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    • 专利摘要:
    Method to obtain atomic force microscopic sensing tips functionalized by activated steam silanization, and the tips obtained by said method. A method for obtaining a functionalized atomic force microscopy sensor tip, characterized in that the functionalization takes place by means of an activated steam silanization process comprising: a) evaporating an organometallic compound containing at least one silicon atom and at least one group functional selected from amino, hydroxyl, carboxyl and sulfhydryl; b) activating the vapor of the organometallic compound of step a) by heating; and c) causing the activated steam of step b) to impinge on a sensor tip for atomic force microscopy to deposit a sheet of the organometallic compound on said sensor tip; where steps b) and c) take place consecutively. As well as the hoisted functional sensing tip obtained by said method. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2684851A1
    申请号:ES201830777
    申请日:2018-07-27
    公开日:2018-10-04
    发明作者:Jose PEREZ RIGUEIRO；Gustavo Victor GUINEA TORTUERO；Rafael DAZA GARCIA；Luis COLCHERO PAETZ
    申请人:Universidad Politecnica de Madrid；
    IPC主号:
    专利说明:

    DESCRIPTION

    Method for obtaining functionalized atomic force microscopy sensor tips by activated steam silanization, and the tips obtained by said method.
     5 FIELD OF THE INVENTION

    The invention is framed within the area of near-field microscopes and, in particular, of atomic force microscopy and of the instrumentation required in this area, by obtaining a functionalized atomic force microscopy sensor tip 10 by steam silanization activated (activated steam silanization, AVS).

    The AVS functionalized tip can be useful for the use of atomic force microscopy as a microstructural characterization technique in Materials Science or Biology, among other applications. fifteen
    STATE OF THE TECHNIQUE

    Atomic force microscopy (MFA) is an extremely versatile microstructural characterization technique that allows the study of a wide range of systems in various environmental conditions. Atomic force microscopy is framed within the group of near-field microscopes in which the sensor element is located at a very short distance from the surface to be observed. In the case of atomic force microscopy, the sensor element is a cantilever, generally equipped with a tip of micrometric dimensions at its end. The atomic force microscope has a positioning system in the three directions of space: X, Y and Z, which allows varying the distance between the tip and the surface of the sample (conventionally identified with the Z direction) at the same time as sweep the surface of the sample (conventionally identified by the X and Y directions). The interactions established between the tip and the surface of the sample flect the cantilever, being able to establish a relationship between the bending of the cantilever and the force to which the tip is subjected due to its interaction with the surface at a given time. The scanning along the X and Y directions, carried out by moving the cantilever along the Z axis so that a constant value of the bending of the cantilever is maintained, allows the isoform lines to be obtained on the surface. When the dominant interaction is the contact interaction due to steric repulsion between the
    tip and surface of the material it is considered that the isofforce lines obtained correspond to the surface topography of the sample.

    The very general principles on which atomic force microscopy is based allows it to be highly versatile compared to other near-field microscopic techniques, optical microscopy and electron microscopy. In particular, the MFA allows working under a wide variety of environmental conditions, including vacuum, air and various liquid media. In particular, the possibility of working in liquid media is a unique opportunity to observe biological systems in in vivo conditions, which are often outside the possibilities of most other microscopic techniques. 10 Additionally, the dependence of the sensor system on the interactions established between the tip and the sample offers a wide range of elements that can be characterized in said sample if these interactions are controlled.

    The combination of the ability to characterize biological systems in vivo within a suitable liquid medium, and the possibility of exploring said biological systems using specific probes, for example, antibodies that recognize a particular biomolecule, has led to the development of the so-called microscopy of chemical atomic force (C-MFA) or affinity. Taking into account the previous discussion, it is found that the critical element to be able to characterize a sample according to the principles of the C-MFA is the availability of MFA tips that are capable of establishing specific interactions with the various elements that may be present in the Sample to analyze. Obtaining MFA tips that can be used for C-MFA conventionally requires two stages: (1) a modification of the tip surface (functionalization) so that functional groups are generated on its surface, and (2) a Stable bonding (in general, through a covalent bond) between the functional groups of the tip surface and the molecule that establishes a specific interaction with certain surface elements. Experience shows that the first stage, consisting of the modification of the tip surface to generate functional groups therein, turns out to be the most complicated from the point of view of the development of viable technologies.

    The strategies commonly used for the functionalization of the MFA tips are based on two alternative procedures (R. Barattin and N. Voyer, Chemical modifications of AFM tips for the study of molecular recognition events, Chem. Commun. 13 (2008), 1513 -35
    1532). On the one hand, a procedure based on the deposition of a thin sheet of gold or the like has been described, so that the posterior union of the recognition molecule takes advantage of some specific interaction between the deposited sheet and some region of the molecule itself. The characteristic example of this procedure is the fixation of molecules that contain a thiol group (also called sulfidryl group, -SH) on a thin gold foil, which takes advantage of the high affinity of the thiol group for the metal surface. An alternative procedure is the use of an organometallic molecule capable of binding to functional groups present spontaneously on the surface of the tip. The typical example of this procedure is the binding of 3-aminopropyltriethoxysilane (APTES) molecules to hydroxyl groups (-OH) present on the surface of the tip or that is generated by exposing the tip to an oxidizing environment.

    The technological development of both alternative procedures is developed in a series of patent documents. Thus, within the technologies corresponding to the first group is the patent application US2007 / 0082352 A1 (Peter Jonathan Cumpson, 15 Microscopy tip) in which a procedure for joining deoxyribonucleic acid (DNA) molecules to MFA tips is indicated. In the described procedure, the DNA molecules are initially modified by binding with an organic molecule that contains a thiol group (-SH). The MFA tip on which it is desired to immobilize the DNA is modified by depositing a thin sheet of gold on it. The fixation of the DNA molecules 20 on the tip is the result of the affinity shown by thiol groups for gold in the metallic phase.

    Among the documents describing technologies based on the second type of procedures is patent application WO2007 / 109689 A2 (Gallardo-Moreno 25 et al., A method for functionalizing an atomic force microscopy tip) in which a coating is deposited of polylysine on an unmodified tip. In this case, the functionalization is achieved through an unspecific interaction that is established between the polylysine molecules, which are positively charged, and the surface of the unmodified tip. Alternatively, a valid procedure for silicon nitride or silicon oxide 30 tips has been proposed, so that an appreciable number of hydroxyl groups are first generated on the surface of the tip, subsequently joining said hydroxyl groups dendrimer type molecules. Said procedure is described in patent application FR2965624 A1 (Dague Etienne, et al., Modified atomic force microscope tip comprises surface covalently grafted by phosphor dendrimers and having at their periphery, 35
    several terminal functions allowing covalent fixing of dendrimers on surface and biomolecules on the dendrimers). The procedure described in patent application KR1020150071876 A (Shim Bong Chushim et al., Method for analyzing nucleic acid sequence using atomic force microscope) is also based on the initial generation of an appreciable density of hydroxyl groups at the MFA tip. The hydroxyl groups are generated by exposure of the tip to a 20% nitric acid solution, subsequently forming a monolayer of APTES molecules on the surface as a result of the reaction of the organometallic molecules with the hydroxyl groups previously generated on the surface . The binding of proteins and / or nucleic acids to the tip was carried out either directly through the amino groups present in the APTES molecule or through a dendrimer molecule that was located between the tip and the biomolecule (protein and / or acid nucleic).

    Among the proposed procedures for the functionalization of MFA tips can also be mentioned patent application WO2012 / 084994 A1 (Polesel-Maris, Jérôme, et 15 al., Atomic force microscope probe, method for preparing same, and uses thereof) which Its main characteristic is that it represents a procedure that shares characteristics of the two families of basic procedures described above. In this case the original tip is modified to expose a graphite surface on it. Subsequently, the graphite surface is chemically modified to generate -OH 20 groups on the surface and, in a final stage, said groups react with various organic molecules to expose different functional groups on the surface capable of covalently binding biomolecules.

    Thus, one of the major drawbacks of the tip functionalization procedures 25 proposed in previous studies is the need to develop a prior surface activation procedure of the material used to manufacture the MFA tip, this prior procedure being different depending on the Type of material used. In addition, the functionalization procedure must be compatible with the biomolecule that is subsequently immobilized on the MFA tip. Consequently, there is a technological need to develop new versatile procedures for the functionalization of MFA tips, which are as general as possible, both in terms of the materials of the tips that can be functionalized and of the biomolecules with which said procedures of Functionalization be compatible.
     35
    In this context, the present invention provides a chemical atomic force microscopy (C-MFA) sensor tip, where functionalization takes place using the activated steam silanization technique (SVA or by its initials in English, AVS, Activated Vapor Silanization ). This technology has already been described previously (RJ Martín-Palma et al., Surface biofunctionalization of materials by amine groups, J. Mater. Res. 19 (2004), 2415-5 2420), in applications focused primarily on the field of Biomaterials and materials for medical use (P. Rezvanian et al., Enhanced biological response of AVS-functionalized Ti-6Al-4V alloy through covalent immobilization of collagen, Scientific Reports 8 (2018), 3337), whose general characteristic is its use in samples with a basically flat surface topography. 10

    SVA technology has demonstrated its ability to deposit a functionalized thin sheet on flat substrates to which it is possible to covalently bind various biomolecules, such as extracellular matrix proteins such as collagen or fibronectin. However, this procedure describes the procedure where the SVA technique is used to efficiently functionalize MFA tips, with a much steeper topography than the flat substrates where the SVA technology has been applied so far, of so that these tips can act as sensing elements in chemical atomic force (or affinity) microscopy procedures.
     twenty BRIEF DESCRIPTION OF THE INVENTION

    The present invention is based on work carried out in the field of thin film deposition and in the field of atomic force microscopy. The inventors have found that the activated steam silanization technique allows to obtain functionalized atomic force microscopy tips, which exhibit a high density of amino (-NH2), carboxyl (-COOH), sulfydryl (-SH) and / or groups hydroxyl (-OH) on its surface. The procedure for the production of functionalized MFA tips using the SVA technique is presented and described below.
     30
    Thus, the present invention provides a method for obtaining a functionalized atomic force microscopy (MFA) sensor tip, characterized in that the functionalization takes place by means of an activated steam silanization process comprising:
    a) evaporate an organometallic compound containing at least one silicon atom and at
    less a functional group selected from the group consisting of amino (-NH2), carboxyl (-COOH), sulfydryl (-SH), hydroxyl (-OH) and a combination of the foregoing;
    b) activating the vapor of the organometallic compound of step a) by heating at a temperature between 400 and 1000 ° C; Y
    c) having the activated vapor of step b) influence a sensor tip for atomic force microscopy 5 to deposit the organometallic compound on said sensor tip;
    where stages b) and c) take place consecutively.

    Obtaining functionalized tips requires in the first stage of evaporation 10 of an organometallic, whose molecule contains at least one silicon atom and at least one amino group (-NH2), carboxyl (-COOH), sulfydryl (-SH) , hydroxyl (-OH) or a combination of the above. Subsequently, the steam is subjected to a heating stage and then the MFA tip to be functionalized is pressed. In order to avoid degradation of the activated organometallic compound, steps b) and c) follow one another without interruption, so that the activation step takes place immediately before the activated vapor has an impact on the MFA tip.

    In the method described in this document, the evaporation and activation steps can take place in different regions of the same equipment, or even of different equipment 20 of the same installation. If so, the steam obtained in the evaporation region is transported hot, preferably at a temperature higher than the evaporation temperature of the organometallic compound in question, to the region where the activation stage b) will take place.
     25
    The present invention also relates to the functionalized MFA tips obtained by the method described herein. These tips are characterized by the base material of the tip, a thickness of the functional sheet preferably located in a range between 50 nm and 1 µm; and a high density of amino, carboxyl, sulfydryl and / or hydroxyl surface groups. In particular, in the case of using an organometallic with 30 amino groups, values close to eight amino groups per nm2 can be reached, corresponding approximately to the theoretical surface density of a monolayer of amino groups on a flat surface. Such density can be measured, based on its ability to covalently bind fluorescent markers.
     35
    Thus, unlike other procedures for functionalizing MFA tips, such as the one described in patent application KR1020150071876 A1, where a monolayer of organometallic molecules is created attached to the hydroxyl groups generated on the surface of the tip material, in The method of the present invention creates a thin sheet, preferably between 50 nm and 1 µm, formed by the decomposition of the 5 organometallic molecules, the formation of this sheet being independent of the surface chemistry of the tip material to be functionalized. .

    DETAILED DESCRIPTION OF THE INVENTION
     10
    The present invention provides a method for obtaining a functionalized atomic force microscopy (MFA) sensor tip, characterized in that the functionalization takes place by means of an activated steam silanization process comprising:
    a) evaporate an organometallic compound containing at least one silicon atom and at least one functional group selected from the group consisting of amino (-NH2), carboxyl (-COOH), sulfydryl (-SH), hydroxyl (-OH) and a combination of the above;
    b) activating the vapor of the organometallic compound of step a) by heating at a temperature between 400 and 1000 ° C; Y
    c) causing the activated vapor of step b) to influence a sensor tip for atomic force microscopy 20 to deposit the organometallic compound on said sensor tip;
    where stages b) and c) take place consecutively.

    The functionalization method described here is based on MFA sensor tips, generally joined in a larger structure called a chip (see figure 1), and includes the functionalization of these tips when they come into contact with the activated vapor of organometallic molecules. The interaction between the tip of MFA and the activated vapor leads to the formation of a sheet on it so that fragments of the organometallic molecule remain active and exposed to the outside environment. Preferably, the thickness of the sheet is between 50 nm and 1 µm, since thicknesses greater than 1 µm could lead to delamination of the sheet deposited on the MFA tip.

    The MFA tips that have active organic fragments on their surface are called functionalized and their main property is that the presence of these
    Active organic fragments modify the interaction of the tip with the medium. The use of functionalized MFA tips modifies the sensing capacity of the MFA technique and the range of measurements that can be obtained with this technique.

    Without being bound to any theory, the inventors consider that the presence of the functionalized sheet 5, preferably with a thickness between 50 nm and 1 µm, is the result of the partial decomposition of the organometallic molecule as a consequence of its thermal activation. The activated molecules that affect the surface interact with it and between them resulting in a solid sheet on the surface, but in which some of the organic fragments of the original organometallic molecule are maintained, 10 keeping said fragments their activity and being exposed to outside of the tip.

    The sensor element in atomic force microscopy is a tip that is located in the vicinity of the surface to be studied and that appears at the end of a cantilever that flecks as a result of the interaction between the tip and the surface. The cantilever, in turn, is located at the end of a more massive structure, generally with a parallelepiped geometry conventionally called chip (see figure 1). The typical dimensions of the chips at commercial points are in the millimeter range, the typical sizes of the overhangs being of the order of tens of microns in the directions perpendicular to that defined by the tip, and of a few microns in the latter direction. Finally, the tip size 20 can vary from tens of nanometers to a tens of microns. There is no limitation, in principle, on the composition of the MFA tips as long as they are compatible with the manufacture of elements with the geometry indicated above. In practice, most commercial MFA tips are formed from silicon (Si) or silicon nitride (Si3N4). The functionalization process of the present invention does not imply any restriction on the geometry of the chips, nor on their composition as long as their size allows the activation of the sensor tip in the activation region.

    On the other hand, the organometallic used in the method of the present invention is formed by molecules that have a common structure in which a silicon atom is attached to one or several hydrocarbon chains, where at least one of these chains comprises one or various amino (-NH2), hydroxyl (-OH), carboxyl (-COOH) or sulfydryl (-SH) groups.

    In preferred embodiments of the present invention, the organometallic compound comprises one or more hydrocarbon chains - (CH2) n-, where n is a number between 1 and 30, preferably between 1 and 6; and at least one functional group selected from the group consisting of hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), amino (-NH2) and a combination of the foregoing. 5

    The hydrocarbon chains of the organometallic compound that is used in the method of the present invention may comprise one or more double or triple bonds between carbon atoms.
     10
    As examples of molecules that can be used as an organometallic compound for the activated steam silanization process described herein are 3-aminopropyltriethoxysilane (APTES) and aminopropyltrimethoxysilane (APTMS), both conducive to the formation of sheets containing groups Not me; mercaptopropylmethoxysilane (MPTMS), with which sheets containing 15 sulphydryl groups are produced; triethoxysilylpropylmalenamic acid, with which sheets containing carboxyl groups are produced; and N-triethoxysilylpropyl-O-polyethylene oxide, with which sheets containing hydroxyl groups are produced. Together with the structure of the molecule itself, an important characteristic of the organometallic is its boiling point. In particular, it is preferred that the boiling temperature of this compound be between 100 ° C and 250 ° C.

    The evaporation stage a) can be carried out by depositing an organometallic fluid in an evaporation chamber located inside an evaporation oven, so that the temperature of the organometallic fluid can be varied. Increasing the temperature of the evaporation chamber above the boiling point of the organometallic fluid leads to the transition of the liquid-vapor phase with the formation of organometallic vapor within the evaporation chamber. Preferably, the range of temperatures at which the evaporation oven is heated is between 50 ° C and 400 ° C, it being even more preferred that this temperature is between 100 ° C and 250 ° C and, 30 being especially preferred than the temperature in the stage a) evaporation is between 130 ° C and 200 ° C. The range of evaporation temperatures will depend on the particular organometallic, with the maximum limit of the temperature of said range being the one in which the organometallic molecule decomposes.
     35
    Preferably, the activation stage b) can take place by heating at a temperature between 400 and 900 ° C, more preferably between 400 and 800 ° C, since too high temperatures favor the appearance of irregularities and inhomogeneities in the deposited sheets.
     5
    The stage of activation of the organometallic vapor can be carried out in an installation comprising a deposition chamber where the MFA tips to be functionalized are located, and another region, preferably in the form of a tube, immediately prior to the deposition chamber and that connects directly with her. According to these embodiments, the activation region corresponds to the region of the tube immediately prior to the deposition chamber and which connects directly with it. Additionally, an activation furnace is located around the activation region, defining its extent and allowing a controlled increase in temperature in said activation region. Thus, in the activation region, the evaporated organometallic vapor in the evaporation chamber crosses a region of elevated temperature before entering the deposition chamber and influencing the MFA tips.

    The efficiency of the method improves when performed under vacuum, avoiding the reaction of the organometallic molecule with atmospheric gases, mainly oxygen. The need to heat the organometallic during the evaporation stage and, later, during the activation stage creates a favorable situation for the oxidation of the organometallic with atmospheric oxygen. Said reaction can decompose the organometallic, preventing the functionalization of the substrate. Thus, carrying out the method, in particular steps a), b) and c) described above, in a vacuum system that allows a residual vacuum between 10-4 and 10-1 mbar is advantageous to avoid the decomposition mentioned above. Said vacuum can be achieved with a rotary pump coupled to a cold trap.

    In the method described in this document, the evaporation and activation stages can take place in different regions of the same installation and, in particular, in different chambers, of the same equipment or even of different equipment. If so, the vapor obtained in the evaporation region is transported hot, preferably at a temperature higher than the evaporation temperature of the organometallic compound in question, to the region where the activation stage will take place.

    In those embodiments of the method of the invention where one or more of the stages of evaporation, activation or deposition of the organometallic on the MFA sensor tip to be functionalized take place in different regions, the use of a carrier gas that facilitates the transit of steam is preferred. of organometallic from one region to another, in particular, from the evaporation chamber to the activation region and, finally, to the deposition chamber, where the activated steam can affect the MFA tip. This carrier gas must be an inert gas with respect to the organometallic gas, so a possible choice is a noble gas such as argon, although the use of molecular nitrogen or carbon dioxide could eventually be considered. The introduction of the carrier gas into the system leads to an increase in the pressure in the system, the pressure range of 10 being found as a result of the introduction of the carrier gas preferably between 10-2 and 100 mbar, more preferably between 5 × 10 -1 and 10 mbar.

    Preferably, in the method for obtaining a functionalized MFA sensor tip described herein, the period of incidence of activated organometallic vapor 15 on the MFA tip is between 1 and 120 minutes.

    As mentioned above, there may be a separation between the evaporation chamber and the activation region, both zones generally joined by a connecting tube. This separation involves the transit of steam along an extension of connecting tube 20 before entering the activation region. To avoid condensation of organometallic vapor during such transit, it is convenient to surround the connection tubes between the evaporation chamber and the activation region with a heating element, which may be a heating tape. Said heating tape must maintain the temperature of the connections at a temperature equal to or greater than the evaporation temperature of step a) of the method of the invention, in order to avoid condensation of organometallic vapor before it reaches the region of activation.

    The deposition chamber may comprise a support for the MFA tips, which allows defining the position of the tips in the deposition chamber and keeping them immobile during the entire process. In particular embodiments of the invention, the geometry of the tip is defined by the distance of the tip with respect to the tube outlet from the activation region (D2; Figure 3) and by the angle formed by the tip with respect to the vertical to said outlet in the direction of the steam flow (α; Figure 3). In particular, in the method described herein, the tips to be functionalized are located so that, in the
    step c), the activated organometallic vapor affects the tips to be functionalized with an angle between 0º and 60º. In particular, when the method takes place in the equipment described in this document (see figure 2), the angle α mentioned above corresponds to the angle of the sensor tip to be functionalized with the opening of the activation region in the chamber of deposition, orifice through which 5 activated organometallic vapor is introduced into the deposition chamber. In this way a compromise is achieved between the flow density (number of molecules / area / time) and the covered area, taking into account that the vapor flow takes an approximate cone shape when entering the deposition chamber.
     10
    Additionally, it is possible to perform the MFA tip functionalization in two alternative orientations: tip support cantilever oriented perpendicularly to the direction of the steam flow (Figure 4A), or tip support cantilever oriented parallel to the direction of the steam flow (figure 4B). With this last orientation, the organometallic mass deposited in the strip and in the rest of the chip is reduced. In particular, the decrease in the organometallic mass deposited in the strip implies a decrease in the offset of the main resonance frequency of the strip, with respect to the value measured before functionalization.

    Without being bound by any theory, it is believed that the functionalization of the MFA tips in 20 particular embodiments of the present invention is the result of the following processes: Formation of the organometallic vapor in the evaporation chamber, entrainment of the organometallic vapor from the evaporation chamber to the activation region, heating of the organometallic molecules within the activation region, incidence of the molecules thus activated to the surface of the MFA tip resulting in the formation of a sheet, preferably with a thickness between 50 nm and 1 m, on the tip. The activation process is sufficient to induce the interaction of the organometallic molecules with each other and with the tip material so that a sheet is deposited on the surface of the MFA tip material, while maintaining part of the organic groups of the molecule before treatment. 30

    The present invention also relates to functionalized MFA sensor tips such as having a sheet deposited on its surface, and said sheet comprises reactive groups selected from the group consisting of amino (-NH2), carboxyl (-COOH), sulfydryl (- SH), hydroxyl (-OH) and a combination of the above. These tips are 35
    obtained by the method described in this document and are characterized in that the functionalization process results in the deposition of a functional sheet, preferably with a thickness in a range between 50 nm and 1 µm. In particular, the tips functionalized by the method of the present invention may have a density of surface amino groups that can reach values close to eight amino groups 5 per nm2, based on their ability to covalently attach fluorescent labels.

    Thus, the functionalized sensor tips of the present invention may have on its surface functionalized sheets, such that said thin sheets have a thickness within the range between 50 nm and 1 µm, and contain reactive groups such as 10 amino (-NH2 ), hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH) or combination of the above, so that these functional groups are exposed to the outside. Said reactive groups may or may not be linked to hydrocarbon chains - (CH2) n-, where n is a number between 1 and 30.
     fifteen
    Additionally, the present invention relates to the use of the functionalized sensor tips described herein in atomic force microscopy.

    The method of obtaining a functionalized MFA tip described in this document can be carried out in a device comprising:
    - An evaporation chamber,
    - An evaporation oven configured to heat the evaporation chamber,
    - An activation region, preferably in the form of a tube, connected to the evaporation chamber,
    - An activation oven configured to heat the activation region, and 25
    - A deposition chamber connected after the activation region.

    In particular embodiments, the activation region has a length in the range between 100 mm and 300 mm, which allows to reach the desired activation temperature in the organometallic vapor at higher working pressures or, equivalently, at greater 30 flow rates. steam.

    In other particular embodiments of the invention, the activation region has a mouth opening to the deposition chamber, and the tip to be functionalized is located at a distance between 1 mm and 50 mm with respect to said hole. As a consequence of dispersion 35
    of the organometallic vapor flow when entering the deposition chamber, this range of distance values results from a compromise between sufficiently high vapor flow density values within the deposition chamber and a sufficiently wide incidence surface that allows the homogenous functionalization of samples with a size of the order of cm. 5

    Preferably, the angle that forms the imaginary line that joins the tip with the opening of the activation region in the deposition chamber with the direction of flow of the organometallic vapor is in the range between 0 ° and 60 °. As in the previous section, this range of angle values results from the compromise between the flow density of the organometallic vapor 10 within the deposition chamber and the area of incidence swept by said flow.

    In particular embodiments of the invention, the support overhang of the MFA tip is oriented perpendicularly to the direction of the flow of organometallic vapor. Alternatively, the MFA tip support overhang is oriented parallel to the direction of the organometallic vapor flow.
    BRIEF DESCRIPTION OF THE FIGURES
     twenty
    To improve the description of the invention it is useful to refer to the Figures included in the document. It should be emphasized that, following common practice, the drawings and schematics of the figures are not to scale. On the contrary, the dimensions of the different elements have been increased or reduced arbitrarily with the sole intention of facilitating the understanding of the indicated details. The figures included in the patent application document 25 are:

    Figure 1. Diagram of the cross section of the functionalized tip. (A) Cross section of a conventional MFA tip with indication of the fundamental elements: (1) Chip, (2) Cantilever and (3) Tip (Tip). (B) Flat view of a conventional 30 MFA tip with indication of the fundamental elements. (C) Cross section of the tip / cantilever functionalized by AVS, showing the functionalized sheet (4). The presence of surface reactive groups is indicated, which in this case are particularized to amino groups (NH2).
     35
    Figure 2. Schematic of the essential elements of an equipment used for the manufacture of functionalized MFA tips by preferred embodiments of the method of the present invention. (1) Carrier gas inlet. (2) Evaporation chamber. (3) Evaporation oven (4) Organometallic compound. (5) Activation oven. (6) Activation region. (7) Deposition chamber. (8) Exit to the vacuum system. 5

    Figure 3. Detail of the activation furnace and the deposition chamber with the definition of the main geometric parameters. D1: Length of the activation furnace, D2, distance between the output of the activation furnace and the MFA tip, and α, angle that forms the line that joins the output of the activation furnace and the MFA tip with the axis of the activation region 10

    Figure 4. Scheme of two possible orientations of the tip with respect to the steam flow. (A) Tip support overhang oriented perpendicular to the direction of steam flow. (B) Tip support overhang oriented parallel to the direction of steam flow. The direction of the activated steam flow at the inlet of the activation chamber is indicated by an arrow.

    Figure 5. The existence of the functionalized thin sheet on the surface of the cantilever and the tip is checked by the use of a fluorescent molecule (fluorescein isothiocyanate) that reacts covalently with amino groups. (A) Shows control over which the functionalized thin sheet has not been deposited. (B) Cantilever system / functionalized tip following the deposition conditions of the Example with a deposition time of 10 minutes. (C) Cantilever system / functionalized tip following the deposition conditions of the Example with a deposition time of 20 minutes.
     25
    Figure 6. Modification of the adhesion of the MFA tips caused by the functionalization and subsequent covalent attachment of an organic molecule. F-z curve of a non-functionalized sample (solid line) and of a functionalized sample to which fluorescein isothiocyanate has been covalently linked (dashed line). An increase in adhesion strength between the tip and the HOPG substrate is observed from 2 nN at the tip without funcinalizing, up to 37 nN with the functionalized tip.

    EXAMPLES

    The following examples are included to provide experts in the area with a complete description of how to make and apply the present invention. They should not be considered in any way to limit the scope of what the inventors consider as their invention, nor should they be supposed to constitute a complete enumeration of all the experiments performed. Unless otherwise indicated, the temperature is indicated in degrees Celsius and 5 the pressure in millibars.

    EXAMPLE 1: The following Table shows the range of the functionalization process parameters used for the deposition of functionalized sheets on silicon nitride MFA tips (Si3N4). 10
     Parameter  Value or Range of values
     Organometallic composition  3-Amino propyltriethoxysilane (APTES) / mercaptopropylmethoxysilane (MPTMS)
     Carrier gas  Argon / Molecular Nitrogen
     Evaporation temperature (ºC)  130-200
     System working pressure during deposition (mbar)  0.5-2
     Steam activation temperature (ºC)  650-800
     Length of the activation region, D1 (mm)  150-200
     Distance from the tip to the exit of the activation region, D2 (mm)  1-10
     Tip angle with the direction of the steam flow at the exit of the activation region, α  0º-45º
     Orientation of the tip with respect to the steam flow at the exit of the activation region  Tip support overhang oriented perpendicular to the direction of steam flow (Figure 4A) / Tip support overhang oriented parallel to the direction of steam flow (Figure 4B)
     Deposition Time (min)  5-30

    EXAMPLE 1.1: In particular, the following implementation of the invention has allowed obtaining functionalized MFA tips through AVS. Organometallic composition: 3-aminopropyltriethoxysilane; carrier gas: argon; Temperature of
    evaporation: 170 ° C; system working pressure: 1 mbar; activation temperature: 750 ° C; activation region length: 150 mm; distance from the tip to the exit of the activation region: 5 mm; tip angle with the direction of flow at the exit of the activation region: 20 °; orientation of the tip with respect to the steam flow at the outlet of the activation region: horizontal (Figure 4A); deposition time: 20 minutes. 5

    SOME PROPERTIES OF THE FUNCTIONALIZED ATOMIC FORCE MICROSCOPY POINTS

    The possibility of functionalizing MFA tips by the procedure described in this document was experimentally validated. Following the details of Example 1.1 presented in the previous Section, the organometallic used for functionalization was 3-aminopropyltriethoxysilane. The use of this organometallic leads to the formation of a sheet whose thickness is between 100-200 nm and whose surface appears a high density of amino groups (-NH2) on the surface. For the verification of the existence of amino groups on the surface of the MFA tip, the molecule isothiocyanate of fluoscein was chosen. Said molecule has a fluorescent region that emits a wavelength corresponding to the green color and an isothiocyanate group, which interacts with amino groups creating a covalent bond. The functionalized MFA tips and unfunctionalized MFA control tips were incubated with a solution of 20 fluorescein isothiocyanate, being subsequently washed to remove the remains of fluorescent molecules that were not covalently bound to the material. The tips were observed in a fluorescence microscope and representative results of the resulting images are shown in Figure 5. All images presented in Figure 5 were obtained under the same observation conditions so that the intensity of the observed fluorescence is a semi-quantitative measure of the density of amino groups on the surface of the MFA cantilever.

    Figure 5A corresponds to the non-functionalized control cantilever, with a very small fluorescence observed and practically indistinguishable from the background fluorescence that is observed outside the contour defined by the cantilever. In contrast, Figures 5B and 5C correspond to functionalized MFA tips under the conditions indicated in the previous Example for 10 minutes and 20 minutes respectively. The increase in fluorescence with respect to the control sample is evident, further showing how said fluorescence appears homogeneously distributed over the entire surface of the cantilever. 35
    It is also observed that the intensity of the fluorescence increases in this case with the deposition time, being greater in the sample in which the deposition has lasted 20 minutes.

    Additionally, the MFA tips to which the fluorescein isothiocyanate molecules were covalently bound were used to verify that such binding could be detected from the different interaction between the tip and the sample after the fluorescein molecule binding to the same. In particular, force curves on the tip versus distance between a graphite model substrate (HOPG) and functionalized tips or control tips (F-z curves) were obtained. A representative Fz curve of the interaction between an un-functionalized control tip and a HOPG model substrate is shown in Figure 6. A representative Fz curve of the interaction of a functionalized tip to which fluorescein isothiocyanate has been covalently bound and the Same HOPG model substrate is shown in Figure 6B. The main difference between the two curves is concentrated in the adhesion region that corresponds to the separation of the tip from the substrate. The adhesion strength of the functionalized tip is greater by a factor of between 2 and 10 to the adhesion strength of the non-functionalized control tip and the substrate itself. Without being bound by any theory, it is assumed that said increase in adhesion strength is the result of an increase in the tip-sample interaction due to the presence of the characteristic organic groups of the fluorescein molecule. twenty

    权利要求:
    Claims (18)
    [1]

    1. A method for obtaining a functionalized atomic force microscopy sensor tip, characterized in that the functionalization takes place by means of an activated steam silanization process comprising:
    a) evaporating an organometallic compound containing at least one silicon atom and at least one functional group selected from the group consisting of amino (-NH2), carboxyl (-COOH), sulfydryl (-SH), hydroxyl (-OH) and a combination of the above;
    b) activating the vapor of the organometallic compound of step a) by heating at a temperature between 400 and 1000 ° C; and 10
    c) having the activated vapor of step b) influence a sensor tip for atomic force microscopy to deposit the organometallic compound on said sensor tip;
    where stages b) and c) take place consecutively.
     fifteen
    [2]
    2. Method for obtaining a functionalized tip according to claim 1, wherein the sensing tip to be functionalized is composed of silicon nitride (Si3N4) or silicon (Si).

    [3]
    3. Method for obtaining a functionalized tip according to any one of claims 1 to 2, wherein the organometallic compound comprises one or more hydrocarbon chains - (CH2) n-, wherein n is a number between 1 and 30; and at least one functional group selected from the group consisting of hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), amino (-NH2) and a combination of the foregoing.

    [4]
    4. Method for obtaining a functionalized tip according to any one of claims 1 to 3, wherein the organometallic compound is selected from the group consisting of 3-aminopropyltriethoxysilane (APTES), aminopropyltrimethoxysilane (APTMS), mercaptopropylmethoxysilane (MPTMS), triethoxysilylpropylmalenamic acid and N-triethoxysilylpropyl-O-polyethylene oxide.
     30
    [5]
    5. Method for obtaining a functionalized tip according to any one of claims 1 to 4, wherein the organometallic compound is 3-aminopropyltriethoxysilane (APTES).

    [6]
    6. Method for obtaining a functionalized tip according to any one of claims 1 to 5, wherein the evaporation stage a) is carried out by heating in a temperature range between 100 ° C and 250 ° C.

    [7]
    7. Method for obtaining a functionalized tip according to any one of the claims 1 to 6, wherein the activation stage b) takes place by heating at a temperature between 400 ° C and 900 ° C.

    [8]
    8. Method for obtaining a functionalized tip according to any one of claims 1 to 7, wherein the evaporation, activation and / or deposition steps take place in a residual vacuum within the range 10-4 and 10-1 mbar.

    [9]
    9. Method for obtaining a functionalized tip according to any one of claims 1 to 7, wherein one or more of the stages of evaporation, activation or deposition of the organometallic take place in different regions, and an inert gas flow is used to transport Organometallic vapor from one region to another.

    [10]
    10. Method for obtaining a functionalized tip according to claim 9, wherein the inert gas is argon or molecular nitrogen.
     twenty
    [11]
    11. Method for obtaining a functionalized tip according to any one of claims 9 to 10, wherein the system pressure is in the range 10-2 and 100 mbar.

    [12]
    12. Method for obtaining a functionalized tip according to any one of claims 1 to 11, wherein the period of incidence of activated organometallic vapor on the MFA tip is between 1 and 120 minutes.

    [13]
    13. Method for obtaining a functionalized tip according to any one of claims 1 to 12, wherein in step c) the activated organometallic vapor strikes 30 on the tips to be functionalized at an angle between 0 ° and 60 °.

    [14]
    14. Method for obtaining a functionalized tip according to any one of claims 1 to 13, wherein the tips are located in a cantilever (2), and this is oriented perpendicularly to the direction of the flow of activated organometallic vapor. 35

    [15]
    15. Method for obtaining a functionalized tip according to any one of claims 1 to 13, wherein the tips are located in a cantilever (2), and this is oriented parallel to the direction of the flow of activated organometallic vapor.
     5
    [16]
    16. A functionalized atomic force microscopy sensor tip, characterized in that it comprises a sheet deposited on its surface comprising reactive groups selected from the group consisting of amino (-NH2) hydroxyl (-OH), carboxyl (-COOH), sulfhydryl ( -SH) and combination of the foregoing, and is obtained by the method described in any one of claims 1 to 15. 10

    [17]
    17.- Sensor tip according to claim 16, wherein the deposited sheet has a thickness between 50 nm and 1 µm.

    [18]
    18. Use of a sensor tip as described in any one of claims 15 16 or 17 in atomic force microscopy.
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    同族专利:
    公开号 | 公开日
    CN112805573A|2021-05-14|
    US11156635B2|2021-10-26|
    ES2684851B2|2019-06-19|
    US20210293851A1|2021-09-23|
    WO2020021136A1|2020-01-30|
    EP3832318A4|2021-09-01|
    EP3832318A1|2021-06-09|
    引用文献:
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    GB0518867D0|2005-09-15|2005-10-26|Secretary Trade Ind Brit|Microscopy tip|
    WO2007109689A2|2006-03-20|2007-09-27|Worcester Polytechnic Institute|A method for functionalizing an atomic force microscope tip|
    EP2237050A1|2009-03-31|2010-10-06|Centro de Investigación Cooperativa En Biomateriales |Apparatus and method for the functionalisation of afm tips|
    FR2965624B1|2010-09-30|2012-10-26|Centre Nat Rech Scient|ATOMIC FORCE MICROSCOPE POINT MODIFIED AND BIOMODIFIED|
    FR2969762B1|2010-12-22|2013-02-08|Commissariat Energie Atomique|ATOMIC FORCE MICROSCOPE PROBE, PROCESS FOR PREPARING THE SAME AND USES THEREOF|
    US9245722B2|2013-09-16|2016-01-26|Georgia Tech Research Corporation|SMS probe and SEM imaging system and methods of use|
    KR20150071876A|2013-12-19|2015-06-29|엘지전자 주식회사|Method for sequencing nucleic acids using atomic force microscope|
    TWI709481B|2014-08-25|2020-11-11|日商東洋紡股份有限公司|Silane coupling agent laminated layer polymer film and its manufacturing method, laminated body and its manufacturing method, and flexible electronic device manufacturing method|CN111610346A|2020-05-07|2020-09-01|浙江大学|Method for measuring hydrophilic and hydrophobic properties of micro-nano scale interface based on atomic force microscope|
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    ES201830777A|ES2684851B2|2018-07-27|2018-07-27|METHOD FOR OBTAINING SOMETHING POINTS OF ATOMIC FORCE MICROSCOPY FUNCTIONALIZED THROUGH ACTIVATED STEAM SILANIZATION, AND THE POINTS OBTAINED BY SUCH METHOD|ES201830777A| ES2684851B2|2018-07-27|2018-07-27|METHOD FOR OBTAINING SOMETHING POINTS OF ATOMIC FORCE MICROSCOPY FUNCTIONALIZED THROUGH ACTIVATED STEAM SILANIZATION, AND THE POINTS OBTAINED BY SUCH METHOD|
    PCT/ES2019/070456| WO2020021136A1|2018-07-27|2019-06-28|Method for obtaining functionalised sensor tips for atomic force microscopy by means of activated vapour silanisation and tips obtained using said method|
    US17/261,066| US11156635B2|2018-07-27|2019-06-28|Method for obtaining functionalised sensor tips for atomic force microscopy by means of activated vapour silanisation and tips obtained using said method|
    CN201980049873.2A| CN112805573A|2018-07-27|2019-06-28|Method for obtaining a functionalized sensor tip of an atomic force microscope by activated vapor silanization and tip obtained with this method|
    EP19841178.7A| EP3832318A4|2018-07-27|2019-06-28|Method for obtaining functionalised sensor tips for atomic force microscopy by means of activated vapour silanisation and tips obtained using said method|
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