JP2005530176A - Coated metal surface on solid support for substitution reaction - Google Patents

Abstract

Coated metal surface on a solid support for substitution reaction A coated metal surface on a solid support, wherein the coating is a self-assembled thiol of an alkyl thiol containing an amide group having an oligo (ethylene glycol) end It consists of a monolayer (SAM) that is tightly attached to the metal surface via the end, and a low molecular weight antigen attached to the SAM-forming OEG molecule via an amide group, where the alkyl moiety is 1-20 methylene groups A coated metal, wherein the oligo (ethylene glycol) moiety has 1 to 15 ethyleneoxy units, and antigens, such as explosives and narcotics, reversibly bind to antibodies specific for the antigen, if desired Disclose the surface. An analytical device for detecting an analyte antigen in an aqueous solution having a higher affinity for the antibody than the antigen of the coating by monitoring the displacement of the antibody from the coating, the coated metal surface on the solid support; For example, it can be used in a method for detecting an analyte antigen as part of a piezoelectric crystal microbalance device or a surface plasmon resonance biosensor.

Description

  The present invention relates to a reversible binding on a solid support for a displacement reaction, particularly for detecting an analyte in an aqueous solution, by substituting an analyte-specific antibody from the metal surface coating. Relates to coated metal surfaces. Detecting the displacement of the analyte in the aqueous solution, and hence its presence, can be achieved by an analytical device, such as a Piezoelectric Crystal Microbalance (PCM) device or a Surface Plasmon Resonance (SPR) biosensor. And do it.

  SPR biosensors are sensitive real-time technologies that can be used to derive information about molecular interactions in the vicinity of specific metal surfaces. This technique allows measurement of concentration, association and dissociation rate constants and affinity, as well as epitope mapping and 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 ", KR Rogers and A. Muchandani (Eds.), Humana Press Inc., Totowa, NJ, pp. 31-53]. One of the components involved in this studied reaction is immobilized on the metal surface before or during the SPR experiment. The immobilized molecules can be exposed to a continuous stream and the interacting species can be injected therein. This method is based on optical detection and the sensing signal reflects a change in dielectric function or refractive index at the surface. These changes are caused by molecular interactions at the surface.

  The PCM technology is based on a vibrating piezoelectric crystal in a microbalance device, the crystal being made of, for example, quartz, aluminum nitride (AlN) or sodium potassium niobate (NKN). If the crystal is a quartz crystal, the device is called a QCM (quartz crystal microbalance). PCM and QCM are weight sensors and are therefore sensitive to mass changes. The QCM includes a piezoelectric quartz crystal plate on which metal electrodes are deposited on both sides. An alternating potential difference applied on such a crystal plate induces shear waves. The crystal resonates at a specific frequency such that the thickness is an odd integer of half wavelength [M. Rodahl, F. Hoeoek, A. Krozer, P. Brzezinski and B. Kasemo, Quartz crystal microbalance setup for frequency and Q- factor measurements in gaseous and liquid environments, Review of Scientific Instruments 66 (1995) pp. 3924-3930]. The most preferable half-wavelength in terms of energy is 1. The resonance frequency varies depending on the crystal thickness, but is usually in the MHz region. The resonance frequency changes due to the mass change on the surface of the plate. Since the frequency change of 0.01 Hz can be easily measured, the QCM becomes a sensitive sensor for measuring mass fluctuations. A number of patents and other documents describe the use of piezoelectric quartz crystals (QCM) as affinity-based chemical sensors / detectors, for example in various immunoassay techniques and in the detection of bacteria and viruses. In most of these applications, QCM instruments are used to analyze the weight gain of crystals after interaction pairs, eg, interaction between antibody and antigen.

  Analyzing small molecules with a conventional immunosensor presents obvious difficulties because of the slow response, i.e. the small change in weight of the sensor crystal. To perform the necessary detection of small molecules, the sensitivity of the instrument must be improved. In order to improve the detectability of small molecules, it is necessary to react small molecules with large molecules and specific interactions between small and large molecules. For example, a small antigen molecule is reacted with an antibody to specifically bind to the antibody to form an antigen-antibody complex that can be easily detected. When antigen derivatives having an affinity for antibodies smaller than the analyte antigen are immobilized on the surface, antibodies specific for these antigens can be reversibly bound to the immobilized antigen. Next, when an analyte antigen is present in the solution, the antibody is displaced from the immobilized antigen, and an antigen-antibody complex with the analyte antigen is formed. When the antibody has a marker, such as a fluorescent label, the complex formed can be detected using that marker. On the other hand, when the immobilized antigen is immobilized on the surface of the biosensor that is sensitive to mass change, the antibody is displaced from the surface, resulting in weight loss. Such substitution reactions are used in the present invention.

  Organic sulfur compounds, such as alkyl thiols, can be used to form well-ordered, closely packed SAMs on gold substrates. A strong chemical bond between sulfur and gold ties the molecule to the surface. After being pinned to the substrate (which occurs in a few seconds), the molecules begin to organize into a tightly packed structure due to van der Waals forces between the alkyl chains. This latter process is time consuming and can take hours or days to complete a well-ordered SAM. The length of the molecule used has a strong influence on the properties of the resulting SAM. All-trans SAMs are fully ordered and closely packed monolayers, whereas poor quality SAMs have defects such as complete or terminal Gauche (more or less spaghetti). Have. Molecules in the SAM exhibit a chain tilt of 25-40 ° due to a mismatch between the carbon chain pinning distance and the van der Waals diameter [B. Liedberg and JM Cooper, Bioanalytical applications of self- assembled monolayers in "Immobilized Biomolecules in Analysis: A Practical Approach", T. Cass and FS Liegler (Eds.), Biosensors, Oxford university press, Oxford, pp. 55-78]. The free end of the molecule can also be attached to the desired group or protein. In this way, surfaces with interesting and useful properties can be designed. Mixing different alkyl thiols further increases diversity. However, it should be noted that certain thiol mixtures in the loading solution do not necessarily form a SAM of the same mixture. Conversely, it is rarer to form because of the complex thermodynamic processes that occur during self-assembly. As far as we know, an antibody specific for an antigen binds reversibly to an immobilized antigen, dissociates in aqueous solution, and binds to an analyte antigen that has a higher affinity for the antibody than the immobilized antigen. For such a substitution reaction, a SAM coupled with a low molecular weight antigen has not been developed.

Detailed Description of the Invention

  The present invention provides an analyzer for detecting an analyte antigen in an aqueous solution by monitoring that the antibody reversibly bound to the antigen on the coating is displaced by dissociation and reaction with the analyte antigen. Useful coated metal surfaces on solid supports are provided.

  In the present description and claims, the term antibody comprises a whole antibody or a portion of an antibody that binds to an antigen or a molecule that binds to a synthesized antigen.

  Accordingly, one aspect of the invention relates to a coated metal surface on a solid support, the coating comprising a self-assembled monolayer (SAM) of an alkyl (OEG) thiol comprising an amide group having an oligo (ethylene glycol) terminus. ). The OEG thiol contains a SH group tightly attached to the metal surface, and a low molecular weight antigen introduced to the SAM-forming OEG thiol molecule via an amide group, the alkyl moiety has 1-20 methylenes, and the OEG moiety Has 1-15 ethyleneoxy units, and the antigen reversibly binds to an antibody specific for the antigen, if desired.

  In one embodiment of the invention, the oligo (ethylene glycol) has 4-6 ethyleneoxy units and the alkyl group has 15 methylene units.

  Low molecular weight antigens are conjugated synthetically to OEG molecules by reacting functional groups on the antigen with terminal functional groups of the OEG thiol prior to SAM formation. These functional groups may be carboxylic acid, amino and hydroxyl types. If the antigen does not have a functional group for the reaction, it may be necessary to introduce a functional group on the low molecular weight antigen prior to the reaction.

  The coated metal surface on the solid support of the invention is usually stored separately from the antibody specific for the antigen before use. However, when used for displacement analysis, the coated metal surface on the solid support comprises a specific antibody reversibly bound to the antigen of the coating.

The metal on the coated metal surface on the solid support of the present invention is preferably selected from the group consisting of gold, silver, aluminum, chromium and titanium. The presently preferred metal is gold.
The antigen of the coating is identical to or a derivative of the analyte antigen, except that it is immobilized through binding to the SAM. For example, the coated antigen can be derivatized to optimize the dissociation of the bound antibody in aqueous solution.

The antigens that are bound to the SAMs of the coating of the invention are the same or different, ie the antigens may bind to the same specific antibody or two or more types that bind to different specific antibodies. There is a mixture of bound antigens and can also detect the presence of several different analyte antigens in aqueous solution. If the antibody has a different marker, such as a fluorescent marker, substitution of the different antibody can be detected. However, a mixture of several different antibodies is usually used when screening the sample for any of the target antigens is sufficient, eg, screening the sample for either a narcotic or an explosive. To avoid interference between different antigens and antibodies with different affinities from each other, it would be necessary to introduce individual patches or microarrays of spots with different antigens on a solid support.
In a preferred embodiment of the invention, the antigen of the coating is selected from the group consisting of optionally derivatized explosives and narcotics. If binding of the selected coating antigen to the specific antibody is too strong to prevent dissociation of the antibody in aqueous solution, for example by denaturation of a functional group such as an ester or amino group (the original group Removing or replacing), or excluding part of the antigen molecule, or introducing new functional groups or side chains into the antigen molecule to chemically modify the antigen molecule and its affinity for the antibody Can be lowered.

  The explosive is preferably trinitrotoluene (TNT), dinitrotoluene (DNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro- Selected from the group consisting of 1,3,5,7-tetrazine (HMX), pentaerythritol tetranitrate (PETN), and nitroglycerin (NG), the narcotic is preferably cocaine, heroin, amphetamine, methamphetamine, cannabi Selected from the group consisting of diol, tetrahydrocannabinol (THC), and methylenedioxy-N-methylamphetamine (ecstasy).

  In a presently preferred embodiment, the solid support on the coated metal surface on the solid support of the present invention is a piezoelectric crystal electrode or a glass plate or prism, for example the coated metal surface on the piezoelectric quartz crystal electrode is a PCM device. Suitable for use, coated metal surfaces on glass plates or prisms are suitable for use in SPR devices.

  Another aspect of the invention is an analyte antigen having a higher affinity for a specific antibody than the antigen of the coating by monitoring the displacement of the coated metal surface on the solid support of the invention from the coating. Relates to the use as part of an analytical device for the detection in water solution.

  Still another embodiment of the present invention is a method for detecting an analyte antigen in an aqueous solution, and if necessary, a coated metal surface on the solid support of the present invention to which an antibody is not bound is used as an antigen. Activation by contacting a specific antibody with a coated metal surface in aqueous solution, binding of the antibody to the antigen of the coating, removal of excess antibody, affinity for the antibody is greater than that of the antigen of the coating. An aqueous solution that may contain high analyte antigens is contacted with an antibody reversibly bound to the coating, the antibody is dissociated and reacted with the analyte antigen, and the metal surface coated using an analytical device It relates to a method comprising detecting mass loss above.

In one embodiment of the method of the present invention, the analytical device is selected from the group consisting of a piezoelectric crystal microbalance device and a surface plasmon resonance biosensor.
In a presently preferred embodiment, the analytical device comprises a flow cell in which is coated a coated metal surface on the solid support of the present invention.
The invention will now be illustrated by the description of several drawings and experiments, but the invention is not limited to the details specifically described.

Experimental Description Contains two types of molecules (first repels proteins and second is a TNT analog), thereby obtaining a SAM containing various amounts of analogs that exhibit low levels of non-specific binding A mixed monolayer capable of being produced.

The first step was to evaluate the protein resistance properties of SAMs constructed from alkylthiols containing amide groups with oligo (ethylene glycol) (OEG) ends. The two types of molecules selected for this purpose are EG ₄ and EG ₆ (FIG. 4). Previous reports have shown the repellent properties of these molecules [P. Harder, M. Grunze, R. Dahint, GM Whitesides and PE Laibinis, Molecular conformation in oligo (ethylene glycol) -terminated self-assembled monolayers on gold and silver surfaces determine their abilitiy to resist protein adsorption, Journal of Physical Chemistry B, 102 (1998) pp. 426-436]. Pure EG ₄ and EG ₆ , respectively, and these two different compositions of SAM were tested by the use of several techniques: null ellipsometry, contact angle goniometry and infrared reflection absorption spectroscopy (IRAS).

  In addition, three TNT analog molecules (ANA1, ANA2 and ANA3), all containing 2,4-dinitrobenzene end groups (FIG. 5), individually and variously as suitable candidates in the above OEG composition Tested with proper mixing. These mixed SAMs were then tested by the same technique used for pure OEGSAM. In addition, SAMs made from 100% analogs (1, 2, and 3 individually) were tested with an X-ray photoelectron microscope (XPS).

  Using null ellipsometry and IRAS, the capacity to bind ABTNT was also measured at various mixing ratios for all TNT analogs. Moreover, the substitution strength of ABTNT in response to exposure to TNT was tested by SPR and QCM.

Compound EG ₄ and EG ₆ molecules were obtained the division of Applied Physics and Chemistry, Department of Physics and Measurement Technology, Linkoeping University, Ueden, from, ANA1, ANA2 and ANA3 is, the division of Chemistry, Department of Physics and Measurement Synthesized by Technology, Linkoeping University, Weden. ABTNT and TNT were obtained from Biosensor Application Aweden AB.

Sample Preparation Silicon wafers were cleaned in TL2 (MilliQ water: 25% hydrogen peroxide: 37% hydrogen chloride 6: 1: 1, 85 ° C. for 10 minutes), rinsed thoroughly in MilliQ water and dried in nitrogen gas After that, a coating of 5% titanium and 2000 gold was applied by electron beam evaporation. The equipment used is a Balzers UMS 500 P system. The evaporation rates were 1 Å / s and 10 Å / s for titanium and gold, respectively. A basic pressure of at least 10 ⁻⁹ was maintained and during evaporation the pressure was always on a low 10 ⁻⁷ scale. This type of surface was used for all experiments except SPR and QCM measurements. SPR surfaces (ordinary gold) were obtained from Biacore AB, Uppsala, Sweden, and gold coated QCM crystals were obtained from Biosensor Applications Sweden. The surface used for the SPR experiment had a surface roughness comparable to that used for the other experiments, whereas the surface coating of the QCM crystal was much rougher.

  Prior to exposure to the thiol loading solution, the sample surface was washed in TL1 (MilliQ water: 25% hydrogen peroxide: 30% ammonia 5: 1: 1 at 85 ° C. for 10 minutes) and rinsed thoroughly in MilliQ water. . The concentration of thiols in the loading solution based on 99.5% ethanol was 20 μM for pure thiol solutions as well as mixed thiol solutions. The surface was incubated for about 40 hours at room temperature. Samples were rinsed twice in 99.5% ethanol, then sonicated for 3 minutes (the gold coating on the QCM crystals was not able to withstand this step, so this treatment was omitted) and in 99.5% ethanol Rinse twice more. Unless otherwise indicated, these surfaces were stored in pure 99.5% ethanol for up to 8 hours before being dried in nitrogen gas and analyzed. A number of samples examined by IRAS and null ellipsometry were also incubated at room conditions for 30 minutes at an ABTNT concentration of 0.02 g / L, prepared in PBS (pH 7.4) and examined again. Samples were always handled with TL1 washed tweezers.

Surface plasmon resonance (SPR)
For the SPR experiment, two different instruments with temperature controlled flow cells, obtained from Biacore AB, were used. A series of experiments were performed on a BiacoreX instrument equipped with 2 flow channels. For these experiments, the flow rate was set at 10 μL / min and the sample surface was loaded by injection of 70 μL ABTNT (0.02 g / L). Subsequent injection of TNT solutions at concentrations of 100 pg / μL and 10 ng // μL into another flow channel caused the substitution to dissociate ABTNT from the surface.

  The second instrument used was a Biacore 2000 with 4 flow channels. The flow rate was 50 μL / min, and all injection volumes were 100 μL. The four flow channels always flowed continuously. As before, the injected ABTNT has a concentration of 0.02 g / L. TNT injection concentrations were 1, 10 and 100 pg / μL.

  In all cases, the flowed buffer was PBS (pH 7.4), and both ABTNT and TNT solutions were prepared in the same medium. The sample surface used for the SPR experiment was a glass plate coated with about 400 mm of gold, and the flow cell temperature was maintained at 25 ° C.

Quartz crystal microbalance (QCM)
QCM measurements were performed at room conditions with a slightly modified flow cell system V2B obtained from Biosensor Applications Sweden AB. The AT-cutQCM crystal used was a thickness shear mode type having a resonance frequency of 10 MHz. The thickness of the deposited titanium and gold layers was 250-300 mm and 400-450 mm, respectively. All parameters were the same as in the experiment with Biacore 2000, ie flow rate 50 μL / min, injection volume 100 μL, ABTNT concentration 0.02 g / L, TNT concentrations 1, 10 and 100 pg / μL, and flushed buffer PBS (pH 7.4) Set to. It should be noted that TNT implantation was performed sequentially in the same flow channel, that is, only 1 pg / μLTNT implantation was performed on the surface that was not actually exposed to TNT.
result

SAMs were produced on gold using the OEG molecules OEG molecules EG ₄ and EG ₆ . In addition to pure EG ₄ and EG ₆ SAM, two mixed SAMs containing both molecules were prepared and tested. The latter contains 75% and 50% EG ₄ with the remainder being EG ₆ . The mixed monolayer was represented by EG ₄ : EG ₆ 3: 1 and EG ₄ : EG ₆ 1: 1. The SAM properties of various compositions of EG ₄ and EG ₆ measured by null ellipsometry and contact angle goniometry are shown in Table 1. The self-assembling method shows good repeatability and the recent findings [R. Valiokas, M. Oestblom S. Svedhem, SCT Svensson, and B. Liedberg; Temperature-driven phase transitions in oligo (ethylene glycol) -terminated self- assembled monolayers, The Journal of Physical Chemistry B, 104 (32) (2000) pp.7565-7569, REF.]. The small angles obtained from contact angle goniometric measurements indicate that these SAMs are less hydrophobic, which is one of the many prerequisites for repelling properties. A slight increase in thickness is observed as the ratio of EG ₆ increases. In general, hydrophobic surfaces attract proteins and cells. Furthermore, low hysteresis values suggest a very homogeneous surface.

TNT analogs Prior to mixing TNT analogs with EG ₄ , the analogs were individually tested with SAM assembled from a loading solution containing pure ANA1, 2 and 3, respectively. The results of ellipsometry and contact angle measurement are shown in Table 2. As expected, the thickness of the analog exceeds the thickness of the OEG molecule. The small difference between the three types of analogs reflects the difference in molecular length, but recalls their chemical structure shown in FIG. The contact angle observed here is generally greater than the angle seen for OEG molecules. Also, the hysteresis between forward and reverse angles is large, representing a rougher surface. This can be expected considering that the relatively bulky dinitrobenzene end groups that could introduce defects into the manufactured SAM are pointing away from the surface.

The ability of ABTNT to bind immobilized ABTNT was evaluated at various different mixing ratios for various TNT analogs. Several techniques were used and they were in good agreement.

Two graphs shown in FIG. 6 is based on the IRAS and ellipsometry measurements, indicating the amount of immobilized ABTNT to various analogs and mixing ratio of these EG _4. For IRAS data, a portion of the amide I band is used as a measure of bound ABTNT and integrated between 1710 and 1665 cm ⁻¹ . Ellipsometry data shows the increase in film thickness after incubation in ABTNT. Here, EG ₄ shows the property to repel the protein. The amount of ABTNT fixed is virtually zero. Furthermore, antibody binding is very similar to the three SAMs, including large amounts of analogs. SAM assembled from a 1% analog solution generally exhibits a low degree of fixation.

Functionality testing The functionality of the three TNT analogs at their different mixing ratios was evaluated in two possible ways. The first is their ABTNT binding capacity and the second is the dissociation of ABTNT in response to TNT exposure. In both cases, two real-time technologies were used: SPR and QCM. In SPR measurements, an increase in response units (RU) corresponds to an increase in the amount of ABTNT bound to the surface, whereas in a QCM experiment, the frequency drop is equal to that. All three analogs have high potential, but ANA1 focused slightly on ANA1 because it performed slightly better than others. For all experiments in this section, running buffer PBS (pH 7.4), both ABTNT and TNT solutions were prepared in the same medium. The ABTNT concentration was always 0.02 g / L.
ABTNT coupling capacity

Two instruments were used for the SPR experiments: Biacore 2000 and Biacore X instruments obtained from Biacore AB. In the measurement performed with the Biacore 2000 apparatus, the flow rate was set at 50 μL / min, and 100 μL of ABTNT was injected and loaded on the sample surface. Figure 7 shows a ABTNT binding capacity of EG ₄ and ANA1, and mixtures thereof. Pure EG ₄ SAM shows very low adsorption, further supporting its protein repellent properties. The same correlation between different mixing ratios was also seen in experiments on a BiacoreX instrument performed at 10 μL / min and an ABTNT injection volume of 70 μL (data not shown).

QCM measurements were performed at room conditions with a slightly modified flow cell system V2B developed by Biosensor Applications Sweden AB. All parameters were set as in the Biacore 2000 experiment, ie, a flow rate of 50 μL / min and an ABTNT injection volume of 100 μL. The curve representing ABTNT binding capacity of EG ₄ and ANA1 and mixtures thereof is shown in FIG. Again, the low ABTNT binding capacity of EG ₄ is clearly demonstrated.

The binding of ABTNT to various SAMs is similar in SPR and QCM experiments. The three surfaces with the most TNT analogs all bind ABTNT very well to the surface and the dissociation of the antibody is very slow. Because of constant exposure to fresh buffer, some degree of ABTNT dissociation is expected, i.e., true equilibrium can never be reached.
ABTNT replacement

SPR curves (Biacore 2000, flow rate 50 μL / min) showing ABTNT desorption in response to TNT injections 1, 10 and 100 pg / μL for various mixing ratios of EG ₄ and ANA1 are shown in FIG. These surfaces were preloaded by injection of 100 μL ABTNT.

The appearance of the curve in FIG. 9 indicates that a SAM with a low ANA1 content has a weak ABTNT bond, and thus a substitution reaction is likely to occur. This may be a result of the antibody valence of 2 and their interaction with the surface. The higher the content of ANA1, the greater the chance that ABTNT will find and bind two (one for each epitope) TNT analog. If EG ₄ : ANA1 is 99: 1, this will not happen much simply because there is less ANA1. This feature is highly correlated because the binding strength of an antibody varies greatly depending on whether only one of its epitopes or both are bound to an antigen.

  10 and 11 show the corresponding results obtained from the QCM measurement (flow rate 50 μL / min, injection volume 100 μL). Mass loss caused by TNT injection is observed as an increase in resonance frequency. A differential df / dt of a frequency proportional to the density is also included, and usually provides a clearer detection signal.

FIG. 1 shows the chemical formulas for several narcotics, namely cocaine, heroin, amphetamine, ecstasy, methamphetamine, cannabinol and tetrahydrocannabinol (THC). FIG. 2 shows the chemical formulas for trinitrotoluene (TNT) and 2,4-dinitrotoluene (2,4-DNT). FIG. 3 schematically shows the displacement mechanism that takes place on the metal surface of a solid support, eg a QCM electrode. This figure is not full scale. In practice, antibodies are much larger than TNT molecules. The sensor surface shown in the figure is formed by self-assembly (SAM) technology. The derivatized TNT molecule, TNT analog, is covalently attached to the metal surface via SAM, and the ABTNT antibody specific for TNT and TNT analog is reversibly loose at first and becomes TNT analog. Once bound and exposed to TNT in solution, 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 structures of ANA1, ANA2 and ANA3. FIG. 6 shows the adsorption of antibodies to various TNT analogs at various mixing ratios with EG ₄ after incubation in ABTNT (0.02 g / L) for 30 minutes. Excellent agreement is observed between the two techniques: IRAS and null ellipsometry. Figure 7 is a real - the time art SPR (Biacore 2000), shows a ABTNT binding capacity observed for EG ₄ and ANA1, and mixtures thereof. The flow rate was set to 50 μL / min, and 100 μL of ABTNT (0.02 g / L) was injected. Note the low adsorption of EG ₄ on the SAM. Figure 8 is a real - the time art QCM (V2B), shows a ABTNT binding capacity observed for EG ₄ and ANA1, and mixtures thereof. The flow rate was set to 50 μL / min, and 100 μL of ABTNT (0.02 g / L) was injected. Note the low adsorption of EG ₄ on the SAM. FIG. 9 shows an experiment with a Biacore 2000 instrument showing SPR responses to TNT injections 1, 10 and 100 pg / μL against SAM of EG ₄ and ANA1, and mixtures thereof. The surface was preloaded by injecting 100 μL of ABTNT. The flow rate was set to 50 μL / min. FIG. 10 shows the QCM measurements made on the modified V2B flow cell mechanism. The flow rate was set to 50 μL / min and all TNT injection volumes were 100 μL. The surface was preloaded with ABTNT (0.02 g / L) by injecting 100 μL. The SAM was assembled from a loading solution containing 100% and 50% ANA1. The TNT concentrations in the injection were 1, 10 and 100 pg / μL, which were performed continuously, and the second and third TNT injections were performed on the surface previously exposed to TNT. Arrow indicates injection. FIG. 11 shows the QCM measurements made on the modified V2B flow cell mechanism. The flow rate was set to 50 μL / min and all TNT injection volumes were 100 μL. The surface was preloaded with ABTNT (0.02 g / L) by injecting 100 μL. The SAM was assembled from a loading solution containing 10% and 1% ANA1. The TNT concentrations in the injection were 1, 10 and 100 pg / μL, which were performed continuously, and the second and third TNT injections were performed on the surface previously exposed to TNT. Arrow indicates injection.

Claims (11)

A coated metal surface on a solid support,
A self-assembled alkylthiol containing an amide group having an oligo (ethylene glycol) terminus, the coating being tightly attached to the metal surface via the thiol terminus, and via the amide group A low molecular weight antigen bound to the SAM-forming OEG molecule,
The alkyl moiety has 1 to 20 methylene groups;
The oligo (ethylene glycol) moiety has 1 to 15 ethyleneoxy units;
A coated metal surface, wherein the antigen is optionally reversibly bound to an antibody specific for the antigen.   The coated metal surface on a solid support according to claim 1, wherein the metal is selected from the group consisting of gold, silver, aluminum, titanium and chromium.   The antigens are the same or different and consist of explosives and narcotics, optionally derivatized, bound to the same monolayer on the solid support or to different monolayers in a patch form Coated metal surface on a solid support according to claim 1 or 2, selected from the group.   The explosive is trinitrotoluene (TNT), dinitrotoluene (DNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1 Coated metal surface on a solid support according to claim 3, selected from the group consisting of 1,3,5,7-tetrazine (HMX), pentaerythritol tetranitrate (PETN), and nitroglycerin (NG). .   4. The solid of claim 3, wherein the narcotic is selected from the group consisting of cocaine, heroin, amphetamine, methamphetamine, cannabinol, tetrahydrocannabinol (THC), and methylenedioxy-N-methylamphetamine (ecstasy). Coated metal surface on support.   Coated metal surface on a solid support according to any one of the preceding claims, wherein the solid support is a piezoelectric crystal electrode or a glass plate or a prism. The oligo (ethylene glycol) has 4-6 ethyleneoxy units,
Coated metal surface on a solid support according to any one of the preceding claims, wherein the alkyl group has 15 methylene units. Use of a coated metal surface on a solid support according to any one of claims 1-7,
Use as part of an analytical device to detect in an aqueous solution an analyte antigen that has a higher affinity for the antibody than the antigen of the coating by monitoring the displacement of the antibody from the coating. A method for detecting an analyte antigen in an aqueous solution,
If necessary, an antibody is not bound to the coated metal surface on the solid support according to any one of claims 1 to 7, and the antigen-specific antibody is coated with the coated metal surface and an aqueous solution. Activating by contacting in,
Binding the antibody to the antigen of the coating;
Removing excess of the antibody;
In contact with the antibody reversibly bound to the coating, moving the aqueous solution that may contain the analyte antigen that has a higher affinity for the antibody than the antigen of the coating;
Dissociating the antibody and reacting with the analyte antigen, and using an analytical device to detect mass loss on the coated metal surface.   The method of claim 9, wherein the analytical device is selected from the group consisting of a piezoelectric crystal microbalance device and a surface plasmon resonance biosensor.   11. A method according to claim 9 or 10, wherein the analytical device comprises a flow cell in which a coated metal surface on a solid support according to any one of claims 1-7 is arranged.

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