Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
  • G01N33/553—Metal or metal coated
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2610/00—Assays involving self-assembled monolayers [SAMs]

Definitions

• the present invention relates to a coated metal surface on solid support for displacement reactions, especially for analyte detection in an aqueous solution by displacement from the metal surface coating of reversibly bound antibodies specific for the analyte. Detection of the displacement, and thus the presence of the analyte in an aqueous solution is performed with a analysis device, such as a Piezoelectric Crystal Microbalance (PCM)device or a Surface Plasmon Resonance (SPR) biosensor.
• PCM Piezoelectric Crystal Microbalance
• SPR Surface Plasmon Resonance
• the SPR biosensor is a sensitive real-time technique, which can be used to extract information about molecular interaction near certain metal surfaces. It offers the possibility to determine concentration, association and dissociation rate constants and affinity as well as epitope mapping and determination of interaction specificity [B. Liedberg and K. Johansen, Affinity biosensing based on surface plasmon detection in "Methods in Biotechnology, Vol. 7: Affinity Biosensors: Techniques and Protocols", K. R. Rogers and A. Muchandani (Eds.), Humana Press Inc., Totowa, NJ, pp. 31-53]. One of the components participating in the studied reaction is immobilized on the metal surface either before or during the SPR experiment.
• the immobilized molecule is exposed to a continuous flow into which one can inject interacting species.
• the method is based on optical detection and the sensing signal reflects changes in dielectric function or refractive index at the surface. These changes can be caused by molecular interaction at the surface.
• the PCM technique is based on an oscillating piezoelectric crystal in a microbalance device, wherein the crystal consists of e.g. quartz, aluminum nitride (A1N) or sodium potassium niobiates (NKN).
• the crystal is a quarts crystal, the device is referred to as a QCM (quartz crystal microbalance).
• QCM quartz crystal microbalance
• the PCM and QCM are gravimetrical sensors and are thus sensitive to mass changes.
• a QCM comprises a piezoelectric quartz crystal plate upon which metal electrodes have been deposited on both sides. An alternating potential difference applied on such a crystal plate induces shear waves. At certain frequencies - such that the thickness is an odd integer of half wavelengths - the crystal will be in resonance [M. Rodahl, F.
• small molecules are reacted with larger molecules by specific interaction between the small molecules and the larger ones.
• small antigen molecules are reacted with antibodies specifically binding to them to form antigen-antibody complexes that are easier to detect. If an antigen derivative with less affinity to the antibody than the analyte antigen is immobilized to a surface, antibodies specific for these antigens may be reversibly bound to the immobilized antigen.
• the antibody when the analyte antigen is present in a solution, the antibody will be displaced from the immobilized antigen and form an antigen-antibody complex with the analyte antigen.
• the antibody carries a marker, such as a fluorescent label, the formed complex can be detected with the aid of the marker.
• the immobilized antigen is immobilized on a surface of a biosensor sensitive for mass changes, then the displacement of the antibody from the surface will result in a weight loss. Such displacement reactions are used in the present invention.
• An organosulphur compound such as an alkyl thiol
• an alkyl thiol can be used to form a well- ordered and densely packed SAM on a gold substrate.
• the strong chemical bond between sulphur and gold couples the molecules to the surface. Once pinned to the substrate, which occurs within seconds, the molecules start to organize themselves into densely packed formations due to the van der Waal forces between the alkyl chains. The latter process is time consuming and it takes hours or even days before a well-ordered SAM is completed.
• the length of the molecules used has a strong influence on the properties of the obtained SAM.
• An all trans SAM is a perfectly ordered and densely packed monolayer, whereas a SAM of poorer quality possesses defects like complete or terminal gauche (more or less spaghettilike).
• the molecules in a SAM will display a chain tilt of 25-40° due to the mismatch between the pinning distance and size of the van der Waal diameter of the carbon chains [B. Liedberg and J. M. Cooper, Bioanalytical applications of self-assembled monolayers in "Immobilized Biomolecules in Analysis: A Practical Approach", T. Cass and F. S. Liegler (Eds.), Biosensors, Oxford university press, Oxford, pp. 55-78].
• the free end of the molecule can be linked to desired groups or even proteins. In this way it is possible to design surfaces with interesting and useful properties. Mixing different alkyl thiols further increases the versatility.
• the present invention provides a coated metal surface on a solid support that is useful in an analysis device for detection of an analyte antigen in an aqueous solution by monitoring displacement of an antibody reversibly bound to an antigen on the coating by dissociation and reaction with the analyte antigen.
• antibody is intended to comprise whole antibodies or antigen-binding parts of antibodies or synthetic antigen-binding molecules.
• one aspect of the invention is directed to a coated metal surface on a solid support, wherein the coating consists of a self-assembled monolayer (SAM) of oligo(ethylene glycol)-terminated amide group-containing alkyl (OEG) thiols.
• SAM self-assembled monolayer
• OEG oligo(ethylene glycol)-terminated amide group-containing alkyl
• the OEG thiols contain a SH group that is firmly attached to the metal surface and a low molecular weight antigen introduced via amide-groups to the SAM-forming OEG thiol molecule, wherein the alkyl portion has 1 to 20 methylenes, the OEG portion has 1 to 15 ethylene oxy units, and wherein the antigens are optionally reversibly bound to antibodies specific for the antigens.
• the oligo(ethylene glycol) has 4-6 ethylene oxy units and the alkyl group has 15 methylene units.
• the low molecular weight antigens are synthetically bound to the OEG molecules prior to SAM formation by reacting functional groups on the antigens with functional groups terminating the OEG thiol. These functional groups can be of the type- carboxylic acid, amino and hydroxyl groups. It may be necessary to introduce a functional group on the low molecular weight antigen prior to the reaction in case the antigen lacks functional groups for the reaction.
• the coated metal surface on a solid support according to the invention will usually be stored separately from the antigen-specific antibodies prior to use. When used in displacement analysis, the coated metal surface on a solid support will, however, comprise the specific antibodies reversibly bound to the antigens of the coating.
• the metal of the coated metal surface on a solid support according to the invention is preferably selected from e.g. the group consisting of gold, silver, aluminum, chromium and titanium. The presently preferred metal is gold.
• the antigen of the coating is the same as or a derivative of the analyte antigen except that it is immobilized through a bond to the SAM.
• the antigen of the coating may thus be derivatized to optimize dissociation of the bound antibody in an aqueous solution.
• the antigens bound to the SAM of the coating according to the invention are the same or different, i.e. the antigens may bind to the same specific antibodies or there may be a mixture of two or more bound antigens binding to different specific antibodies enabling the detection of the presence of several different analyte antigens in an aqueous solution.
• the antibodies carry different markers, such as fluorescent markers, it will be possible to detect displacement of the different antibodies.
• the antigen of the coating is selected from the group consisting of optionally derivatized explosives and narcotics.
• the antigen molecule may be chemically modified, e.g. by modification of functional groups such as ester or amino groups (by removal of, or replacing the original groups) or by eliminating a part of the antigen molecule, or introducing new functional groups or side chains to the antigen molecule, to reduce its affinity to the antibody.
• the explosives are preferably selected from the group consisting of trinitrotoluene (TNT), dinitrotoluene (DNT), hexahy ⁇ Yo-l,3,5-trinitro-l,3,5-triazine (RDX), octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazine (HMX), pentaerythritol tetranitrate (PETN), and nitroglycerine (NG), and the narcotics are preferably selected from the group consisting of cocaine, heroine, amphetamine, methamphetamine, cannabinols, tetrahydrocannabinols
• the solid support of the coated metal surface on a solid support according to the invention is a piezoelectric quarts crystal electrode or a glass plate or prism, and thus the coated metal surface on the piezoelectric crystal electrode is suitable for use in a PCM device and the coated metal surface on a glass plate or prism is suitable for use in a SPR apparatus.
• Another aspect of the invention is directed to the use of the coated metal surface on a solid support according to the invention as part of an analysis device for detection in an aqueous solution of analyte antigens with higher affinity to specific antibodies than the antigens of the coating by monitoring the displacement of the antibodies from the coating.
• Yet another aspect of the invention is directed to a method of detecting analyte antigens in an aqueous solution comprising activating, if necessary, the coated metal surface on a solid support according to the invention lacking bound antibodies by bringing antigen- specific antibodies into contact with the coated metal surface in an aqueous solution, allowing binding of the antibodies to the antigens of the coating, removing excess antibodies, bringing the aqueous solution possibly containing the analyte antigens that have higher affinity to the antibodies than the antigens of the coating into contact with the antibodies reversibly bound to the coating, allowing the antibodies to dissociate and react with the analyte antigens, and detecting the loss of mass on the coated metal surface by means of an analysis device.
• the analysis device comprises a flow cell in which the coated metal surface on a solid support according to the invention is placed.
• Fig.l shows the chemical formula of some narcotics, Cocaine, Heroine,
• Fig. 2 shows the chemical formula of trinitrotoluene (TNT) and 2,4- dinitrotoluene (2,4-DNT).
• Fig. 3 shows schematically the displacement mechanism taking place on the metal surface of a solid support, e.g. a QCM electrode. Note that the representation is not to scale. In reality an antibody is much larger than a TNT molecule.
• the sensor surface displayed is based on the self-assembly (SAM) technique.
• SAM self-assembly
• the derivatized TNT molecules, TNT-analogs are covalently bound to the metal surface via the SAM, the ABTNT antibodies specific for the TNT and TNT-analog is at first reversibly, loosely, bound to the TNT- analogs, and at exposure to TNT in solution, the ABTNT dissociates and forms a complex with TNT.
• Fig. 4 shows the chemical structures of EG 4 and EG 6 .
• Fig. 5 shows the chemical structure of ANA1, ANA2 and ANA3.
• Fig. 6 shows adsorption of antibodies, after 30min incubation in ABTNT (0.02g/L), to different TNT-analogues at different mixing ratios with EG . An excellent agreement is observed between the two techniques, IRAS and null ellipsometry.
• Fig. 7 shows the ABTNT-binding capacity observed for EG and ANA1, and mixtures thereof, by the real-time technique SPR (Biacore2000). The flow rate was set to
• Fig. 8 shows ABTNT-binding capacity observed for EG 4 and ANA1, and mixtures thereof, by the real-time technique QCM (V2B).
• the flow rate was set to 50 ⁇ L/min and a volume of lOO ⁇ L of ABTNT (0.02g/L) was injected. Note the low adsorption onto the SAM of EG 4 .
• Fig. 9 shows experiments on a Biacore2000 instrument showing SPR response to TNT injections of 1, 10 and lOOpg/ ⁇ L for SAMs of EG 4 and ANA1, and mixtures thereof.
• the surfaces had previously been loaded by injections of lOO ⁇ L ABTNT.
• the flow rate was set to 50 ⁇ L/min.
• Fig. 10 shows QCM measurements performed on a modified V2B flow cell system.
• the flow was set to 50 ⁇ L/min and all TNT injection volumes were lOO ⁇ L.
• the surfaces had previously been loaded with ABTNT (0.02g/L) by an injection of lOO ⁇ L.
• the SAMs were assembled from loading solutions containing 100% and 50% ANA
• the concentrations of TNT in the injections were 1, 10 and lOOpg/ ⁇ L and they were made in series, leaving the surface previously exposed to TNT for the second and third TNT injection.
• the arrows show the injections.
• Fig. 11 shows QCM measurements performed on a modified V2B flow cell system.
• the flow was set to 50 ⁇ L/min and all TNT injection volumes were lOO ⁇ L.
• the surfaces had previously been loaded with ABTNT (0.02g/L) by an injection of lOO ⁇ L.
• the SAMs were assembled from loading solutions containing 10% and 1% ANAL
• the concentrations of TNT in the injections were 1, 10 and lOOpg/ ⁇ L and they were made in series, leaving the surface previously exposed to TNT for the second and third TNT injection.
• the arrows show the injections. Description of experiments
• a mixed monolayer was produced that contained two kinds of molecules, the first being protein repellent and the second being a TNT-analogue, thereby making it possible to obtain SAMs containing a varying amount of analogue that displays low levels of non-specific binding.
• the initial step was to evaluate the protein resistant properties of a SAM constituted of oligo(ethylene glycol) (OEG)-terminated amide group-containing alkyl thiols.
• the two molecules chosen for this purpose were EG and EG 6 (Fig 4).
• Previous reports have shown repellent qualities for these molecules [P. Harder, M. Grunze, R. Dahint, G. M. Whitesides and P. E. Laibinis, Molecular conformation in oligo(ethylene glycol)-terminated self-assembled monolayers on gold and silver surfaces determines their ability to resist protein adsorption, Journal of Physical Chemistry B, 102 (1998) pp. 426-436].
• SAMs of pure EG and EG 6 , respectively, as well as different compositions of the two were characterized by use of several techniques, namely null ellipsometry, contact angle goniometry and infrared reflection absorption spectroscopy (IRAS).
• null ellipsometry contact angle goniometry
• IRAS infrared reflection absorption spectroscopy
• TNT-analogue molecules ANA1, ANA2 and ANA3 ( Figure 5), all containing a 2,4-dinitrobenzene end group, were examined separately and in different mixings with a suitable candidate among the OEG compositions mentioned above. These mixed SAMs were then characterized by the same techniques used for the pure OEG SAMs. In addition, SAMs made from 100% analogue - 1,2 and 3 separately - were examined with X-ray photoelectron spectroscopy (XPS).
• XPS X-ray photoelectron spectroscopy
• the EG 4 and EG 6 molecules were obtained from the divisions of Applied Physics and Chemistry, Department of Physics and Measurement Technology, Link ⁇ ping University, Sweden and the analogues ANA1, ANA2 and ANA3 were synthesized at the division of Chemistry, Department of Physics and Measurement Technology, Linkoping University, Sweden.
• ABTNT and TNT were from Biosensor Applications Sweden AB. Sample preparation
• Silicon wafers were cleaned in TL2 (MilliQ water:25% hydrogen peroxide:37% hydrogen chloride 6: 1 : 1 at 85°C for lOmin) and rinsed thoroughly in MilliQ water and dried in nitrogen gas prior to coating of 25 A of titanium and 2000A of gold by electron beam evaporation.
• the equipment used was a Balzers UMS 500 P system.
• the evaporation rate was lA/s and lOA/s for titanium and gold, respectively.
• a base pressure of at least 10 "9 was kept and during evaporation the pressure was noted to be on the low 10 "7 scale at all times. This type of surfaces was used for all experiments except SPR and QCM measurements.
• the SPR surfaces (plain gold) were obtained from Biacore AB, Uppsala, Sweden and the gold-coated QCM crystals were obtained from Biosensor Applications Sweden. It should be noted that the surfaces used for SPR experiments had a similar surface roughness compared to the ones used for the rest of the experiments, whereas the surface coating of the QCM crystals were of a much rougher nature.
• the surfaces were stored in pure 99.5% ethanol for a maximum of 8h before they were dried in nitrogen gas and analysed. A number of samples examined with IRAS and null ellipsometry were also subsequently incubated at room conditions for 30min in ABTNT at a concentration of 0.02g/L, prepared in PBS (pH7.4) and examined again. At all time the samples were handled with TL1 -cleaned forceps.
• SPR Surface plasmon resonance
• the second instrument used was a Biacore2000 equipped with four flow channels.
• the flow rate was 50 ⁇ L/min and all injection volumes were lOO ⁇ L.
• the four flow channels were run in series at all times.
• the injected ABTNT had the concentration 0.02g/L.
• the concentrations of the TNT injections were 1, 10 and lOOpg/ ⁇ L.
• the running buffer was PBS (pH7.4) and both ABTNT and TNT solutions were prepared in the same medium.
• the sample surfaces used for SPR experiment were glass plates coated with about 400 A gold and the flow cell temperature was kept at 25°C.
• the QCM measurements were performed at room conditions on a slightly modified flow cell system V2B from Biosensor Applications Sweden AB.
• the AT-cut QCM crystals used were a thickness shear mode type with a resonance frequency of 10MHz.
• the thickness of the deposited titanium and gold layer were 250-300A and 400-450A, respectively. All parameters were set as in the experiments with Biacore2000, i.e. flow rate 50 ⁇ L/min, injection volume lOO ⁇ L, ABTNT concentration 0.02g/L, TNT concentrations 1, 10 and lOOpg/ ⁇ L and running buffer PBS (pH7.4). It should be noted that the TNT injections were made one after another in the same flow channel, which means that only the lpg/ ⁇ L TNT injection was in fact performed on a TNT-non-exposed surface.
• the OEG molecules EG 4 and EG 6 were used to produce SAMs on gold. Besides pure EG 4 and EG 6 SAMs, two mixed SAMs containing both molecules, were prepared and examined. The latter ones were assembled from loading solutions containing 75% and 50% of EG and the rest EG 6 . The mixed monolayers were denoted EG :EG 6 3:1 and EG 4 :EG 6 1:1.
• the SAM characterization of different compositions of EG 4 and EG 6 with null ellipsometry and contact angle goniometry are given in Table 1. The self-assembly process displays good repeatability and the obtained results agree with recent findings [R. Valiokas, M. Ostblom, S. Svedhem, S. C. T.
• TNT- analogues were mixed with EG 4 , they were examined separately in SAMs assembled from loading solutions containing pure ANA1, 2 and 3, respectively.
• the results from ellipsometry and contact angle measurements are summarized in Table 2. As expected, the thickness of the analogues exceeds that of the OEG molecules.
• the two diagrams, illustrated in Figure 6, are based on IRAS and ellipsometric measurements and show the amount of immobilized ABTNT for the different analogues and their mixing ratios with EG 4 .
• IRAS data part of amide I band was used as a measure of bound ABTNT, integrating between 1710-1665cm _1 .
• the ellipsometric data shows the increase in film thickness after incubation in ABTNT.
• EG 4 shows its protein repellent properties.
• the amount of ABTNT immobilized is virtually zero.
• the binding of the antibody is quite similar for the three SAMs containing high amounts of analogue.
• the SAMs assembled from 1% analogue solutions generally displays a lower degree of immobilization. Functionality test
• TNT-analogues The functionality of the three TNT-analogues in their different mixing ratios has been evaluated with two aspects in mind. First, their ABTNT-binding capacity has been considered and second, the dissociation of ABTNT in response to TNT exposure. In both cases the two real-time techniques, SPR and QCM, have been employed. For the SPR measurements an increase in response units (RU) corresponds to an increased amount of bound ABTNT on the surface, while for QCM experiments a frequency drop is the equivalent. All three analogues possess a high potential, but focus has been on ANA1, since it displayed a slightly better performance than the others. For all experiments in this section the running buffer was PBS (pH7.4) and both ABTNT and TNT solutions were prepared in the same medium. The ABTNT concentration was always 0.02g/L. ABTNT-binding capacity
• ABTNT binding of ABTNT to the different SAMs is similar for SPR and QCM experiments.
• a certain release of ABTNT is expected due to the constant exposure to fresh buffer, i.e. a true equilibrium can never be reached.

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