 run and less than 20 nm. The increased metal roughness results in defect sites in the resulting biosensing film. These defect sites promote the adsorption of the aggregates from the colloidal dispersion. This results in a greater aggregate loading density and increased capacity.

[0048] Whether or not plasma cleaning is practiced, the substrate is immersed in the diluted dispersion of amphiphilic compound. The metal substrate incubates in the amphophilic compound for a time period sufficient to permit binding of a layer of amphiphilic compound to the metal. At room temperature, the incubation period requires about 0.5 to about 18 hours. Typically, room temperature incubation requires about 14 hours. More preferably, the substrate is placed in the dispersion of amphiphilic compound and placed in an oven preheated to 50°C for a period of 20 minutes. Following incubation, the substrate, now carrying a non-planar amphiphilic layer, is removed from the dispersion and washed with copious amounts of water. The rinsed substrate is allowed to dry.

[0049] While the substrate now carries a non-planar layer of amphiphilic compound, unbound amphiphilic compounds may be present. To remove the unbound compounds, the substrate is washed or immersed in a bath of alcohol and acid. Treatment in the alcohol/acid bath for about 5 to about 10 minutes will remove any unbound amphiphilic compound. The preferred alcohols suitable for use in the bath include but are not limited to: 2-propanol, ethanol, methanol, or any aromatic alcohol. The preferred acids include, but are not limited to: hydrochloric acid, phosphoric acid, or acetic acid. The preferred alcohol/acid bath is a 5OmM hydrochloric acid/2- propanol blend where the components are present in equal amounts by volume. [0050] Following the alcohol/acid treatment, the amphiphilic compound is rinsed with water and allowed to dry. The resulting biosensing surface has a three dimensional self-assembled layer of amphiphilic compound suitable for binding ligands or other biomolecules of interest. Figure 6 shows the three dimensional self-assembled layer that can be expected from using a colloid containing only the amphiphile of Figure 4w. In this case, for the micelles binding to the metal and biosensing film 26 formation occurs concurrently. The alternative structure of Figure 5 is generated when the Y component of the constituent amphiphiles contain reactive groups capable of cross-linking. Although not shown in Figure 5 such cross-linking can also form internal linkages within a micelles that provide greater stabilization of the micelle and removes the dependence on the X component to chemisorb to the gold while also enabling linkages from the micelle to the barrier film. Alkene groups located anywhere in the compound provide this linkage flexibility as exposure to ambient light triggers cross-linking. However any reactive group(s) capable of generating stable linkage bonds may be chosen including alkyne, alkene, disulfide, hydrazide-aldehyde, amine-n-hydroxysuccinimide, epoxy, carbidimide. Method 2 - Preparing a Non-Planar Amphiphilic Layer by Self Assembly of Barrier Layer followed by Active Linkage of Aggregates.

[0051] In another preferred embodiment, the current invention provides a biosensing surface that is assembled in two separate incubation steps as opposed to a single incubation step as outlined in method 1. The result is a three dimensional interaction surface wherein the aggregates are bound to the exposed surface of the barrier layer as shown in Figure 7. This surface possesses equivalent performance to the three dimensional interaction surface produced from method 1. To achieve greater flexibility in terms of the chemical properties of the sensing surface it is possible to prepare this surface from a single compound or a mixture. [0052] Such a surface may be prepared by first constructing a self-assembled monolayer as follows. A stock dispersion of amphiphilic compound is prepared in a solvent that does not promote micelle formation but instead provides a solution as opposed to a dispersion of the biosensing film forming compound. In a preferred embodiment the biosensing film forming compound is dissolved in DMSO. A further dilution in DMSO is made to yield a final concentration of amphiphilic compound of about 30μg/mL to about 70μg/mL, with a preferred concentration of about 50 μg/mL, The resulting working solutions contain dissolved amphiphilic compound that is not present in the form of aggregates. A clean gold coated substrate is then exposed to this solution and incubated in the solution for 20 minute in an oven pre-heated to about 50°C. The optional cleaning procedure is described in METHOD 1. The resulting biosensing film is then activated in order to facilitate covalent attachment of micelles or micelle- like aggregates. Any coupling chemistry that produces a stable bond between the aggregate and the biosensing film may be chosen. In a preferred embodiment the biosensing film surface may be chemically activated by exposure to a bifunctional cross-linker such as butanediol diglycidyle ether. A solution containing 10% butane dioldiglycidyl ether in 0.1M NaOH is exposed to the biosensing film surface for 0.5-6 hours at room temperature. Alternatively an accelerated activation can be performed by carrying out this incubation step at elevated temperatures (e.g. 10 minutes at 100°C). The activated surface possesses reactive epoxy groups capable of forming covalent bonds with common functional groups such as amine, thiol hydrazide, aldehyde and carboxyl groups. The colloidal dispersion of amphiphilic compound as prepared in Method 1 is then incubated on the epoxy activated biosensing film surface for 12 hours at 60°C resulting in the covalent linkage of the aggregates and formation of the three dimensional sensing surface. In a preferred embodiment the amphiphilic compound has a general structure of X_n-R_m-Y_w or X_n- R_m-Aryl-Y_w or X_n-Aryl-R_m-Y_w , In addition, in a preferred embodiment, both the biosensing film forming the barrier layer and the subsequently attached micelles are composed of the same compound.

[0053] To remove the unbound compounds, the substrate is washed or immersed in a bath of alcohol and acid. Treatment in the alcohol/acid bath for about 5 to about 10 minutes will remove any unbound amphiphilic compound. The preferred alcohols suitable for use in the bath include but are not limited to: 2-propanol, ethanol, methanol, or any aromatic alcohol. The preferred acids include, but are not limited to: hydrochloric acid, phosphoric acid, or acetic acid. The preferred alcohol/acid bath is a 5OmM hydrochloric acid/2-propanol blend where the components are present in equal amounts by volume. Following the alcohol/acid treatment, the amphiphilic compound is rinsed with water and allowed to dry.

[0054] Optionally the surface of the metal may be deposited such that the average surface roughness (root mean square) is greater than 3nm and less than 20nm. The biosensing film barrier film is assembled as described above but the increased metal roughness results in defect sites in the resulting biosensing film. The surface is then activated as described above and the defect sites promote the adsorption of the aggregates from the colloidal dispersion. This results in a greater aggregate loading density and Increased capacity.

[0055] One skilled in the art will recognize that procedures of Method 2 may be used to prepare a homogeneous sensing surface as depicted in Figure 5 wherein the barrier layer 32 and attached micelle 34 are the same amphiphilic compound 28. To prepare the homogeneous sensing surface, the subsequent steps of attaching micelle 44 to barrier layer 32 simply utilizes a second dispersion of the same amphiphilic compound prepared as described above. [0056] Figure 8 demonstrates the enhanced sensitivity of a biosensing surface using the ligand binding monolayers of the current invention. Figure 8 is a response curve reflecting the accumulation of protein on a biosensing surface prepared according to the description of Method 1. The biosensing surface has a ligand binding layer prepared according to method 1 using the amphiphile in Figure 4x. The curve depicted was generated by injecting a 1OmM sodium acetate buffer solution containing 25 μg/mL NeutrAvidin, pH 4.5, onto the electro negative self- assembled monolayer. Under these conditions the electropositive protein is attracted to the surface. As shown in Fig. 8, the pre-concentration response exceeded 12,000 RU. This response is equivalent to at least four monolayers of neutravidin at the surface. Under these buffer conditions, a planar surface would yield an electrostatic field penetration depth of only 1.5 monolayer equivalents of neutravidin. Therefore, the results demonstrate that the self-assembled monolayer surface of the current invention must possess complex topography. [0057] Other embodiments of the current invention will be apparent to those skilled in the art from a consideration of this specification or practice of the invention disclosed herein. Thus, the foregoing specification is considered merely exemplary of the current invention with the true scope and spirit of the invention being defined by the following claims.

Claims

I claim:

1. A sensing surface comprising: a solid substrate; a metal layer carried by said solid substrate; a sensing surface comprising a self-assembled monolayer of amphiphilic compounds, said self-assembled monolayer bound to said metal and having a non-planar topography.

2. A sensing surface comprising: a solid substrate; a metal layer carried by said solid substrate; a sensing surface comprising a self-assembled monolayer of amphiphilic compounds, said self-assembled monolayer bound to said metal and having a non-planar topography; and, said amphiphilic compounds having the general structure of X_n-R_m-Y_w, wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands; and, n, m and w are integer numbers from 1 to 10.

3. A sensing surface comprising: a solid substrate; a metal layer carried by said solid substrate; a sensing surface comprising a self-assembled monolayer of amphiphilic compounds, said self-assembled monolayer bound to said metal and having a non-planar topography; and, said amphiphilic compounds having a structure selected from the group consisting of X_n- R_m-Y_w, X_n-Aryl-R_m-Y_w and X_n-R_m- Aryl-Y_w, where Aryl is at least one aromatic group, X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide, R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring, Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatom chains, said chain carrying an active group for covalently binding ligands; and, n, m and w are integer numbers from 1 to 10.

4. The sensing surface of claims 1-3, wherein a portion of said amphiphilic compounds are cross-linked to one another thereby forming micelles, said micelles bound to said self-assembled monolayer.

5. The sensing surface of claims 1-3, wherein said self-assembled monolayer further comprises of aggregates of said amphiphilic compounds which are not covalently bound to said metal layer.

6. The sensing surface of claims 1-3, wherein said self-assembled monolayer further comprises an organic compound having the general structure of R-Y, wherein said organic compound is bound to said amphiphilic compound thereby forming said non-planar topography.

7. The sensing surface of claims 1-3, further comprising a second amphiphilic compound having a structure selected from the group consisting of X_n-R_m-Y_w, X_n-Aryl-R_m-Y_w and X_n-R_m- Aryl-Y_w.

8. The sensing surface of claims 1-3, wherein said amphiphilic compounds are capable of forming at least two chemisorption bonds with said metal.

9. The sensing surface of claims 1-3, wherein said amphiphilic compound has a structure of X₂--R₂-Aryl-Y.

10. The sensing surface of claim 1-3, wherein said organic compound is a surfactant.

11. The sensing surface of claims 1-3, wherein said the non-planar portions of said self- assembled monolayer extend outward from said substrate a distance of about 0.5 nm to about 100 nm.

12. The sensing surface of claim 1-3, wherein R is a saturated or unsaturated hydrocarbon chain comprising from about 2 carbon atoms to about 30 carbon atoms.

13. The sensing surface of claims 1-3, wherein the active group carried by the Y chain is selected from the group consisting of: carboxylic acids, hydroxyl groups, amine groups, carboxyl, aldehyde, hydrazide, carbonyl, epoxy, alkyne, thiol, vinyl groups, cyano, azide, maleimide.

14. The sensing surface of claims 1-3, wherein Y is from about one to about six ethylene oxide units.

15. A method for preparing a sensing surface comprising: preparing a dispersion comprising an organic solvent and an amphiphilic compound having the general structure of X_n-R_m-Y_w, wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non- aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands; and; n, m and w are integer numbers from 1 to 10; and, treating a metal coated substrate with said dispersion for a period of time sufficient to deposit a self-assembled monolayer of said amphiphilic compound on the surface of said metal.

16. A method for preparing a sensing surface comprising: preparing a dispersion comprising an organic solvent and an amphiphilic compound having a general structure selected from the group consisting of X_n-R_m-Y_w, X_n-Aryl-R_m-Y_w and X_n-R_1n- Aryl-Y_w wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands, where Aryl is an aromatic group; and; n, m and w are integer numbers from 1 to 10; agitating said dispersion for a period of time sufficient to uniformly disperse said organic compound; treating a metal coated substrate with said dispersion for a period of time sufficient to deposit a self-assembled monolayer of said amphiphilic compound on the surface of said metal, wherein said self-assembled monolayer has a non-planar topography.

17. The method of claims 15-16, wherein each amphiphilic compound having a general structure selected from the group consisting of X_π-R_m-Y_w, X_n- ArVl-R_1n-Yw and X_n-R_n,- Aryl- Y_w forms at least two chemisorption bonds with said metal.

18. The method of claims 15-16, wherein said step of treating said metal coated substrate with said dispersion comprises immersing said substrate in said dispersion for a period of time of about 3 hours to 18 hours.

19. The method of claims 15-16, wherein the active group carried by the Y chain is selected from the group consisting of: carboxylic acids, hydroxy! groups, amine groups, carboxyl, aldehyde, hydrazide, carbonyl, epoxy, alkyne, thiol, vinyl groups, cyano, azide, maleimide.

20. The method of claim 19, following said immersion step, further comprising the steps of: washing said substrate carrying said self-assembled monolayer with water; allowing said substrate carrying said self-assembled monolayer to dry; removing unbound organic compounds from said metal coated substrate; and, washing said substrate carrying said self-assembled monolayer with water, thereby yielding a planar monolayer on said metal coated substrate.

21. The method of claim 20, wherein said step of removing unbound organic compounds from said metal coated substrate comprises treating said self-assembled monolayer with a solution comprising an alcohol and an acid.

22. The method of claim 21, wherein said alcohol is selected from the group consisting of: 2- propanol, ethanol, methanol, or an aromatic alcohol, and said acid is selected from the group consisting of hydrochloric acid, phosphoric acid, and acetic acid.

23. The method of claim 22, further comprising the step of cleaning and oxidizing the surface of said metal coated substrate.

24. The method of claim 23, wherein said step of treating said metal coated substrate with said dispersion of organic compound occurs immediately following said step of cleaning and oxidizing the surface of said metal coated substrate.

25. The method of claim 15-16, further comprising the steps of: oxidizing said self-assembled monolayer; treating said substrate carrying said oxidized self-assembled monolayer with a second dispersion of said amphiphilic compound in an organic solvent for a period of time sufficient to bind additional organic compounds to said self-assembled monolayer, thereby yielding a non-planar monolayer of organic compounds on said metal coated substrate.

26. The method of claims 15-16, wherein said organic solvent used in said dispersion of organic compound is an alcohol.

27. The method of claims 15-16, wherein said solvent is selected from the group consisting of ethanol, 2-propanol or methanol.

28. The method of claims 15-16, wherein said step of treating said metal coated substrate with said dispersion comprises immersing said substrate in said dispersion and placing said substrate in said dispersion in an oven pre-heated to a temperature between about 40° C to about 100° C for a period of time of about one minute to about 100 minutes.

29. The method of claims 15-16, wherein said step of treating said metal coated substrate with said dispersion comprises immersing said substrate in said dispersion, and placing said substrate in said dispersion in an oven pre-heated to a temperature of about 50° C for a period of time of about 20 minutes.

30. A method for preparing a sensing surface comprising the steps of: preparing a first dispersion comprising a polar solvent and an amphiphilic compound having a general structure selected from the group consisting of X_π-R_m-Y_w, X_n-Aryl-R_m-Y_w and X_n-R_1n- Aryl-Y_w wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from the group consisting of oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands, where Aryl is an aromatic group; and; n₅ m and w are integer numbers from 1 to 10; uniformly dispersing the amphiphilic compound throughout the dispersion; immersing a metal coated substrate in the dispersion of amphiphilic compound for a period of time sufficient to deposit a self-assembled monolayer of said amphiphilic compound on the surface of said metal, wherein said self-assembled monolayer has a non-planar topography; allowing the self-assembled monolayer of said amphiphilic compound to dry; removing unbound amphiphilic compound; rinsing the self-assembled monolayer of said amphiphilic compound with water; and, allowing the self-assembled monolayer of said amphiphilic compound to dry, thereby yielding a substrate carrying a non-planar monolayer of said amphiphilic compound.

31. The method of claim 30, further comprising the step of washing the self-assembled monolayer of amphiphilic compound with water following removal of the metal coated substrate from said dispersion.

32. The method of claim 30, further comprising the steps of: preparing a second dispersion comprising a polar solvent and an amphiphilic compound having a general structure selected from the group consisting of X_n-R_m-Y_w? X_τrAryl-R_m-Y_w and X_n-R_m-Aryl-Y_w wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carhoxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands, where Aryl is an aromatic group; and; n, m and w are integer numbers from 1 to 5; uniformly dispersing the amphiphilic compound throughout the dispersion; immersing the substrate in the second dispersion; sealing the substrate in the second dispersion and incubating for a period of time sufficient to attach aggregates of said amphiphilic compound from said second dispersion to said non-planar monolayer of said amphiphilic compound from said first dispersion; subsequently rinsing the substrate in water; drying the substrate; immersing the substrate in an alcohol/acid solution to remove unbound amphiphilic compound; rinsing the substrate with water; and, allowing the substrate to dry thereby yielding a substrate carrying a non-planer monolayer of amphiphilic compound from said first dispersion and carrying aggregates of said amphiphilic compound from said second dispersion.

33. A method for preparing a sensing surface comprising the steps of: preparing a first dispersion comprising a polar solvent and an amphiphilic compound having a general structure selected from the group consisting of X_n-R_m-Y_w, X_n-Aryl-R_m-Y_w and X_n-R_m-Aryl-Y_w wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands, where Aryl is an aromatic group; and; n, m and w are integer numbers from 1 to 10; uniformly dispersing the amphiphilic compound throughout the dispersion; immersing a metal coated substrate in the dispersion for a period of time sufficient to deposit a self-assembled monolayer of said amphiphilic compound on the surface of said metal, wherein said self-assembled monolayer has a non-planar topography; washing the metal coated substrate with water: allowing the metal coated substrate to dry; removing unbound amphiphilic compound; rinsing the wafer with water; and, allowing the wafer to dry, thereby yielding a substrate carrying a non-planar monolayer of said amphiphilic compound.

34. The method of claims 30 and 33, wherein said period of time sufficient to deposit a self- assembled monolayer of said amphiphilic compound is between about 3 hours and about 18 hours.

35. The method of claims 30 and 33, wherein said period of time sufficient to deposit a self- assembled monolayer of said amphiphilic compound is about 14 hours.

36. The method of claims 30 and 33, wherein said step of removing unbound amphiphilic compound comprises immersing the metal coated substrate in an alcohol/acid solution.

37. The method of claim 33, further comprising the steps of: preparing a second dispersion comprising a polar solvent and an amphiphilic compound having a general structure selected from the group consisting of X_n-R_m-Y_w, X_n-Aryl-R_m-Y_w and X_n-R_m-Aryl-Y_w wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands, where Aryl is an aromatic group; and; n, m and w are integer numbers from 1 to 5; uniformly dispersing the amphiphilic compound throughout the dispersion; immersing the substrate in the second dispersion; sealing the substrate in the second dispersion and incubating for a period of time sufficient to attach aggregates of said amphiphilic compound from said second dispersion to said non-planar monolayer of said amphiphilic compound from said first dispersion; subsequently rinsing the substrate in water; drying the substrate; removing unbound amphiphilic compound; rinsing the substrate with water; and, allowing the substrate to dry thereby yielding a substrate carrying a non-planer monolayer of amphiphilic compound from said first dispersion and carrying aggregates of said amphiphilic compound from said second dispersion.

38. The method of claim 37, wherein said step of removing unbound amphiphilic compound comprises immersing the metal coated substrate in an alcohol/acid solution.

39. The method of claims 30 and 33, wherein said step of treating said metal coated substrate with said dispersion comprises immersing said substrate in said dispersion and placing said substrate in said dispersion in an oven pre-heated to a temperature between about 40° C to about 100° C for a period of time of about one minute to about 100 minutes.

40. The method of claims 30 and 33, wherein said step of treating said metal coated substrate with said dispersion comprises immersing said substrate in said dispersion, and placing said substrate in said dispersion in an oven pre-heated to a temperature of about 50° C for a period of time of about 20 minutes.

41. A method for preparing a sensing surface comprising the steps of: preparing a dispersion of X₂-R₂-ATyI-Y having a concentration of between about 30μg/mL to about 70μg/mL by dissolving X₂-R₂- Aryl- Y in alcohol; uniformly dispersing the X₂-R₂-Aryl-Y compound; immersing a metal coated substrate in the dispersion Of X₂-R₂-ATyI-Y; immersing the metal coated substrate in said dispersion of X₂-R₂-Ar^l-Y for about 10 to about 18 hours thereby depositing a self-assembled monolayer of said amphiphilic compound on said metal coated substrate; subsequently washing the self-assembled monolayer of said amphiphilic compound with water: allowing the self-assembled monolayer of said amphiphilic compound to dry; removing unbound X₂-R₂-ATyI-Y by immersing the metal coated substrate in an alcohol/acid solution; rinsing the wafer with water and allowing the wafer to dry.

42. The method of claim 41 , wherein said dispersion has a concentration of about 50μg/mL.

43. The method of claim 41, wherein said metal coated substrate remains in said dispersion of X₂-R₂-ATyI-Y for about 14 hours.

44. The method of claim 41, wherein said alcohol/acid solution is 2-propanol and 5OmM hydrochloric acid.

45. The method of claim 41, wherein said dispersion of X₂-R₂-ATyI-Y is prepared in ethanol.

46. A method for preparing a sensing surface comprising the steps of: preparing a first dispersion comprising a non-polar solvent and an amphiphilic compound having a general structure selected from the group consisting of X_n-R_m- Y_w, X_n-Aryl-R_m-Y_w and X_n-Rj_n- Aryl-Y_w wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands, where Aryl is an aromatic group; and; n, m and w are integer numbers from 1 to 10; uniformly dispersing the amphiphilic compound throughout the dispersion; immersing the metal coated substrate in the dispersion for about 10 hours to about 18 hours thereby depositing a non-planar monolayer of amphiphilic compound on said metal coated substrate; removing the metal coated substrate from said dispersion and washing the metal coated substrate with water: allowing the metal coated substrate to dry; following drying, immersing the metal coated substrate in an alcohol/acid solution to remove unbound amphiphilic compound; rinsing the wafer with water; allowing the wafer to dry, thereby yielding a substrate carrying a planar monolayer of said amphiphilic compound; preparing a second dispersion comprising a polar solvent and an amphiphilic compound having a general structure selected from the group consisting of X_n-R_1n- Y_w, X_n-ATyI-R_7n-Y_w and X_n-R_1n- Aryl-Y_w wherein X is selected from the groups consisting of alkyne, thiol, phosphorous, nitro, selenol, carboxylate, phosphane, and sulfide; R is selected from the group consisting of saturated hydrocarbon chains, unsaturated hydrocarbon chains, saturated hydrocarbon chains with heteroatoms and unsaturated hydrocarbon chains with heteroatoms, conjugated rings, aromatic and non-aromatic hydrocarbon rings, rings with two or more hydrocarbon chains attached to the ring; Y is selected from oligoethylene oxides, glycerol, oligosaccharides and branched heteroatoms chains, said chain carrying an active group for covalently binding ligands, where Aryl is an aromatic group; and; n, m and w are integer numbers from 1 to 5; uniformly dispersing the amphiphilic compound throughout the dispersion; immersing the substrate in the second dispersion; sealing the substrate in the second dispersion and incubating for a period of time sufficient to attach aggregates of said amphiphilic compound from said second dispersion to said non-planar monolayer of said amphiphilic compound from said first dispersion; subsequently rinsing the substrate in water; drying the substrate; immersing the substrate in an alcohol/acid solution to remove unbound amphiphilic compound; rinsing the substrate with water; and, allowing the substrate to dry thereby yielding a substrate carrying a planer monolayer of amphiphilic compound from said first dispersion, said planar monolayer carrying aggregates of said amphiphilic compound from said second dispersion.

47. A method for preparing a sensing surface comprising the steps of: preparing a stock dispersion of X₂-R₂-ATyI-Y by dissolving lOOmg of purified compound X₂-R₂-AIyI-Y with DMSO; diluting the stock dispersion with DMSO to a concentration of 50μg/mL dispersion OfX₂-R₂-AIyI-Y; agitating the 50μg/mL dispersion for 60 seconds to ensure uniform dispersion of the X₂-R₂-Aryl-Y compound; obtaining a metal coated substrate; immersing the metal coated substrate in the dispersion of X₂-R₂-Aryl-Y and allowing the metal coated substrate to incubate for 14 hours; following the incubation period, washing the metal coated substrate with copious amounts of water; allowing the wafer to dry immersing the metal coated substrate in a 50:50 (v/v) solution of 2-propanol and 5OmM hydrochloric acid for 10 minutes to remove unbound X₂-R₂-Aryl-Y; rinsing the metal coated substrate with water; and, allowing the metal coated substrate to dry, thereby yielding a wafer carrying a planar monolayer OfX₂-R₂-ATyI-Y; preparing a 50μg/mL dispersion Of X₂-R₂-ATyI-Y in ethanol; gently agitating the dispersion for 60 seconds to uniformly disperse said X₂-R₂- Aryl-Y in the ethanol; immediately immersing the metal coated substrate in the newly prepared 50μg/mL dispersion of X₂-R₂-Aryl-Y; sealing the metal coated substrate carry the planar monolayer of X₂-R₂-AIyI-Y in the dispersion and incubating for 3-5 hours subsequently rinsing the metal coated substrate in water; drying the metal coated substrate; immersing the metal coated substrate in a 50:50 (v/v) solution of 2-propanol and 5OmM hydrochloric acid for 10 minutes to remove unbound X₂-R₂-ATyI-Y; rinsing the metal coated substrate with water; and, allowing the metal coated substrate to dry thereby yielding a wafer carrying a non-planer monolayer Of X₂-R₂-ATyI-Y carrying aggregates of X₂-R₂-Aryl-Y.