阅读说明：本技术 通过活化蒸汽硅烷化获得原子力显微镜的官能化传感器针尖的方法和用该方法获得的针尖 (Method for obtaining a functionalized sensor tip of an atomic force microscope by activated vapor silanization and tip obtained with this method ) 是由 何塞·佩雷斯·里盖罗 古斯塔沃·维克托·几内亚·托尔图艾罗 拉斐尔·达扎·加西亚 路易斯·科尔 于 2019-06-28 设计创作，主要内容包括：本发明涉及一种获得用于原子力显微镜的官能化传感器针尖的方法,其特征在于官能化通过活化蒸汽硅烷化工艺进行,包括：a)蒸发包含至少一个硅原子和至少一个官能团的有机金属化合物,所述至少一个官能团选自由胺基、羟基、羧基及硫醇基；b)通过加热,活化步骤a)中所述有机金属化合物的蒸汽；以及c)使步骤b)中形成的活化蒸汽撞击原子力显微镜的传感器针尖,以将所述有机金属化合物的膜沉积在所述传感器针尖上,连续地进行步骤b)和c)。本发明还涉及使用该方法获得的官能化传感器针尖。(The invention relates to a method for obtaining a functionalized sensor tip for atomic force microscopy, characterized in that the functionalization is carried out by an activated vapor silanization process comprising: a) evaporating an organometallic compound comprising at least one silicon atom and at least one functional group selected from the group consisting of amine groups, hydroxyl groups, carboxyl groups, and thiol groups; b) activating the vapor of the organometallic compound in step a) by heating; and c) impinging the activated vapor formed in step b) against a sensor tip of an atomic force microscope to deposit a film of the organometallic compound on the sensor tip, steps b) and c) being performed successively. The invention also relates to a functionalized sensor tip obtained using this method.)

1. A method of obtaining a functionalized sensor tip for atomic force microscopy, characterized in that the functionalization is performed by an activated vapor silylation process comprising:

a) vaporizing an organometallic compound comprising at least one silicon atom and at least one functional group selected from the group consisting of amine groups (-NH)₂) A carboxyl (-COOH), a thiol (-SH), a hydroxyl (-OH), and combinations thereof;

b) activating the vapour of the organometallic compound in step a) by heating to a temperature between 400 and 1000 ℃;

c) impinging the activated vapor formed in step b) on a sensor tip of an atomic force microscope to deposit the organometallic compound on the sensor tip to form a film having a thickness of 50nm to 1 μ ι η;

steps b) and c) are carried out continuously.

2. The method of obtaining a functionalized tip of claim 1, wherein the sensor tip to be functionalized is made of silicon nitride (Si)₃N₄) Or silicon (Si).

3. A method for obtaining functionalized needle tips according to any of claims 1 to 2, wherein the organometallic compound comprises one or more hydrocarbon chains- (CH)₂)_n-, where n is a number between 1 and 30; and at least one functional group selected from the group consisting of hydroxyl (-OH), carboxyl (-COOH), thiol group(s) ((R))-SH), amino (-NH)₂) And combinations thereof.

4. A method of obtaining a functionalized needle tip according to any of claims 1 to 3, wherein the organometallic compound is selected from the group consisting of 3-Aminopropyltriethoxysilane (APTES), Aminopropyltrimethoxysilane (APTMS), mercaptopropylmethoxysilane (MPTMS), triethoxysilylpropylmaleic acid and N-triethoxysilylpropyl-O-polyethylene oxide.

5. A method of obtaining a functionalized needle tip according to any of claims 1 to 4, wherein said organometallic compound is 3-Aminopropyltriethoxysilane (APTES).

6. Method for obtaining a functionalized needle tip according to any of the claims 1 to 5, wherein said evaporation step a) is carried out by heating in a temperature range between 100 ℃ and 250 ℃.

7. Method for obtaining functionalized needle tips according to any of the claims 1 to 6, wherein the activation step b) is carried out by heating to a temperature between 400 ℃ and 900 ℃.

8. Method for obtaining functionalized needle tips according to any of the claims 1 to 7, wherein said evaporation, activation and/or deposition step is carried out at 10^-4To 10^-1Residual vacuum in the range of mbar.

9. A method of obtaining a functionalized needle tip according to any of claims 1 to 7, wherein one or some of said steps of evaporation, activation or deposition of said organometallic compound are carried out in different zones and a flow of inert gas is used to transport said organometallic vapour from one zone to another.

10. A method of obtaining a functionalized needle tip according to claim 9, wherein said inert gas is argon or molecular nitrogen.

11. A method of obtaining a functionalized needle tip according to any of claims 9 to 10, wherein the pressure of the system is at 10^-2To 100 mbar.

12. A method of obtaining a functionalized tip according to any of claims 1 to 11, wherein the time of impinging said atomic force microscope tip with said activated organometallic vapour is from 1 to 120 minutes.

13. A method of obtaining a functionalized needle tip according to any of claims 1 to 12, wherein in step c) the activated organometallic vapour impinges on the needle tip to be functionalized at an angle between 0 ° and 60 °.

14. A method to obtain a functionalized needle tip according to any of claims 1 to 13, wherein the needle tip is located in a cantilever (2) and it is perpendicular to the direction of the activated organometallic vapour flow.

15. A method to obtain a functionalized needle tip according to any of claims 1 to 13, wherein said needle tip is located in a cantilever (2) and it is parallel to the direction of the activated organometallic vapour flow.

Technical Field

The invention belongs to the field of near-field microscopes, and particularly relates to the field of atomic force microscopes and instruments required in the field.

Activated vapor silanization functionalized tips can be used to use atomic force microscopy as a microstructure characterization technique in materials science or biology, among other applications.

Background

An atomic force microscope (AFM or MFA for short) is a microstructure characterization technology with wide application, and can be used for researching various systems under various environmental conditions. Atomic force microscopes belong to the class of near-field microscopes, in which the sensor element is very close to the surface to be observed. When using an atomic force microscope, the sensor element is a cantilever, typically equipped with a micrometer-sized tip at its end. The atomic force microscope has a positioning system in both X, Y and Z spatial directions that changes the distance between the tip and the surface of the sample (typically defined by the Z direction) as the sample surface (typically defined by the X and Y directions) is scanned. The interaction established between the tip and the sample surface causes the cantilever to bend, and a relationship between the curvature of the cantilever and the pressure applied to the tip is established due to the interaction with the surface at a given time. Scanning in the X and Y directions can obtain isodynes of the surface by moving the cantilever along the Z axis to maintain the cantilever bending at a constant value. When the primary interaction is a contact interaction due to spatial repulsion between the tip and the surface of the material, the resulting isodyne is considered to correspond to the surface topography of the sample.

The general principles on which atomic force microscopy is based make it highly versatile compared to other near-field microscopy, optical microscopy and electron microscopy techniques. In particular, atomic force microscopes make it possible to work under a variety of environmental conditions, including in vacuum, in air, and in various liquid media. In particular, the possibility of being able to work in liquid media provides unprecedented opportunities for viewing biological systems under in vivo conditions, beyond the scope of most other microscopic techniques. Furthermore, the sensor system has a dependency on the interaction established between the tip and the sample, which is characterized by a plurality of elements if the interaction can be controlled.

The development of chemical atomic force (C-AFM or C-MFA for short) or affinity microscopy is facilitated by the ability to characterize biological systems in vivo by binding in a suitable liquid medium and the possibility to explore said biological systems using specific probes (e.g. antibodies recognizing certain biomolecules). It was found from the previous discussion that a key factor enabling the characterization of samples according to the C-AFM principle is the availability of the atomic force microscope tip, which can establish specific interactions with the various elements that may be present in the sample to be analyzed. In general, obtaining an atomic force microscope tip that can be used in C-AFM requires two steps: (1) modifying (functionalizing) the tip surface to generate functional groups on its surface; (2) stable binding (usually by covalent bonds) between functional groups on the tip surface and molecules, while specific interactions are established between molecules and certain surface elements. Experience has shown that the first step (involving modification of the tip surface to generate functional groups) is the most complex from the point of view of feasible technological development.

The usual strategy for atomic force microscope tip functionalization is based on two alternative approaches (r. barattin and n. voyer, study of chemical modification of atomic force microscope tips at molecular recognition events, chem. comm. 13(2008), 1513-. In one aspect, methods have been described based on the deposition of thin gold films or the like, the subsequent binding of recognition molecules taking advantage of the specific interaction between the deposited film and the regions of the molecule itself. A typical example of this method is the immobilization of molecules containing thiol groups (also called thiol groups, -SH) on thin gold films, taking advantage of the high affinity of thiol groups for metal surfaces. Another approach is to use organometallic molecules that can spontaneously bind to functional groups on the tip surface. A typical example of this approach is to combine 3-Aminopropyltriethoxysilane (APTES) molecules with hydroxyl (-OH) groups on the surface of the tip or generated by exposure of the tip to an oxidizing environment.

The technological developments of two alternative methods are presented in a series of patent documents. Thus, a first group of corresponding technologies is patent application US2007/0082352 a1(Peter Jonathan Cumpson, microscope tip), which shows a method of binding deoxyribonucleic acid (abbreviated DNA or ADN) molecules to atomic force microscope tips. In the method, a DNA molecule is first modified by binding it to an organic molecule comprising a thiol group (-SH). For atomic force microscope tips that immobilize DNA, modification can be performed by depositing a gold film on them. The ability of the DNA molecule to be immobilized at the tip of the needle is due to the affinity of the thiol group for gold in the metallic phase.

Documents describing techniques based on the second approach include patent application WO2007/109689 a2(Gallardo-Moreno et al, a method of atomic force microscope tip functionalization), in which a polylysine coating is deposited on an unmodified tip. In this case, functionalization is achieved by establishing non-specific interactions between the positively charged polylysine molecules and the unmodified tip surface. Alternatively, effective methods for silicon nitride or silicon oxide tips have been proposed, first generating a large number of hydroxyl groups on the tip surface, and then binding dendrimer-type molecules to the hydroxyl groups. Said method is described in patent application FR2965624a1 (Dague etinne et al, modified atomic force microscope tips comprising a surface grafted by covalent bonds with a fluorescent dendrimer and having a plurality of terminal functions at its periphery, making it possible to covalently immobilize the dendrimer on the surface and the biomolecules on the dendrimer). The process described in patent application KR1020150071876A (Shim Bong Chushim et al, a method for analyzing nucleic acid sequences using atomic force microscopy) is also based on the initial generation of hydroxyl groups at considerable density on the tip of an atomic force microscope. Hydroxyl groups are generated by exposing the tip to a 20% nitric acid solution, and a monolayer of APTES molecules is subsequently formed on the surface as organometallic molecules react with the hydroxyl groups previously generated on the surface. The protein and/or nucleic acid may be bound to the needle tip directly through amine groups in the APTES molecule, or through dendrimers between the needle tip and the biomolecule (protein and/or nucleic acid).

Among the proposed methods for the functionalization of atomic force microscope tips, mention may be made of the patent applications WO 2012/084994A 1(Polesel-Maris, Mass,Et al, atomic force microscope probe, preparation method and use thereof), which is mainly characterized by having the characteristics of the two basic methods. In this case, the original needle tip is modified to expose the graphite surface thereon. Then, the graphite surface is chemically modified to generate-OH groups on the surface. Finally, the groups react with various organic molecules, exposing different functional groups on the surface that are capable of covalently binding biomolecules.

Therefore, one of the major drawbacks of the tip functionalization methods proposed in earlier studies is the need to develop in advance a method for activating the surface for the fabrication of atomic force microscope tip materials, which varies from material type to material type. Furthermore, the functionalization method must be compatible with the biomolecules to be subsequently immobilized on the atomic force microscope tip. Therefore, there is a need in the art to develop new general methods for the functionalization of atomic force microscope tips, either as tip materials that support functionalization or as biomolecules compatible with the functionalization methods.

In this regard, the present invention provides a sensor tip for a chemical atomic force microscope (C-AFM) wherein functionalization is performed using an activated vapor silylation technique (AVS). This technique has been described in the previous article (rjmart i in-Palma et al, using amine groups for surface biofunctionalization of materials, j. mater. res.19(2004), 2415-2420), mainly in the field of biomaterials and medical materials (p. rezvanian et al, enhancement of the biological reaction of AVS-functionalized Ti-6AI-4V by covalent immobilization of collagen, scientific report 8(2018), 3337), the general feature of which is that it is used for samples with a substantially smooth surface morphology.

Activated vapor silylation techniques demonstrate the ability to deposit functionalized films on smooth substrates to which various biomolecules, such as extracellular matrix proteins (e.g., collagen or fibronectin), can be covalently bound. However, the method described in this article for the efficient functionalization of atomic force microscope tips using activated vapor silylation techniques has a much steeper topography than the smooth substrates to which activated vapor silylation techniques have hitherto been applied, and therefore the tips can be used as sensor elements for chemical atomic force (or affinity) microscopy.

Brief summary of the invention

The present invention is based on work performed in the field of thin film deposition and in the field of atomic force microscopy. The inventors have found that the activated vapor silylation technique makes it possible to obtain functionalized tips for atomic force microscopy that exhibit a high density of amine groups (-NH) on their surface₂) A carboxyl group (-COOH), a thiol group (-SH) and/or a hydroxyl group (-OH). A method for producing functionalized tips for atomic force microscopy by activated vapor silylation techniques is presented and described below.

Accordingly, the present invention provides a method of obtaining a functionalized sensor tip for Atomic Force Microscopy (AFM), characterized in that the functionalization is performed by an activated vapor silylation process comprising:

a) vaporizing an organometallic compound comprising at least one silicon atom and at least one functional group selected from the group consisting of amine (-NH)₂) Carboxyl (-COOH), thiol (-SH), hydroxyl (-OH), and combinations thereof;

b) activating the vapor of the organometallic compound in step a) by heating to a temperature of 400 ℃ to 1000 ℃;

c) impinging the activated vapor formed in step b) against a sensor tip of an atomic force microscope to deposit the organometallic compound on the sensor tip;

steps b) and c) are carried out successively.

Obtaining a functionalized tip first requires organometallic evaporation, the molecules of which contain at least one silicon atom and at least one amine group (-NH)₂) A carboxyl group (-COOH), a thiol group (-SH), a hydroxyl group (-OH), or combinations thereof. Subsequently, a heating steam step is performed, which is then impinged on the atomic force microscope tip to be functionalized. In order to avoid degradation of the activated organometallic compound, steps b) and c) are carried out continuously one after the other, so that the activation step is carried out immediately before the activating vapor is impinged on the atomic force microscope tip.

In the process described in this document, the evaporation and activation steps can be performed in different zones of the same unit, or even in different units of the same device. In this case, the vapour obtained in the evaporation zone is thermally conveyed to the zone where the activation step b) will take place, preferably at a temperature higher than the evaporation temperature of the organometallic compound in question.

The invention also relates to atomic force microscope functionalized tips obtained by the methods described herein. These tips are characterized by a base material of the tip, the thickness of the functional film preferably being in the range between 50nm and 1 μm; and a high density of amine, carboxyl, thiol and/or hydroxyl groups on the surface. In particular, in the case of using an organometallic compound having amine groups, values close to 8 amine groups/nm can be achieved²Approximately corresponding to the theoretical surface density of the amine based monolayer on a flat surface. The density can be measured based on the function of covalently binding a fluorescent label.

Thus, unlike other methods for functionalizing atomic force microscope tips, for example the method described in patent application KR1020150071876a 1, a monolayer of organometallic molecules is made that adhere to hydroxyl groups generated on the surface of the tip material. In the method according to the invention, a film, preferably between 50nm and 1 μm, is formed by the decomposition of organometallic molecules, independently of the chemical nature of the surface of the tip material to be functionalized.

Detailed description of the invention

The invention provides a method for obtaining a functionalized sensor tip for atomic force microscopy, characterized in that the functionalization is carried out by an activated vapor silylation process comprising:

a) vaporizing an organometallic compound comprising at least one silicon atom and at least one functional group selected from the group consisting of amine groups (-NH)₂) A carboxyl (-COOH), a thiol (-SH), a hydroxyl (-OH), and combinations thereof;

b) activating the vapour of the organometallic compound in step a) by heating to a temperature between 400 ℃ and 1000 ℃;

c) impinging the activated vapor formed in step b) against a sensor tip of an atomic force microscope to deposit the organometallic compound on the sensor tip;

steps b) and c) are carried out continuously.

The functionalization methods described herein are based on the sensor tips of atomic force microscopy, commonly employed in larger structures called chips (see fig. 1), which include functionalization of these tips when contacted with activated vapor of organometallic molecules. The interaction between the atomic force microscope tip and the activating vapor causes a film to form thereon, thereby leaving the organometallic molecular fragments active and exposed to the external medium. Preferably, the film has a thickness of between 50nm and 1 μm, since a thickness greater than 1 μm may result in delamination of the film deposited on the tip of the atomic force microscope.

The presence of active organic fragments on the surface of the atomic force microscope tip is called functionalization and its main property is that the presence of said active organic fragments alters the interaction of the tip with the medium. The use of an atomic force microscope functionalized tip changes the sensing capabilities of the atomic force microscope technique and the range of measurements available through the technique.

Without being bound by any theory, the inventors believe that the presence of the functionalized film (preferably 50nm to 1 μm thick) is a result of partial decomposition of the organometallic molecules due to thermal activation. The activated molecules impinging on the surface interact with the surface and with each other to form a solid film on the surface, but retain some organic fragments of the original organometallic molecules that remain active and exposed outside the tip.

The sensor element in an atomic force microscope is a tip located near the surface to be investigated and at the end of a cantilever, which is bent due to the interaction between the tip and the surface. While the cantilever arms are located at the end of a larger structure of parallelepiped geometry, commonly referred to as a chip (see fig. 1). The standard size of the chip in a commercial tip is in the order of millimeters, with the standard size of the cantilever in the direction perpendicular to the tip being on the order of tens of microns, and in the latter direction being a few microns. The size of the tip varies from tens of nanometers to ten micrometers. Theoretically, there is no limitation on the composition of the atomic force microscope tip, so long as it is compatible with the fabrication of components having the geometries described above. In practice, most commercial atomic force microscope tips are made of Silicon (SI) or silicon nitride (SI)₃N₄) And (3) preparing. The functionalization process of the present invention does not impose any restrictions on the geometry of the chip and its composition, as long as it is of a size that enables the activation of the sensor tip in the activation region.

Furthermore, the organometallic compounds used in the process of the invention consist of molecules having a common structure in which the silicon atoms are bound to a single or to several hydrocarbon chains, at least one of these chains comprising one or more amine groups (-NH)₂) Hydroxyl (-OH), carboxyl (-COOH) or thiol (-SH).

In a preferred embodiment of the invention, the organometallic compound comprises one or more hydrocarbon chains- (CH)₂)_n-, where n is a number between 1 and 30, preferably 1 to 6; at least one functional group selected from hydroxyl (-OH), carboxyl (-COOH), thiol (-SH), and amine (-NH)₂) And combinations thereof.

The hydrocarbon chain of the organometallic compound used in the process of the invention may comprise one or more double or triple bonds between carbon atoms.

Examples of molecules of organometallic compounds that can be used in the activated vapor silylation process described herein include: 3-Aminopropyltriethoxysilane (APTES) and Aminopropyltrimethoxysilane (APTMS), both of which result in the formation of a film containing amine groups; mercaptopropylmethoxysilane (MPTMS), which produces thiol-group-containing membranes; triethoxysilylpropylmaleic acid (triethoxysilpropylmalemic acid), which produces a film containing carboxyl groups; N-triethoxysilylpropyl-O-polyethylene oxide (N-triethoxysilylpropyloxide), which produces a film containing hydroxyl groups. In combination with the molecular structure itself, an important characteristic of the organometallic compound is its boiling point. In particular, the boiling temperature of the compound is preferably between 100 ℃ and 250 ℃.

The evaporation step a) may be performed by depositing the organometallic fluid in an evaporation chamber inside the evaporation furnace, thereby changing the temperature of the organometallic fluid. The temperature of the vaporization chamber is increased above the boiling point of the organometallic fluid causing a liquid-vapor phase transition of the organometallic vapor to occur within the vaporization chamber. Preferably, the temperature to which the evaporation oven is heated is in the range of 50 ℃ to 400 ℃, more preferably 100 ℃ to 250 ℃, and particularly preferably the temperature of the evaporation step a) is best in the range of 130 ℃ to 200 ℃. The range of vaporization temperatures depends on the particular organometallic compound, fixed as the maximum temperature limit of the range that results in decomposition of the organometallic molecule.

Preferably, the activation step b) may be carried out by heating to between 400 ℃ and 900 ℃, more preferably between 400 ℃ and 800 ℃, since too high a temperature may lead to irregularities and non-uniformities in the deposited film.

The apparatus for performing the organometallic vapor activation step comprises a deposition chamber in which the atomic force microscope tip to be functionalized is located and another zone, preferably tubular, located before and directly connected to the deposition chamber. According to these embodiments, the activation zone corresponds to the region of the conduit that precedes and is directly connected to the deposition chamber. Further, the activation oven is located around the activation zone, defining its extension and allowing a controlled temperature rise in said activation zone. Thus, in the activation zone, the organometallic vapor that evaporates in the evaporation chamber passes through a high temperature zone before entering the deposition chamber and striking the tip of the atomic force microscope.

The efficiency of the process is improved in a vacuum environment, avoiding the reaction of organometallic molecules with atmospheric gases (mainly oxygen). Heating of the organometallic compound during the evaporation and activation steps is required to facilitate subsequent oxidation of the organometallic compound with atmospheric oxygen. The reaction may decompose the organometallic compound, thereby preventing functionalization of the substrate. Therefore, in permission 10^-4To 10^-1It is advantageous to carry out the above-described process, in particular the above-described steps a), b) and c), in a vacuum system with a residual vacuum in millibars (mbar) to avoid the above-described decomposition. The vacuum may be achieved by a rotary pump coupled to a cold trap.

In the process described in this document, the evaporation and activation steps can be carried out in different zones of the same device, and in particular, in different chambers of the same unit or even of different units. In that case, the vapour obtained in the evaporation zone is transported at a high temperature, preferably at a temperature higher than the evaporation temperature of the organometallic compound in question, to the zone where the activation step is to be carried out.

In an embodiment of the method according to the invention, at least one or more evaporation, activation or deposition steps of the organometallic compound on the atomic force microscope sensor tip to be functionalized are carried out in different zones, preferably using a carrier gas which facilitates transport of the organometallic vapour from one zone to another, in particular from the evaporation chamber to the activation zone and finally to the deposition chamber, wherein the activation vapour can impinge on the atomic force microscope tip. Although molecular nitrogen or carbon dioxide may be considered as the end, the carrier gas is necessarily an inert gas for the organometallic compound, and therefore a rare gas such as argon gas may be selected. If a carrier gas is introduced into the system, it will cause an increase in pressure within it; because of the introduction of carriersThe working pressure range generated by gas is preferably 10^-2To 100 mbar, but more preferably 5x10^-1To 10 mbar.

Preferably, in the methods of obtaining an atomic force microscope functionalized sensor tip described in this document, the time for the activated organometallic vapor to impinge on the atomic force microscope tip is from 1 to 120 minutes.

As mentioned before, there may be a gap between the evaporation chamber and the activation zone, which are usually attached by a connecting tube. Such spacing involves the transport of steam along the extension of the connecting tube before entering the activation zone. In order to prevent condensation of the organometallic vapor during the above-mentioned transport, the connecting line between the evaporation chamber and the activation zone can be conveniently surrounded by a heating element, which can be a heating belt. The heating zone must maintain the junction at a temperature equal to or higher than the vaporization temperature in step a) of the process of the invention to prevent condensation of the organometallic vapor before it reaches the activation zone.

The deposition chamber may contain an atomic force microscope tip holder, which allows the position of the tip in the deposition chamber to be determined and held stationary throughout the process. In a particular embodiment of the invention, the geometry of the needle tip is such that the needle tip passes from the outlet of the connecting tube to the activation zone (D)₂(ii) a Fig. 3) and the angle (a; fig. 3). In particular, in step c) of the method described herein, the tip to be functionalized is arranged: the activated organometallic vapor impinges on the tip to be functionalized at an angle between 0 ° and 60 °. In particular, when the method is employed in the unit described in this document (see fig. 2), the aforementioned angle α corresponds to the angle of the sensor tip to be functionalized with respect to the opening of the activation zone in the deposition chamber. The activated organometallic vapor is introduced into the deposition chamber through the opening. In this way, a coordination of the flow density (number of molecules/area/time) and the coverage area is achieved, taking into account that the vapor flow is approximately conical when entering the deposition chamber.

Further, atomic forces may be performed in two alternative directionsFunctionalization of microscope tips: the needle tip holder cantilever is perpendicular to the direction of the steam flow (fig. 4A), or the needle tip holder cantilever is parallel to the direction of the steam flow (fig. 4B). With the latter orientation, the amount of organometallic compound deposited on the remainder of the tape and chip is reduced. In particular, the deposition on the strip is reduced with respect to the measurement before functionalization(strap)The quality of the organometallic compound needs to be reduced(strip)Motion at the primary resonance frequency.

Without being bound by any theory, the tip functionalization of an atomic force microscope in certain embodiments of the present invention is believed to be the result of the following process action: forming an organometallic vapor in the evaporation chamber, drawing the organometallic vapor from the evaporation chamber to an activation zone, heating the organometallic molecules in the activation zone, impinging the molecules thus activated on the surface of the atomic force microscope tip, thereby forming a film on the tip, preferably having a thickness of between 50nm and 1 μm. The activation process is sufficient to induce interaction of the organometallic molecules with each other and with the tip material such that a film is deposited on the surface of the atomic force microscope tip material, but a portion of the organic groups of the molecules remain prior to processing.

The invention also relates to a functionalized sensor tip for an atomic force microscope, having a film deposited on its surface, and said film comprising a moiety selected from the group consisting of amine groups (-NH)₂) A carboxyl group (-COOH), a thiol group (-SH), a hydroxyl group (-OH), and combinations thereof. The tip is obtained by the method described in this document, characterized in that the functionalization process results in the deposition of a functional film, preferably with a thickness between 50nm and 1 μm. In particular, based on the ability to covalently bind fluorescent labels, the density of amine groups on the tip surface functionalized by the method of the invention can reach values close to 8 amine groups/nm²。

Thus, the functionalized sensor tip of the present invention may have a functionalized membrane formed on its surface such that the membrane has a thickness in the range of 50nm to 1 μm and comprises a reagent group, such as an amine group (-NH)₂) Hydroxyl (-OH), carboxyl (-COOH), thiol (-SH), or a combination thereof, thereby exposing these functional groups to the outside. The reagent group may or may not be linked to a hydrocarbon chain- (CH)₂)_n-binding, wherein n is a number between 1 and 30.

Furthermore, the present invention relates to the use of a functionalized sensor tip in an atomic force microscope as described herein.

The methods described in this document for obtaining a functionalized tip of an atomic force microscope can be performed in one unit, comprising:

-an evaporation chamber for evaporating the liquid,

-an evaporation furnace configured for heating the evaporation chamber,

an activation zone connected to the evaporation chamber, preferably in the form of a tube,

an activation furnace configured for heating the activation zone,

-a deposition chamber connected downstream of the activation zone.

In particular embodiments, the length of the activation zone is between 100mm and 300mm, such that the desired activation temperature can be achieved in the organometallic vapor at higher operating pressures, or equivalently, at higher vapor flows.

In other particular embodiments of the invention, the activation zone has an opening to the deposition chamber, the tip of the needle to be functionalized being located at a position of 1mm to 50mm relative to said opening. Due to the dispersion of the organometallic vapor stream as it enters the deposition chamber, which is obtained by sufficiently high vapor stream density values and sufficiently large impingement surfaces within the deposition chamber, uniform functionalization can be performed using samples having dimensions on the order of centimeters.

Preferably, an angle between an imaginary line connecting the tip and an opening of the activation region in the deposition chamber and a flow direction of the organometallic vapor is between 0 ° and 60 °. As described in the previous section, this range of angular values is derived from the flow density of the organometallic vapor within the deposition chamber and the impingement area swept by the flow.

In certain embodiments of the present invention, the atomic force microscope tip holder cantilevers are oriented perpendicular to the direction of organometallic vapor flow. Alternatively, the direction of the cantilever of the atomic force microscope tip holder is parallel to the direction of the organometallic vapor flow.

Drawings

The invention is described in connection with the drawings of this document to facilitate a better understanding of the invention. It must be emphasized that, by convention, the drawings and drawing figures are not to scale. Rather, the dimensions of the various elements are scaled accordingly to facilitate understanding of the details shown. The drawings of this document include:

FIG. 1, cross-sectional view of a functionalized tip. (A) Atomic force microscope conventional tip cross-sectional view showing the basic elements: (1) a chip, (2) a cantilever, and (3) a needle tip. (B) Atomic force microscope conventional tip plan view showing the basic elements. (C) Tip/cantilever cross-section functionalized by activated vapor silylation, wherein functionalized membrane (4) is shown. Also shown are reagent groups on the surface, in this case embodied as amine groups (NH)₂)。

FIG. 2 is a diagram of the basic elements of a unit for fabricating an atomic force microscope functionalized tip by a preferred embodiment of the method of the present invention. (1) A carrier gas inlet. (2) An evaporation chamber. (3) And (4) an evaporation furnace. (4) An organometallic compound. (5) And (5) an activation furnace. (6) An activation zone. (7) And a deposition chamber. (8) And (4) a vacuum system outlet.

FIG. 3, the activation furnace and the deposition chamber are detailed, including the definition of the main geometrical parameters. D₁: length of activation furnace, D₂: the distance between the outlet of the activation furnace and the tip of the atomic force microscope, and the alpha: the angle between the line between the outlet of the activation furnace and the tip of the atomic force microscope and the axis of the activation zone.

Fig. 4, two possible patterns of needle tip with respect to vapor flow. (A) The needle tip holder cantilever is perpendicular to the direction of the steam flow. (B) The needle tip holder cantilever is parallel to the direction of the steam flow. The direction of the activating vapor flow at the inlet of the activation chamber is shown by the arrow.

FIG. 5 is a graph showing the confirmation of the presence of a functionalized thin film on the surface of the cantilever and the tip by using a fluorescent molecule (fluorescein isothiocyanate) which covalently reacts with an amine group. (A) Control samples with no functionalized film deposited. (B) The cantilever/tip system was functionalized, following the deposition conditions of the examples, with a deposition time of 10 minutes. (C) The cantilever/tip system was functionalized, following the deposition conditions of the examples, with a deposition time of 20 minutes.

FIG. 6 is a graph of atomic force microscope tip adhesion modification resulting from functionalization and subsequent covalent bonding of organic molecules. Fz curves for the unfunctionalized sample (solid line) and the functionalized sample (dashed line). For functionalized samples, fluorescein isothiocyanate was covalently bound. The adhesion between the tip and the HOPG substrate increased from 2nN for the non-functionalized tip to 37nN for the functionalized tip.

Detailed Description

The following examples are put forth so as to provide those of ordinary skill in the art with a complete description of how the invention may be made and used. It should not be construed as limiting the scope of the invention in any way, nor constituting all the tests to which the invention relates. Unless otherwise indicated, temperatures are expressed in degrees celsius (degrees centroide) and pressures are expressed in millibars (millibars).

Example 1: the following table shows silicon nitride (Si) for use in atomic force microscopy₃N₄) Range of functionalization process parameters for depositing functionalized films on the tip.

Example 1.1:in particular, by the following embodiments of the present invention, the functionalized tip of an atomic force microscope can be obtained by activated vapor silylation. Organometallic composition: 3-aminopropyltriethoxysilane; carrier gas: argon gas; evaporation temperature: 170 ℃; the working pressure of the system is as follows: 1 mbar; activation temperature: 750 ℃; length of active region: 150 mm; distance from needle tip to activation zone exit: 5 mm; the included angle between the needle tip and the flow direction at the outlet of the activation zone: 20 degrees; direction of the needle tip with respect to the activation zone outlet steam flow: level (fig. 4A); deposition time: for 20 minutes.

Characteristics of atomic force microscope functionalized needle tip

The feasibility of functionalizing atomic force microscope tips by the methods described in this document has been experimentally verified. According to example 1.1 in the preceding paragraphIn said detail, the organometallic compound used for the functionalization is 3-aminopropyltriethoxysilane. The use of the organometallic compound results in the formation of a film having a thickness of between 100-200nm and a high density of surface amine groups (-NH) present on the surface of the film₂). And selecting and using fluorescein isothiocyanate molecules when verifying the existence of amine groups on the surface of the needle point of the atomic force microscope. The molecule has a fluorescent region with emission wavelengths corresponding to green and isothiocyanate groups that interact with amine groups to produce covalent bonds. The functionalized and unfunctionalized control afm tips are incubated with fluorescein isothiocyanate solutions and then washed to remove fluorescent molecular residues not covalently bound to the material. Representative results of the images obtained by observing the tip under a fluorescence microscope are shown in FIG. 5. All images shown in fig. 5 were obtained under the same observation conditions, and the fluorescence intensity observed was therefore a semi-quantitative measure of the density of amine groups on the surface of the cantilever of the atomic force microscope.

Fig. 5A corresponds to the unfunctionalized control cantilever, and very weak fluorescence was observed, with little difference from background fluorescence observed outside the cantilever-defined profile. In contrast, fig. 5B and 5C correspond to atomic force microscope tips functionalized under the 10 minute and 20 minute conditions shown in the previous examples, respectively. The increase in fluorescence is more pronounced relative to the control sample, which also shows how the fluorescence is evenly distributed over the entire cantilever surface. It was also observed that in this case the fluorescence intensity increased with the deposition time, the fluorescence intensity of the sample was greater at a deposition duration of 20 minutes.

In addition, an atomic force microscope tip covalently bound to a fluorescein isothiocyanate molecule was used to verify that after the fluorescein molecule was bound to the tip and sample, the binding could be detected by the different interactions between the tip and sample. In particular, a force curve (F-z curve) of the force on the tip versus the distance between the model graphite substrate (HOPG) and the functionalized or control tip was obtained. FIG. 6 shows a typical F-z curve for the interaction between a non-functionalized control tip and a model HOPG substrate. FIG. 6B shows a typical F-z curve of fluorescein isothiocyanate covalently bound functionalized tips interacting with the same model HOPG base. The main difference of the two curves is concentrated in the adhesion zone, which corresponds to the separation of the tip from the base. The adhesion of the functionalized tips was 2-10 times greater than the adhesion of the unfunctionalized control tips and the substrate itself. Without being bound by any theory, it is assumed that the increase in adhesion is a result of an increase in tip-sample interactions due to the presence of the characteristic organic groups of fluorescein molecules.