Molecular sensor preparations and uses thereof

Abstract

The present invention relates to a method of preparing a molecular sensor that is specific for a target molecule having a saccharide or peptide region. The method comprises using the target molecule as a template and incubating the template with a receptor to form a template-receptor complex. A molecular scaffold is formed on a surface around the template-receptor complex such that the receptor and at least a portion of the template are embedded in the scaffold, and the template is removed to produce a cavity defined by the scaffold, such that the cavity is complementary to at least a portion of the saccharide or peptide region of the target molecule.

Claims

1. A method of preparing a molecular sensor that is specific for a target molecule having a saccharide or peptide region, the method comprising, in a stepwise manner: forming a template-receptor complex using the target molecule as a template, comprising: forming a mixture comprising a solution of the target molecule, comprising one or more of the target molecules, and a solution of a receptor, comprising one or more receptor molecules, capable of selectively and reversibly binding the target molecule; wherein the one or more receptor molecules in the mixture are in an amount such that substantially all the receptor molecules bind to the target molecules, thus avoiding an excess of the receptor molecules; and forming a molecular scaffold on a surface around the template-receptor complex, in the absence of uncomplexed receptors, such that the receptor and at least a portion of the template are embedded in the scaffold; and removing the template from the template-receptor complex of the molecular scaffold to produce a cavity defined by the molecular scaffold, wherein: the cavity is complementary to at least a portion of the saccharide or peptide region of the target molecule and comprises the one or more receptor molecules at a surface thereof, the spatial arrangement of the one or more receptor molecules being specific for binding of the target molecule, and wherein: the receptor molecules comprise: a recognition motif comprising an acid group, a phosphate group, an aromatic group, a conjugated group, or a hydrogen bonding group; and a first binding moiety for binding the template-receptor complex to the molecular scaffold, and wherein the first binding moiety is a polymerizable group or is capable of reacting with one or more of the molecules which form the molecular scaffold so as to form a covalent bond between the receptor and the scaffold molecules.

2. The method of claim 1, wherein the step of removing the template comprises dissociating the template from the receptor, thereby producing the cavity having the receptor molecules at the surface thereof.

3. The method of claim 1, wherein the target molecule is a glycoprotein and the recognition motif binds to the saccharide or the peptide region of the glycoprotein.

4. The method of claim 1, wherein the recognition motif comprises a boronic acid group.

5. The method of claim 1, wherein the molecular scaffold is formed from a first type of molecules, each of the first type of molecules comprising a tether moiety for tethering the molecular scaffold to the surface, and wherein the tether moiety is a thiol, a disulfide, an organosilane, a dialkyl sulfide, an alcohol, an amine or a carboxylic acid group.

6. The method of claim 5, wherein the step of forming the molecular scaffold comprises exposing the surface to the first type of molecules so as to allow adsorption of the first type of molecules onto the surface and wherein the first type of molecules form a self-assembled monolayer (SAM) on the surface.

7. The method of claim 6, wherein the surface is exposed to the first type of molecules in the presence of the template-receptor complex.

8. The method of claim 7, further comprising cross-linking the first type of molecules after adsorption of the molecules onto the surface.

9. The method of claim 7 , wherein each of the first type of molecules comprises a second binding moiety capable of binding to other of the first type of molecules, to the receptor and/or to further molecules, and wherein the second binding moiety is a polymerizable group.

10. The method of claim 1, wherein the molecular scaffold is formed from a first type of molecules and a second type of molecules.

11. The method of claim 10, wherein at least one of the first and second types of molecules comprises an elongate moiety comprising ethylene glycol, or an oligomer thereof.

12. The method of claim 10, comprising: forming a SAM on a surface from a first type of molecules; binding the template-receptor complex to the SAM; immobilizing the second type of molecules on the SAM so as to form the molecular scaffold around the bound template; and removing the template from the template-receptor complex of the molecular scaffold to produce the cavity defined by the molecular scaffold, wherein the cavity is complementary to at least a portion of the saccharide or peptide region of the target molecule.

13. The method of claim 12, wherein each of the first type of molecules comprises a first coupling moiety for coupling to the second type of molecules, wherein each of the second type of molecules comprises a second coupling moiety for coupling to the first type of molecules, to other of the second type of molecules and/or to the receptors, and wherein the second type of molecules are immobilized on the SAM by a reaction between the first coupling moieties of the first type of molecules and the second coupling moieties of the second type of molecules.

14. The method of claim 13, wherein the second type of molecules are immobilized on the SAM by a click reaction between the first coupling moieties of the first type of molecules and the second coupling moieties of the second type of molecules.

15. The method of claim 10, comprising: forming a SAM on a surface using the first type of molecules; exposing the SAM-functionalized surface to a mixture of the second type of molecules, the template-receptor complex and a catalyst so as to effect atom transfer radical polymerization (ATRP) between the first molecules, the second molecules and the receptor, thereby forming the molecular scaffold around the template; and removing the bound template from the template-receptor complex of the molecular scaffold to produce the cavity defined by the molecular scaffold, wherein the cavity is complementary to at least a portion of the saccharide or peptide region of the target molecule.

16. The method of claim 15, wherein each of the first type of molecules comprises an ATRP initiator and wherein each of the second type of molecules comprises at least one second cross-coupling moiety which is a polymerizable group.

17. The method of claim 1, method further comprising: exposing a surface to a first type of molecules in the presence of the template-receptor complex, each of the first type of molecules comprising a tether moiety and a polymerizable group; initiating polymerization between the polymerizable groups so as to form the molecular scaffold on the surface around the template-receptor complex, wherein the receptor is covalently bound to the scaffold and at least a portion of the template is embedded in the scaffold; and removing the template from the template-receptor complex of the molecular scaffold to produce the cavity defined by the molecular scaffold, wherein the cavity is complementary to at least a portion of the saccharide or peptide region of the target molecule.

18. The method of claim 1, wherein the surface is a surface of a nanoparticle.

19. A method of preparing a molecular sensor that is specific for a target molecule having a saccharide or peptide region, the method comprising, in a stepwise manner: forming a template-receptor complex using the target molecule as a template, comprising: forming a mixture comprising a solution of the target molecule, comprising one or more target molecules, and a solution of a receptor, comprising on one or more receptor molecules, capable of selectively and reversibly binding the target molecule; wherein the one or more receptor molecules in the mixture are in an amount such that substantially all the receptor molecules bind to the target molecules, thus avoiding an excess of the receptor molecules; forming a molecular scaffold on a surface around the template-receptor complex, in the absence of uncomplexed receptors, such that the receptor and at least a portion of the template are embedded in the scaffold; and removing the template from the template-receptor complex of the molecular scaffold to produce a cavity defined by the molecular scaffold, wherein: the cavity is complementary to at least a portion of the saccharide or peptide region of the target molecule and comprises spatially arranged sets of the receptor molecules on the surface that are specific for the target molecules, and the receptor molecules comprise a boronic acid group or a diphosphate.

20. The method of claim 1, wherein the recognition motif comprises a boronic acid or boronic acid derivative and the target molecules comprises a glycan.

Description

(1) Embodiments of the invention will now be described by way of example and with reference to the accompanying Figures, in which:

(2) FIG. 1 is a schematic diagram showing the steps in the formation of a molecular sensor for a target protein according to an embodiment of the present invention;

(3) FIG. 2 is a schematic diagram showing the steps in the formation of a molecular sensor for a target protein according to an alternative embodiment of the present invention;

(4) FIG. 3 is a schematic diagram showing the steps in the formation of a molecular sensor for a target protein according to a further embodiment of the present invention;

(5) FIG. 4 is a plot of the surface plasmon resonance signals, in response units (RU), representing protein binding to a molecular sensor prepared using RNase B as a template over a number of cycles; and

(6) FIG. 5 is a plot of the surface plasmon resonance signals, in response units (RU), representing protein binding to a molecular sensor prepared using lysozyme as a template.

(7) FIG. 6 is a plot of the surface plasmon resonance signals, in response units (RU), representing disaccharide binding to a non-inventive BA-terminated surface (panel A) and a molecular sensor prepared using maltose as a template (Panel B).

(8) With reference to FIG. 1, a molecular sensor specific for a target molecule may be built in a stepwise manner from molecular building blocks using both self-assembly and molecular imprinting techniques. In step (1) a self-assembled monolayer (10) is formed on a gold surface (12) from molecules of AAM-SS (14). Each AAM-SS molecule provides two SAM molecules, each comprising a disulfide tether moiety for binding to the gold surface (12), an acrylamide polymerizable group for binding to the template-receptor complex and to other SAM molecules, and a terminal alkyne as a first cross-coupling moiety.

(9) In step (2), receptor molecules (16) are incubated with a glycoprotein target molecule, which functions as a template (18). The receptor molecules (16) are acrylamide boronic acid monomers (AM-BA) comprising a boronic acid recognition moiety and an acrylamide polymerizable group, connected by an aryl linker. The BA recognition moiety binds to the glycosylation site of the glycoprotein template (18), forming a template-receptor complex (20).

(10) In step (3), the template-receptor complex (20) is cross-linked to the SAM (10) via the acrylamide groups of the receptor molecules and the SAM molecules (14).

(11) In step (4), a molecular scaffold is built around the template (18) using azide-terminated hepta(ethylene) (Az-OEG) scaffold molecules (22). The azide groups function as second cross-coupling moieties which react via click chemistry with the terminal alkynes of the SAM molecules, thereby immobilizing the scaffold molecules (22) on the SAM (10).

(12) In step (5), the glycoprotein template (18) is removed by washing with water, leaving behind a cavity (24). The shape of the cavity (24) is complementary to and specific for the shape of the template (18), and thus provides a recognition or binding site that is selective for the target glycoprotein (18).

(13) With reference to FIG. 2, a molecular sensor specific for a target molecule may alternatively be built using ATRP to produce an imprinted cross-linked film on a surface. In step (1), a SAM (30) is formed on a gold surface (32) using ATRP initiator-functionalized thiol molecules (34).

(14) In step (2), receptor molecules (36) comprising a BA recognition motif and a vinyl polymerizable group are incubated with a target glycoprotein (38) in an aqueous medium to form a template-receptor complex (40).

(15) In step (3), the SAM-functionalized gold surface (30, 32) is immersed in an aqueous solution containing EG dimethacrylate scaffold monomers (42), a catalyst (CuCl, CuBr.sub.2, and/or 2,2′-bipyridine) and the template-receptor complex (40). The mixture is incubated for a period of time sufficient for polymerization to occur, resulting in a cross-linked film or scaffold (44) around the template.

(16) In the final step (not shown), the template is removed by washing using a suitable solution, leaving behind an imprinted cross-linked film.

(17) FIG. 3 shows a further method of preparing a molecular sensor specific for a target molecule. In step (1), receptor molecules (50) comprising a BA recognition motif and an acrylamide first binding moiety are incubated with a target glycoprotein (52) in an aqueous medium to form a template-receptor complex (52). Isothermal titration calorimetry (ITC) is used to determine the protein: receptor molar ratio required for saturation binding. It is preferred that no excess of receptors is used since unbound receptors could interfere with formation of the cavity and subsequent binding.

(18) In step (2), the template-receptor complex (52) is simultaneously combined with a planar gold surface (56) and first molecules (58) comprising acrylamide binding moieties in an aqueous solution. By virtue of a tether moiety on the first molecules (58), the first molecules (58) and the template-receptor complex are adsorbed onto to the gold surface (56) to form a molecular scaffold (60).

(19) In step (3), polymerization is initiated between the acrylamide binding moieties of the first molecules (58) and the receptor molecules (50), thereby forming a rigid network.

(20) In step (4), the template is dissociated from the complex by washing, leaving behind a cavity (62) in the molecular scaffold (60) with receptors (50) at the surface thereof. The cavity (62) and the receptors (50) are thus available to bind target proteins in solution.

EXAMPLES

(21) Methodology

(22) Contact Angle

(23) Contact angles were determined using a home-built contact angle apparatus, equipped with a charged coupled device (CCD) KP-M1E/K camera (Hitachi) that was attached to a personal computer for video capture. The dynamic contact angles were recorded as a micro-syringe was used to quasi-statically add liquid to or remove liquid from the drop. The drop was shown as a live video image on the PC screen and the acquisition rate was 4 frames per second. FTA Video Analysis software v1.96 (First Ten Angstroms) was used for the analysis of the contact angle of a droplet of UHP H.sub.2O at the three-phase intersection. The averages and standard errors of contact angles were determined from five different measurements made for each type of SAM.

(24) Ellipsometry

(25) The thickness of the deposited monolayers was determined by spectroscopic ellipsometry. A Jobin-Yvon UVISEL ellipsometer with a xenon light source was used for the measurements. The angle of incidence was fixed at 70°. A wavelength range of 280-820 nm was used. The DeltaPsi software was employed to determine the thickness values and the calculations were based on a three-phase ambient/SAM/Au model, in which the SAM was assumed to be isotropic and assigned a refractive index of 1.50. The thickness reported is the average and standard error of six measurements taken on each SAM.

(26) X-Ray Photoelectron Spectroscopy (XPS)

(27) Elemental composition of the SAMs were analysed using an Escalab 250 system (Thermo VG Scientific) operating with Avantage v1.85 software under a pressure of ˜5×10.sup.−9 mbar. An Al Kα X-ray source was used, which provided a monochromatic X-ray beam with incident energy of 1486.68 eV. A circular spot size of ˜0.2 mm.sup.2 was employed. The samples were attached onto a stainless steel holder using double-sided carbon sticky tape (Shintron tape). In order to minimise charge retention on the sample, the samples were clipped onto the holder using stainless steel or Cu clips. The clips provided a link between the sample and the sample holder for electrons to flow, which the glass substrate inhibits. Low resolution survey spectra were obtained using a pass energy of 150 eV over a binding energy range of 0 eV to 1250 eV obtained using 1 eV increments. The spectra recorded were an average of 3 scans. The high resolution spectra were obtained using a pass energy of 20 eV and 0.1 eV increments over a binding energy range of 20-30 eV, centred on the binding energy of the electron environment being studied. A dwell time of 50 ms was employed between each binding energy increment. The spectra recorded were an average of between 5-250 scans (N (1s)=100, Au (4f)=5, S (2p)=150, B (1s)=250, O (1s)=50, C (1s)=50). Sensitivity factors used in this study were: N (1s), 1.8; Au (4f), 17.1; S (2p), 1.68; B (1s), 0.486; O (1s), 2.93; C (1s), 1.0.

(28) Surface Plasmon Resonance (SPR)

(29) SPR experiments were performed with a Reichert SR7000DC Dual Channel Spectrometer (Buffalo, N.Y., USA) at 25° C. Prior to the binding studies, a baseline for the SAMs was established by running degassed PBS pH 8.5 through the machine at a flow rate of 25 μl/min.

Example 1: SAM Preparation

(30) Polycrystalline gold substrates were purchased from George Albert PVD., Germany and consisted of a 50 nm gold layer deposited onto a glass covered with a thin layer of chromium. The Au substrates were cleaned by immersion in piranha solution (7:3, H.sub.2SO.sub.4:H.sub.2O.sub.2) at room temperature for 10 min. Samples removed from the piranha solution were immediately rinsed with Ultra High Pure (UHP) H.sub.2O, followed by HPLC grade methanol (Fischer Scientific) for 1 min. Immediately after cleaning, the substrates were immersed in freshly prepared 0.1 mM methanolic solutions of AAM-SS molecules for 18 hours. Post-immersion in the SAM forming solution, the substrates were rinsed with HPLC MeOH and dried with a stream of argon.

(31) The AAM-SS SAMs were characterized by ellipsometry and contact angle, exhibiting a thickness and wetting properties consistent with the formation of a stable, sparsely packed monolayer (Table 1). The presence of AAM-SS SAMs was further confirmed by X-ray photoelectron spectroscopy, producing the expected surface elemental ratios (Table 1).

(32) In order to confirm that the alkyne and acrylic groups on the AAM-SS SAM are accessible and able to participate in surface reactions with AM-BA via acrylic polymerisation or Az-OEG via click chemistry, respectively, the AAM-SS SAMs were independently modified with AM-BA or Az-OEG.

(33) Crosslinking of AAM-SS SAMs with AM-BA was initiated using ammonium persulphate. SAMs of AAM-SS were placed in 1 ml 1 mM of AM-BA monomers in UHQ H.sub.2O, to which 100 μl of ammonium persulphate was added (40 mg/ml). The resulting mixture was then allowed to react for 15 minutes. After this time, the chips were removed from the crosslinking solution and rinsed for one minute with UHQ H.sub.2O. Samples were then dried under a stream of argon before being characterized by contract angle, ellipsometry and XPS.

(34) Click reactions were carried out between AAM-SS SAMs and Az-OEG. 1.2 ml of a 5 mM solution of Az-OEG was mixed with 150 □l of both copper sulphate (40 mM) and sodium ascorbate (100 mM). AAM-SS SAMs were placed in click reaction solutions and allowed to react for between 0.5 to 24 hours. After reaction chips were removed from the click solution and rinsed well with UHQ water and sonicated with 0.1 mM EDTA solution to remove any copper, prior to surface characterization. Again all surfaces were characterised by contact angle, ellipsometry and XPS.

(35) Preparation of BA-AAM-SS monolayers by crosslinking at room temperature AAM-SS SAM with AM-BA led to a decrease in wettability and, as expected, to an increase in thickness of the film (Table 1).

(36) TABLE-US-00001 TABLE 1 Ellipsometric thickness, advancing and receding contact angles and nitrogen/sulphur (N/S) XPS ratio of the different monolayers investigated. Thickness (nm) Contact angle (°) N/S XPS ratio SAM Observed Theoretical Advancing Receding Observed Expected AAM-SS 0.42 ± 0.2  0.91 65.1 ± 1.15 46.94 ± 3.84 1.9 2 BA-AAM-SS 0.82 ± 0.3  0.85 44.9 ± 5.4  39.4 ± 7.7 2.95 3 OEG-AAM-SS 1.95 ± 0.12 2.9 39.9 ± 3.5  31.3 ± 3.2 5.38 5

Example 2: Sensor Fabrication

(37) To form the sensor, a one pot multi-step template procedure was used. A solution of AM-BA was mixed with a 10 fold excess of the target protein, all adjusted to pH 8.5. This was allowed to incubate for 30 minutes, after which the SAMs were placed in the resultant solution. To this 100 μl of ammonium persulfate solution (40mg/ml) was added to initiate cross-linking. The solution was then incubated for a further 5 minutes. To this solution 1 ml of a 5 mM solution of O-(2-Azidoethyl)heptaethylene glycol was added. After 10 minutes the click reaction was initiated by the addition of a 30 μl solution of pre-prepared catalyst (15 μl of 40 mM Copper sulphate and 15 μl of 100 mM sodium ascorbate). The mixture was then allowed to react for a total for 4 hours. After the reaction time had been reached the SPR chips were removed from the reaction solution and rinsed with liberal amount of UHQ H.sub.2O for 3 minutes to remove bound template protein.

(38) Following rinsing of the template compounds from the cavities, SPR binding experiments were used to confirm the selectivity of molecularly imprinted sensors. All SPR experiments were conducted at 25° C., with a SPR flow rate of 25 μl/min, using a 150 μl loop. Stock protein solutions were prepared from freeze dried proteins, using an accurate balance to produce a final concentration of 1 mg/ml. For SPR experiments, protein samples were prepared by a 1:10 dilution of the stock solution, followed by a serial dilution to produce samples of 100, 50, 25 and 12.5 μg/ml of each protein. Molar concentrations of each protein were then calculated from published mass data. Protein samples were injected for a 5 minute association phase before switching back to buffer for the dissociation phase for up to 10 minutes. The same injection protocol was used to regenerate the surface in between sample injections, using an acidic regeneration solution.

(39) It was found that the surfaces displayed a much higher equilibrium binding response to target proteins than to non-target proteins (see below). This is indicative of the nano-cavities adopting a surface conformation which offers a complementary binding site to the target protein, which results in an increased affinity.

Example 3: PSA Sensor

(40) A sensor for the detection of PSA was fabricated using the method described in Example 2. Once formed, the ability of the sensor to bind proteins was investigated using SPR and from the SPR data dissociation constants (K.sub.d) were calculated.

(41) The PSA-imprinted surface exhibited excellent selectivity towards PSA, with all other proteins showing significantly reduced affinity (Table 2). A dissociation constant of 1.8 □M is comparable to the value for other antibodies specific for PSA (typically with values in the nM-□M range).

(42) TABLE-US-00002 TABLE 2 Affinity data for PSA (target molecule) and other proteins Protein K.sub.d (μM) PSA 1.8 ± 0.1 Lysozyme 4.9 ± 0.1 □-1-acid glycoprotein (α1-AGP) 5.3 ± 0.1 RNAse B 6.7 ± 0.5 Bovine serum albumin (BSA) 21.6 ± 0.6  □-1-antitrypsin α1-AT 30.9 ± 0.9  horseradish peroxidase (HRP) 52.5 ± 2.0 

(43) The PSA-imprinted surface revealed a 3-30 fold selectivity to PSA over other glycosylated and non-glycosylated proteins. The difference in the magnitude of the binding affinity between the non-targeted proteins appears to be primarily attributed to their molecular size (Table 3), in which proteins of similar or smaller size to that of the target PSA displayed higher binding affinities than other larger proteins examined. There is no observable general trend in the amount of non-target protein bound to the imprinted surface with isoelectric point. It is noted however that positively charged proteins at pH 8.5 are more prone to interact with the negatively charged boronate ion species present in the imprinted surfaces. Thus, it is reasonable to explain the higher affinity of lysozyme among the non-target proteins for the PSA-imprinted surfaces based on Coulombic interactions.

(44) Although it could have been hypothesised that a higher degree of glycosylation would induce a higher non-specific binding from the non-targeted glycoproteins due to the interaction of the sugar residues with the BA containing-nanocavities, such trend is not established by the data. Remarkably, ribonuclease (RNAse) B, which is a smaller glycoprotein than PSA with similar degree of glycosylation, produced a very low SPR response when evaluated at concentrations as high as 650 nM.

(45) TABLE-US-00003 TABLE 3 Protein PSA Lysozyme α1-AGP RNAse B BSA α1-AT HRP size 4.4 × 2.8 × 5.9 × 3.8 × 14 × 7 × 4.0 × (nm × nm × 4.1 × 5.1.sup.a 3.2 × 3.sup.b 4.2 × 3.9.sup.a 2.8 × 2.2.sup.b 4 × 4.sup.b 3 × 3.sup.b 6.7 × 11.7.sup.b nm) glycosylation 8.3 0 45 9 0 5 21 (%) Isoelectric 6.2-7.5 11.1 2.8-3.8 9.2-9.6 4.7 4.5-5.5 9 point .sup.aestimated using ChemBio Ultra 3 D .sup.bliterature values

(46) It is important to note that OEG-terminated surfaces created without the glycoprotein-AM-BA complex displayed minimal non-specific protein binding, with SPR responses below 20 response units. The low binding of RNAse B to the PSA-imprinted surface provides evidence that spatially arranged sets of BAs on the surface that are specific for the target PSA glycoprotein have been created.

(47) Furthermore, SPR analysis revealed the detection of PSA at nM levels and excellent reproducibility of the imprinted surfaces. The surface coverage for PSA was found to range between 0.024 ng/mm.sup.2 and 0.140 ng/mm.sup.2 (100 response units (RUs)˜0.1 ng/mm), depending on the concentration of PSA to which the sensor was exposed to. By converting surface coverage mass into molecular units of PSA (Mw=28.4 kDa) per mm.sup.2, 81 nM has led to a coverage of 5.1×10.sup.8 PSA molecules/mm.sup.2 whereas a 8 fold increase in concentration (i.e. 650 nM) raised the coverage to 3.0×10.sup.9 PSA molecules/mm.sup.2. This value is below the maximum amount of PSA that can be captured on the imprinted surface as determined by R.sub.max (Equation 1) and identified to occur at 9.8×10.sup.9 PSA molecules/mm.sup.2. Taking dimensions of PSA to be 4.4 nm×4.1 nm×5.1 nm (Table 3), and assuming ellipsoidal projection onto a plane, the ideal PSA surface coverage is approximated to be 1.4×10.sup.10 PSA molecules/mm.sup.2. These results established that the imprinted surfaces can attain high surface coverage (70%) by PSA, with the remaining OEG non-nanocavity areas on the surface providing the desired interprotein distance for efficient binding affinity and selectivity.

(48) $\begin{array}{cc}{R}_{\mathrm{eq}}=\left(\frac{C_p}{C_p+K_D}\right)R_{\max }& \mathrm{Equation}1\end{array}$

(49) where

(50) R.sub.eq is the SPR response at equilibrium

(51) C.sub.p is the concentration of injected protein

(52) K.sub.d is the dissociation constant for binding of the protein to the survace

(53) R.sub.max is the maximum response if all available binding sites are occupied

Example 4: RNAse B Sensor

(54) A sensor for the detection of RNAse B was fabricated using the method described in Example 2. Once formed, the ability of the sensor to bind proteins was investigated using SPR. It was observed that the response to RNase B (the template protein) was significantly higher than that to non-template proteins. Dissociation constants calculated from the SPR data for RNase B and various non-target proteins are given in Table 4 below. These data demonstrate that the molecularly imprinted surface was able to distinguish between the target protein and non-target proteins with a high degree of selectivity (10-200 fold selectivity to RNase B over other glycosylated and non-glycosylated proteins). Although RNAse B and lysozyme have not so dissimilar dimensions and isoelectric points (Table 3), the RNAse B-imprinted surface revealed a 8-fold enhanced selectivity for RNAse B over lysozyme, supporting the notion that BA carbohydrate receptors on the glycoprotein-imprinted surfaces contribute to the selectivity and affinity of the imprinted surface.

(55) TABLE-US-00004 TABLE 4 Affinity data for RNase B (target molecule) and other proteins Protein K.sub.d (μM) RNase B 3.1 ± 0.1 Lysozyme 24.3 ± 0.1  BSA 33.8 ± 0.6  HRP 119 ± 2  α1-AGP 201 ± 7  α1-AT 570 ± 50 

(56) One goal of this work was to develop a system which is able not only to distinguish between different proteins, but to be able to distinguish between different forms of the same protein, specifically the saccharide on the surfaces. To this end the fabrication process was repeated to create more RNase selective surfaces. Solutions of RNase A and RNase B were injected onto these surfaces, and the binding responses were monitored. It was observed that the RNase B solutions produced a higher response at the same concentrations than the RNase A. Furthermore, kinetic analysis demonstrated that the surfaces were able to demonstrate a higher affinity for the RNase B (Table 5), indicating that the stronger interactions are dictated by the presence of the glycan on RNAse B, and in turn its specific covalent bond formation with the spatially immobilised BA moieties on the surface. The weaker RNAse A interactions are considered to have arisen to some extent from Coulombic interactions between the known positively charged RNAse A domain along its longest axis and the negatively charged boronate ion species present in the imprinted surfaces.

(57) TABLE-US-00005 TABLE 5 Affinity data for binding of RNAse A and RNAse B to a molecular sensor produced using RNAse B as the template. Protein K.sub.d RNAse B 3.1 ± 0.1 μM RNase A 8.0 ± 0.1 μM

(58) Bare RNAse B-imprinted surfaces (absence of BA molecules in the nanocavities) exhibited lower affinity and rather poor specificity, capturing the glycoprotein template and its non-glycosylated form in a similar fashion. The bare RNAse B-imprinted surfaces resulted in about 7-fold reduced affinity to RNAse B compared with the BA-containing RNAse B-imprinted surface. These observations further highlight that the overall binding strength and selectivity of the imprinted surface towards the target glycoprotein arises from two distinct effects: shape matching and specific covalent interactions between the active tetrahedral boronate ion and the sugar residues in the glycoprotein.

(59) Sensitivity of the imprinted surfaces for the target glycoprotein in complex biological conditions such as serum was also investigated. Simultaneous adsorption of RNAse B (ranging from 0.01 mg/ml to 0.1 mg/ml) and 0.5% serum (i.e. 0.32 mg/ml) on RNAse B-imprinted surfaces was monitored by SPR. In order to eliminate the background signal, the RNAse-B imprinted surfaces were initially blocked with 0.5% serum, thereby allowing it to bind to all potential sites of non-specific interaction. The blocked RNAse-B imprinted surfaces were shown to provide highly sensitive detection for RNAse B at levels as low as 3% (w/w). The slightly reduced affinity of RNAse B towards the blocked RNAse-B imprinted surfaces (K.sub.d=6.5 □M±0.2) in comparison with bare RNAse-B imprinted surfaces (K.sub.d=3.1 □M±0.1) can be explained as a result of the blocking and elimination of the non-specific contribution to the overall binding affinity of RNAse B to the imprinted surface and/or serum competition for binding sites.

(60) Reusing a functionalised sensing surface is a highly desirable feature. For this reason, the performance of the RNAse B-imprinted surface in the serial capture and acidic regeneration of RNAse B was evaluated. The cycles were recorded by repeatedly injecting 0.85 □M solutions of RNAse B for 5 min, followed by PBS buffer for 10 min, regeneration with an acidic solution for 5 min and PBS buffer for 10 min. As illustrated in FIG. 4, the imprinted surfaces were shown to be remarkably stable for more than 10 cycles of binding and regeneration of the surface.

Example 5: Maltose Sensor

(61) A sensor for the detection of maltose was fabricated using the method described in Example 2 (save for the omission of the O-(2-Azidoethyl)heptaethylene glycol addition) with maltose as the template. FIG. 5 (panel A) shows SPR plots at varying concentrations for 3 disaccharides (palatinose, maltose and cellobiose) for a standard boronic acid terminated surface (i.e. not in accordance with the invention) and panel B shows SPR plots for the maltose sensor. Boronic acid preferentially binds fructose, hence the stronger affinity shown for palatinose (glucose-fructose, as opposed to cellobiose and maltose which have differently configured glucose-glucose structures) with the unimprinted surface (Panel A). When a maltose template is used and the boronic acids are fixed, the surface binds more strongly to maltose (Panel B). This result demonstrates that selectivity for a particular oligosaccharide can be created using the methods of the invention.