EP2656085A1

EP2656085A1 - Atomic force microscope probe, method for preparing same, and uses thereof

Info

Publication number: EP2656085A1
Application number: EP11799102.6A
Authority: EP
Inventors: Jérôme Polesel-Maris; Thomas Berthelot; Pascal Viel
Original assignee: Commissariat a lEnergie Atomique CEA; Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2010-12-22
Filing date: 2011-12-20
Publication date: 2013-10-30
Also published as: US20130276176A1; US8959661B2; FR2969762A1; FR2969762B1; WO2012084994A1

Abstract

The present invention relates to an atomic force microscope probe which includes: (i) a piezoelectric resonator provided with two electrodes and coated with an insulating layer; and (ii) a tip attached to said coated resonator and functionalised by at least one grouping or molecule of interest. The present invention also relates to the method for preparing same and to the various uses thereof.

Description

ATOMIC FORCE MICROSCOPE PROBE,

ITS PREPARATION METHOD AND USES THEREOF

DESCRIPTION

TECHNICAL AREA

The present invention belongs to the field of sensors and, more particularly, to the field of Atomic Force Microscope (AFM) probes which are particularly useful in biology.

More particularly, the present invention relates to the use of an AFM probe such as a piezoelectric integrated sensor probe and methods for preparing this probe for the fine chemical analysis of molecular links through the chemical force spectroscopy in a liquid medium and especially in a biological liquid medium.

Direct applications of this type of probe typically cover DNA sequencing, personalized diagnosis, pharmacological engineering and molecular testing in the nanotoxicological field.

STATE OF THE PRIOR ART

The use of microleviers (or

Standard AFM, combined with detection by an external optical lever involving a laser or photodiode, for chemical force spectroscopy in molecular linkage analysis has already been demonstrated and implemented routinely (Janshoff et al., 2000, Angew Chem, Int Ed, vol 39, pp. 3212-3237).

Several patents and patent applications relate to chemical force spectroscopy using an AFM tip. US Patent 7,013,717 in the name of Veeco Instruments Inc. is contemplating grafting a single-stranded DNA on the tip of an AFM cantilever and complementary single-stranded DNA on the probed surface. International Application WO 2007/087653 in the name of Universitat Linz proposes a method for detecting 5-methylcytosine involving anti-5-methylcytosine antibodies grafted onto the tip of an AFM cantilever.

In addition, some recent commercial systems of chemical force spectroscopy are beginning to appear on the market as the automated machine proposed by the nAmbition or JPK companies.

In parallel, the use of piezoelectric integrated sensor AFM probes has already been demonstrated by several teams around the world.

In particular, in US Pat. No. 5,641,896, Karraï uses, for applications in near-field optical microscopy (SNOM), the force feedback provided by the piezoelectric probe to approach a fiber. optical glued on the probe a few tens of nanometers from the surface. US Pat. No. 6,240,771 describes the use of such a piezoelectric integrated sensor AFM probe with a sharp metal tip glued to the end of the probe to scan crystals with atomic resolution. Kaghesima and al. (2002, Applied Surface Science, vol 188, pp. 440-444) and Rensen and van Hulst (2000, Appl Phys Lett, vol 77, pages 1557-1559) have also demonstrated the use of this probe for AFM in a liquid medium with total immersion of the probe, respectively in an organic solution or in ultrapure water. Other teams used the piezoelectric probe outside the liquid, but with the tip immersed in a meniscus of liquid to probe the surface below (eg, Koopman et al., 2003, Appl. 83, pages 5083-5085).

Finally, some work has illustrated the use of these sensor probes integrated piezo electric ^¬ for analyzing liquid properties such as viscosity and density (US Patent 6,393,895 in the name of Symyx Technologies Inc.) or as chemical noses for detecting molecules selectively grafted (US Patent 6,393,895 and US Patent Application 2010/0068697 in the name of Drexel University).

Integrated sensor probes such as piezoelectric sensors have several advantages over a standard AFM cantilever because they make it possible to dispense with the conventional optical detection system of the latter. They are easier to integrate and use in extreme environments. But an essential point about their use in biological applications is the need to be able to dive into a biological solution keeping a good mechanical and electrical behavior. This condition is not obvious to satisfy because saline solutions used in biology like HEPES, Tris or PBS are extremely conductive due to the presence of many ions in the aqueous medium. Thus, the probe can not be used as such in these liquids because of the inevitable problems of electric leakage, short circuit or corrosion effects on electrodes of the piezo electric sensor ^¬.

The inventors therefore set themselves the aim to provide a system comprising a probe of AFM including an integrated sensor AFM probe piezo electric ^¬ useful for the analysis of molecular bonds in spectroscopy and chemical force in solutions buffers especially in solutions implemented in the field of biology. The AFM probe must also maintain good mechanical and electrical properties, have a good quality factor, when the latter is used in buffer solutions and be obtained by a simple method of implementation. As a reminder, the quality factor is a description of the damping of an oscillating system. It is given by the ratio between the energy transmitted and the energy lost. In the case of a probe, the quality factor represents the energy transmitted in the bandwidth around the resonance with respect to the energy contained outside the bandwidth.

STATEMENT OF THE INVENTION

The inventors solved the aforementioned technical problems and achieved the goal. Indeed, the present invention relates to an atomic force microscope probe comprising: i) a piezoelectric resonator provided with two electrodes and coated with an insulating layer; and

ii) a point fixed on said resonator coated and functionalized with at least one group or a molecule of interest.

The AFM probe according to the present invention is a piezoelectric integrated sensor probe. Indeed, it is based on a piezoelectric material associated with two electrodes. Advantageously, the body of the AFM probe according to the present invention is made of a single piezoelectric material. The two electrodes associated with the piezoelectric material make it possible to excite the resonator by applying an oscillating voltage. These same two electrodes are also used to analyze the response of the piezoelectric resonator ^¬ by a current-voltage amplifier converting the displacement current into a voltage. In other words, the AFM probe according to the present invention comprises only two electrodes for actuation and motion detection. Figure 1A illustrates the equivalent electrical model of the piezoelectric resonator in the air. C_quartz is the capacity proportional to the mechanical rigidity, L_quartz is the inductance proportional to the effective mass of the probe and R_quartz is the resistance proportional to the damping of the probe (because of the losses of energy). C_stray_quartz is a parasitic capacitance formed by the two electrodes around the piezoelectric material. Therefore, if one of the three mechanical properties previously mentioned, ie mass, stiffness and damping, is modified by the external environment of the probe, this causes a change in the electrical response detected by the amplifier.

In addition, since the resonator used in the context of the present invention is coated with an insulating layer and can be used in a biological solution. The liquid medium, but also the coating modify the mechanical properties and therefore the electrical properties of the piezoelectric resonator ^¬ as illustrated by the circuit of Figure 1B.

The piezoelectric resonator implemented in the context of the present invention is advantageously a piezoelectric material which has the advantage of being directly piezoelectric. Thus, no high pressure polarization step is necessary to align the domains and obtain piezoelectric properties, such polarization being necessary, for example, for piezoelectric polycrystalline ceramics. In addition, the piezoelectric material used in the context of the present invention retains the properties piezo electric ^¬ stable over time. This piezoelectric material is chosen in particular from quartz, topaz, tourmaline, berlinite, aluminum nitride, zinc oxide, cadmium sulphide, lead titanate, lead titano-zirconate, lithium niobate, lithium tantalate, potassium niobate, langasite, gallium arsenide, sodium barium niobate, bismuth germanium oxide, indium arsenide, antimonide, a piezoelectric polymer such as polyvinylidene fluoride (PVDF) and its derivatives, and mixtures thereof.

More particularly, the piezoelectric resonator used in the context of the present invention is made of quartz.

The electrodes used in the context of the present invention are advantageously metallic and in particular of a metal chosen from among gold, nickel, aluminum, copper, tin and silver.

The piezoelectric resonator provided with two electrodes implemented in the context of the present invention can be in all the configurations and geometries particularly envisaged in the AFM probes. More particularly, the piezoelectric resonator provided with two electrodes is in the form of a piezoelectric tuning fork (or "tuning fork" in English) with an electrode arranged on each of the branches of the tuning fork, or in an interdigitation configuration electrodes arranged on the two branches of the tuning fork.

The insulating layer used to coat the resonator of the probe according to the present invention corresponds to a passivation layer necessary to protect the electrodes of this probe from any contact with a liquid. It is present on the whole of the resonator and on a part of the electrodes of which it is provided. Indeed, the ends of the non-passivated electrodes ensure good electrical contact of the resonator and in particular the piezoelectric tuning fork to a connector connected to the downstream amplification chain. The critical part to be isolated and protected from the biological liquid medium via the deposition of an insulating layer is the resonator and in particular the piezoelectric tuning fork in operation. This coating must satisfy several conditions which are:

be chemically and mechanically stable in saline,

present a wide electrical resistance barrier to prevent current leakage of less than a few pico-amps (the displacement current or the probe have an order of magnitude of tens of picoamperes),

- have a low dielectric constant with respect to the frequency to avoid a strong parasitic capacitance effect (see Figure 1B) with the added parasitic capacitance C_stray_coating due to the coating layer,

- be consistent with the geometry of the probe where acute and singular angles may be present,

- be thin to avoid affecting too much the mechanical properties of the probe,

- be biocompatible and non-toxic.

Any insulating layer meeting the above conditions can be used to coat the resonator of the probe according to the invention.

Advantageously, this insulating layer may be a commercial electrophoretic cathodic or anodic paint used especially in the automobile industry for coating the structure of the car and protecting it against corrosion, but also in the food industry to coat the inner part of canned foods to avoid metal contact with food. In addition, this type of paint has already been used to isolate the tip of a tunneling microscope and a scanning electrochemical microscope. Examples of such a paint include BASF GLASSOPHOR electrophoretic paint and CLEARCLAD COATINGS Inc. CLEARCLAD electrophoretic paint.

As a variant, this insulating layer may be made of a material chosen from silicon dioxide, parylene or another [2, 2] paracyclophane derivative, polyimidine, aluminum oxide, aluminum nitride, a polymer vinyl and a hydrophobic silane including a hydrophobic silane as defined in the patent application US 2010/0068697. More particularly, in the context of the present invention, the insulating layer is a parylene layer.

The insulating layer, as previously indicated, must be thin to avoid overly affecting the mechanical properties of the probe. Therefore, its thickness will depend on the size of the piezoelectric ^¬ resonator and in particular the size of the tuning fork. Thus, the insulating layer implemented in the invention advantageously has a constant or substantially constant thickness on the piezoelectric resonator. This thickness is typically between 50 nm and 20 μπι, in particular between 250 nm and 10 μπι and, in particular, between 750 nm and 5 μπι. In the context of the present invention and atomic force microscopy in general, the fixed tip and in particular glued on the resonator probe the surface to be scanned, studied, characterized and / or analyzed. This tip is in the form of a wire, a fiber or a nanotube. In addition, it has an apex defined as the end portion of the tapered tip or nanotube subjected to interaction via the group or the grafted molecule (e) for the analysis of molecular links in chemical force spectroscopy.

The tip used in the context of the present invention comprises at least one material selected from platinum, chromium, chromium-gold, platinum-iridium, titanium-platinum, titanium nitride, tungsten carbide, tungsten, carbon or a mixture thereof.

Advantageously, the tip according to the invention may consist solely of only one of the previously listed materials. Depending on the constituent material, the tip may be more or less flexible, or more or less rigid. It may especially be platinum-iridium or platinum.

In a variant, the tip according to the invention may have two distinct zones, each consisting of one of the previously listed materials.

In fact, the work of the inventors has shown that, when immersed in a solution, a tip that is too soft will be distorted by the fluidic and electrostatic interaction with the liquid while avoiding its contact with the surface. The reduction of the length of this very flexible tip will increase its rigidity and therefore, its stability to stay upright during operation in liquid medium. But to fix such a flexible tip with a specific chemical functionalization, with a reduced length to increase sufficiently its rigidity, directly to the resonator and in particular directly to a branch of the tuning fork, will not be compatible with the operation of the tip alone immersed in a solution. In this configuration, the body of the piezoelectric resonator is kept outside the liquid to obtain the highest sensitivity with a high quality factor. The inventors have therefore proposed to add a long rigid tip between the resonator and in particular the branch of the tuning fork and the flexible tip with a specific chemical functionalization which will be in contact with the surface. In this variant, the tip presents:

a ^1st fixed element and in particular glued to the resonator (rigid portion) and

a ^2nd member (flexible portion) fixed and in particular bonded to the element ¹ and functionalized with a group or molecule of interest.

Advantageously, the element ¹ is a platinum wire / iridium and ^2nd a platinum wire, a graphite fiber or carbon nanotube. This 2 ^nd element constitutes the apex as previously defined.

Whatever the variant envisaged, the tip used in the context of the present invention has a total length of between 2 μπι and 5 mm, in particular between 3 μπι and 1 mm, and in in particular, between 4 μπι and 100 μπι. In the case of the variant of a tip with two distinct elements, the length of the element ¹ is comprised between 2 μπι and 4 mm and in particular between 3 μπι and 1 mm and the length of the 2 ^nd element is between 400 nm and 50 μπι and, in particular, between 800 nm and 10 μπι.

The apex of the tip of the probe according to the present invention is functionalized by at least one group of interest or at least one molecule of interest.

By "group of interest" (or "molecule of interest") is meant, in the context of the present invention, a group (or a molecule) capable of chemically interacting with another group or another molecule possibly present. (e) at a surface to be scanned, studied, characterized and / or analyzed by atomic force microscopy. This chemical interaction may involve a covalent bond between the molecule or a non-covalent bond such as a hydrogen or ionic bond.

In the context of the present invention, the group of interest may be a simple or complex chemical group. It is in particular selected from -OH, -C (0) OH, -NH _2, -CH _3, -C (0) 0 ^", -NH ₃ ^+, -C (0) H, -N _3, or alkene terminal.

In the context of the present invention, the molecule of interest is a molecule, natural or synthetic, especially biological. Advantageously, it is chosen from peptides, oligopeptides, proteins such as enzymes, antibodies or antibody fragments, cell or membrane receptors, polysaccharides, cells or cell parts such as organelles or cell membranes, nucleic acids such as DNA and RNA, and natural or synthetic molecules for therapeutic purposes.

The functionalization of the tip by the group or the molecule of interest consists in grafting, via at least one covalent bond, the group or the molecule of interest on the surface of the apex of the tip. This grafting can be direct or indirect. Advantageously, the functionalization of the tip by the group or the molecule of interest is indirect and involves a connecting arm covalently bonded, on the one hand, to the tip and in particular to the surface of the apex of the tip and, on the other hand, the group or the molecule of interest.

Any connecting arm commonly used in chemistry is usable in the context of the present invention. However, the linker implemented in the context of the present invention advantageously comprises a (hetero) arylene group. The linker may comprise only one (hetero) arylene group. Alternatively, the linker may correspond to a (hetero) arylene group linked to another group of the mobile molecular chain type such as a polyethylene glycol (PEG) group. This variant makes it possible to confer on the group or on the molecule of interest a greater degree of freedom with respect to the same group or to the same molecule. directly linked to (hetero) arylene grouping. This increase in the degree of freedom actually favors possible interactions with the surface to be scanned, studied, characterized and / or analyzed.

In the context of the present invention, the term "arylene group" means an aromatic hydrocarbon structure consisting of one (or more) aromatic ring (s) each comprising from 3 to 10 carbon atoms, in particular from 3 to 10 carbon atoms. to 8 carbon atoms and in particular 6 carbon atoms.

In the context of the present invention, the term "heteroarylene group" means a heteroaromatic hydrocarbon structure consisting of one (or more) heteroaromatic ring (s) each containing from 3 to 10 carbon atoms, in particular from 3 to 10 carbon atoms. to 8 carbon atoms and, in particular, 6 carbon atoms, the (or) heteroatom (s) may be N, 0, P, Si or S and in particular N, O or S. The heteroatoms can in particular interrupt an aromatic ring and / or separate two aromatic rings from an arylene group.

The present invention also relates to a method for preparing an atomic force microscope probe as previously described. The method according to the invention comprises the steps of:

a) depositing on a resonator provided with two electrodes an insulating layer whereby a resonator is obtained; b) fixing, on the resonator provided with two electrodes and coated with an insulating layer obtained in step (a), a tip;

c) optionally cut and / or engrave the tip set in step (b);

d) functionalizing the fixed and refined peak in steps (b) and (c) by a group or a molecule of interest. Step (a) of the process according to the invention consists in depositing an insulating thin layer as defined above on the surface of the resonator. This deposit can be performed by any technique to obtain a thin layer. Advantageously, said deposition is carried out by immersion ("dip coating"), by spray ("spray coating"), by spin coating ("spin coating"), by pyrolytic or electrochemically physical deposition.

More particularly, the deposition during step (a) of the process according to the invention is a pyrolytic physical deposit or an electrodeposition. Figure 3 and the experimental section below describe a system used for the implementation form involving electrodeposition. The person skilled in the art will know, depending on the nature and the composition of the insulating layer to be produced, the most suitable electrophoretic bath and operating mode. The experimental part below also shows a protocol involving a pyrolytic physical deposition of monomers, the corresponding insulating layer, in this case, the polymer obtained. It should be noted that known passivation methods for intrinsically conductive materials of simple geometry may be more difficult to apply when the piezoelectric resonator is in the form of a tuning fork. Indeed, passivation can alter the mechanical properties, particularly via a meniscus effect, of this system provided with electrodes and with a more complex geometry.

Results obtained for piezoelectric tuning fork resonators and described in the experimental part, it appears that:

when the insulating layer is an electrophoretic paint, the latter is preferably electrodeposited by implementing a voltage ramp of 0 V at a voltage of between 5 V and 50 V, in particular between 10 V and 25 V, and particular, of the order of 17 V (ie 17 V ± 2 V) and this, in a time between 1 min and 20 min, especially between 2 min and 10 min and, in particular, of the order of 5 min (ie 5 min ± 2 min). Such a method makes it possible to obtain a conformal deposit on the tuning fork without leakage of current, without loss of sensitivity and without altering the mechanical properties.

the passivation with known parylene for systems of simple shape of the beam or disk type and involving a pyrolytic physical deposition of monomers has never been described on piezoelectric resonator systems with a tuning fork type complex geometry, and done, the results improved obtained with such an insulating layer on tuning forks have never been described or suggested.

Step (a) may be repeated several times and in particular at least two, three or four times until an insulating layer having a satisfactory thickness is obtained.

Similarly, step (a) may comprise a step of annealing the insulating layer, in particular when the insulating layer is an electrophoretic paint, once the latter has been deposited. This annealing step is carried out advantageously between 80 ° C and 180 ° C and in particular between 110 ° C and 160 ° C for 5 min to 60 min.

Several studies have described different methods of attaching a tip to the end of a piezoelectric resonator (US Patent 5,641,896 and Giessibl, 2003, Rev. Mod Phys., Vol 75, pages 949-983), these The last ones can be used during step (b) of the method.

Advantageously, the tip is fixed on the resonator by means of a conductive adhesive such as conductive epoxy glue and in particular Epotek H21D glue. In the previously described embodiment where the tip is made up of two distinct elements, a conductive adhesive, identical or different, is used to adhere the element ¹ to the resonator on the one hand, and the 2 ^nd element ^1, element d 'somewhere else.

The tip is glued to be perpendicular to the surface to be scanned, studied, characterized and / or analyzed. For this reason, it must be glued to a certain angle on the resonator and in particular on the branch of the tuning fork, this angle being a function of the geometrical configuration of the probe holder of the atomic force microscope used thereafter.

The length of the tip, whether it includes one or two elements, is critical for accurate and artifact-free measurements. In particular, in the case of a tip with two elements, the length of the element ¹ (rigid portion) and also the length of the 2 ^nd (soft or flexible part) are very critical to retain good properties and for oscillation Avoid artefacts in the measurements due to deformation of the flexible tip during the approach of the tip to the surface. Therefore, it may be necessary to cut and / or engrave the tip of the probe or the various elements constituting it. In this case, step (c) is not optional.

Step (c) consists more particularly in a step of electrochemical etching of the tip comprising one or two element (s).

Electrochemical etching is a well known method in the field of STM to prepare sharp points whose radius has a few nanometers, necessary to obtain images with high lateral resolution. An electrochemical cut of the tip (from a few millimeters to several tens of micrometers long), but also a fine etching of the apex (below 100 nm) of the tip fixed on the resonator can be obtained. The geometry proposed by Fotino (1993, Rev. Sci., Instruction 64, pages 159- 167) for etching tungsten tips, allows more efficient etching by using the shear force of the bubble stream produced during the etching reaction.

The experimental part below proposes protocols for chemical etching to be used for platinum / iridium and / or platinum tips. On the basis of this teaching and on the basis of the knowledge of the state of the art, a person skilled in the art will be able to determine the optimum operating conditions for the chemical etching to be implemented during step (c) of the process. depending on the nature of the tip of the probe. The chemical protocol for functionalizing the tip of the probe with a group or a molecule of interest, during step (d) of the method may consist of any protocol for grafting, covalently and directly or indirectly, a such group or molecule at the surface of the tip of the probe.

Advantageously, this protocol implements the methods described in international applications WO 2008/078052 and WO 2009/121944 in the name of the CEA. These applications describe the grafting of molecules derived from cleavable aryl salts and in particular from aryl diazonium salts by using nonelectrochemical conditions.

More particularly, step (d) of the process according to the invention implements at least one element chosen from an aryl diazonium salt bearing a group of interest or a precursor of such a group, an aryl diazonium salt carrying a molecule of interest or a precursor of such a molecule, an aryl diazonium salt bearing an amino function or a precursor of such salts aryl diazonium.

By "precursor" is meant, in the context of the present invention, an entity separated from the group of interest, the molecule of interest or the aryl diazonium salt by a single process step and easy to implement. The precursors may have greater stability than the interest group, the molecule of interest or the aryl diazonium salt under the same environmental conditions. For example, arylamines are precursors of aryl diazonium salts. Indeed, by simple oxidation reaction, for example, with NaNO ₂ in an acidic aqueous medium, or with NOBF ₄ in an organic medium, it is possible to form the corresponding aryl diazonium salts. Similarly, an aryl diazonium salt bearing a -N¾ group may be used to functionalize the tip with an -OH group as shown in 111.2 (C) below.

Likewise, an aryl diazonium salt carrying a group --N¾ may be grafted onto the tip of the probe and then rediazotized so as to react with a molecule of interest or with a molecule carrying a group of interest. The molecule of interest and the molecule carrying a group of interest in the context of the present invention correspond to the molecule of interest of the international application WO 2009/121944. The example in point 111.2 (D) illustrates this particular form of implementation.

Those skilled in the art will find information on operating protocols and in particular as to the non-electrochemical conditions to be implemented in international applications WO 2008/078052 and WO 2009/121944 and other teachings resulting from the processes described in the latter. The present invention also relates to the use of a probe as defined above or capable of being prepared by a process as defined previously by atomic force microscopy and in particular for chemical force spectroscopy in biological solution and for imaging. AFM in biological solution. More particularly, these probes are used in dynamic mode at an excitation frequency close to or equal to their resonance frequency. As a variant, these probes are used in off-resonance dynamic mode at an excitation frequency remote from their resonance frequency or at a frequency corresponding to a harmonic different from their fundamental resonance frequency.

It should be noted that, until now, no piezoelectric piezoelectric type resonator has been used with a functionalized tip and / or in biology, so the use of this system for force spectroscopy chemical has never been described or even considered. At present, the three main methods for conducting AFM dynamic probes are (1) AFM-Frequency Modulation (AFM), also known as "AFM Non-Contact", (2) AFM Amplitude Modulation (or AM-AFM for Amplitude Modulation-AFM), also known as AFM Tapping, and (3) AFM Phase Mode (or PM-AFM for Phase Mode). AFM "). Depending on the method used, the different dynamic output variables changing (frequency change, amplitude, phase change) acquired during the AFM can be converted into interaction force values between the tip end and the surface. to scan, study, characterize and / or analyze.

In the context of the present invention, the probe is advantageously used in AM-AFM mode. In the latter, the signals acquired at the output by approach / withdrawal spectroscopy are the amplitude of oscillation and the phase shift relative to the relative distance tip-surface. These two pieces of information (amplitude and phase versus distance) can then be used to recover the chemical interaction forces between the tip and the surface using the formalism proposed elsewhere (Sader et al., 2004, Appl. 84, pages 1801- 18003, Katan et al., 2009, Nanotechnology, Vol 20, 165703, Uchihashi et al., 2004, Appl Phys Lett, vol 85, pp 3575-3577). .

The chemical interaction between the surface to be analyzed and the tip attached to the end of the resonator and in particular to the end of one of the branches of the Piezoelectric tuning fork will modify the resonance properties of the piezoelectric resonator. In other words, the detected changes in the resonance properties of the piezoelectric probe are directly related to the chemical information of the chemical bonds between the tip of the tip and the surface as shown in Figure 2. In this schematization the probe comprises a piezoelectric tuning fork (1) having a tip (2) functionalized by a group or a molecule of interest (3). It is brought into contact with a solid substrate (6) on which a receptor (5) is grafted, thus making it possible to assess the chemical interactions (4) between this receptor and the group or the molecule of interest.

The final chemical bond analysis uses several chemical force curves acquired with the probe for different charge rates (or approach-withdrawal rate) to obtain a Bell-Evans plot. This Bell-Evans scheme, described by Muller and Dufrêne, 2008 (Nature Nanotechnology, vol 3, pages 261-269 and Muller et al., 2009, Nature Chem Biol., Vol 5, pages 383-390) allows to obtain quantitative and qualitative chemical information on the energy landscape of chemical links. The dissociation rate k _{Q ff} , the potential barriers (free enthalpies), the transition distances can be directly obtained against the charge rate, but also against the temperature or the pH of the solution.

It is thus possible to observe if there is a phenomenon of attraction, repulsion or harpooning between the moiety or molecule of interest functionalizing the tip of the probe and the surface to be scanned, studied, characterized and / or analyzed.

Therefore, the present invention finds applications in the field of diagnosis, DNA sequencing by detecting hybridization between complementary DNA or RNA strands between a target strand attached to the tip of the probe according to the invention. present invention and a strand in solution or attached to a surface and / or the detection of compounds of therapeutic interest such as sugars, proteins, antibodies or oligopeptides.

Indeed, it is possible to graft onto a surface such as a surface of a solid support made of silicon, glass, metal, polymer or plastic, a membrane receptor particularly involved in a pathological state. The grafting can implement a method as described in international applications WO 2008/078052 and WO 2009/121944. The receptor thus grafted is maintained in a biological medium or a buffer adapted in particular so that it is in a native configuration. The probe according to the invention is functionalized by a synthetic or natural molecule which one wishes to know a potential potential in the treatment of the pathological state. The present invention will make it possible to know if there is a phenomenon of attraction, repulsion or harpooning between this molecule and the grafted receptor.

Other features and advantages of the present invention will become apparent upon reading the examples given below by way of illustration and not limitation and referring to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the electrical circuits, equivalent to the Butterworth-Van Dyke circuits, of a quartz: in vacuum or in air (Figure 1A), with a coating and immersed in a liquid (Figure 1B).

Figure 2 is a schematic diagram of the integrated piezoelectric sensor probe for determining chemical interactions at the molecular level.

Figure 3 illustrates the device for electrolytic deposition to obtain a resonator coated with an insulating layer.

Figure 4 shows a photograph of the tuning fork obtained during an electrochemical deposition of the electrophoretic paint either without optimization of the voltage parameters (no voltage ramp, the voltage applied directly) (Figure 4A) or by applying a slow voltage ramp (Figure 4B).

Figure 5 shows the voltage ramp (13) via the ramp voltage generator to optimize the electrophoretic paint layer (Figure 5A), with the simultaneous tracking of the current during electroplating during the first electroplating (14). ) and during the second electrodeposition (15) (Figure 5B).

Figure 6 shows the amplitude resonance curves in three different pipe configurations obtained for the same tuning fork coated with electrophoretic paint: in the air, with the only tip immersed in the solution and with the entire probe immersed in the solution.

Figure 7 shows the amplitude (thin lines) and the phase (thick lines) of the same type of tuning fork but coated by a layer of parylene: in the air (Figure 7A) and the whole probe immersed in a solution ( Figure 7B).

Figure 8 shows a detailed view of the arrangement of the tip carried by the tuning fork.

Figure 9 is a schematization of the configuration used to perform the electrochemical section but also the fine engraving of the apex of the tip fixed on a branch of the tuning fork piezoelectric ^¬ electric.

Figure 10 shows the optical microscope image of a platinum / iridium tip with a diameter of 50 μπι after the engraving of an apex end (radius of the end of the tip less than 100 nm).

Figure 11 shows the optical microscope images of a graphite tip with a diameter of 10 μπι before (Figure 11A) and after (Figure 11B) the engraving of a fine apex (radius of the tip of the lower tip). at 1 μπι).

Figure 12 shows different chemical functionalizations of the tip tip presented as a triangle, based on the chemistry of the diazonium salt.

Figure 13 shows the chemical functionalization by BSA of a surface based on diazonium salt chemistry. Figure 14 shows three approach-shrink curves performed on a surface of mica, coated with gold, clean without chemistry, with tips whose end is functionalized by φ-Ν¾ (Figure 14A), (p-CH ₃ (Figure 14B) and φ-ΟΗ (Figure 14C) No specific chemical grafting appears.

Figure 15 shows three approach-shrink curves performed on a surface of mica, coated with gold and functionalized by BSA, with tips whose end is functionalized by φ-Ν¾ (Figure 15A), (p- CH ₃ (Figure 15B) and φ-ΟΗ (Figure 15C) A specific chemical grafting signature appears clearly on the withdrawal curves The graph in Figure 15D shows a tip whose non-functional end is used to probe the surface of mica, coated with gold and functionalized by BSA.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

I. Piezoelectric tuning fork

The piezoelectric probe used is a quartz piezoelectric resonator with two metal electrodes obtained by physical deposition (sputtering or evaporation of a metal target) of a chromium or titanium bonding layer (thickness of the order of 50 nm) followed by a second deposit of nickel or gold (thickness of the order of 400 nm) in the form of a commercially available tuning fork (eg CITIZEN AMERICA CFS206). II. Insulating coating.

II.1. Electrophoretic paint coating. After an ultrasonic bath of the quartz resonator in ethanol and acetone, the quartz probe is coated in a bath of BASF GLASSOPHOR, electrophoretic cathodic paint, followed by an ultrapure water rinse Millipore and finally an annealing.

The device of Figure 3 comprises a piezoelectric tuning fork (1) and its two electrodes connected together (7) to an ammeter (8). The mass (9) is connected to the other connector of the ammeter with the negative electrode of the voltage ramp generator (10). The positive electrode of the voltage ramp generator (10) is connected to the platinum counter electrode (11) of the electrodeposition bath (12) containing the cathodic electrophoretic paint mixture diluted in ultrapure water. The use of anodic electrophoretic paint is possible and involves inverting the polarities of the voltage generator of the ramp.

The difficulty remains with the standard tuning fork because of the effects of the meniscus between the branches as shown in Figure 4A. But after several tests varying the dilution of the paint in the bath (40% in ultrapure Millipore water) and the annealing temperature (140 ° C for 15 min), a thin homogeneous layer, without meniscus effect and without bubbles trapped in the cured paint, is reproducibly obtained as shown in Figure 4B.

In addition, the voltage ramp rate is essential for controlling thickness and uniformity electrodeposited polymer layer of electrophoretic paint (see Figure 5). A ramp from 0 V to 17 V in 300 sec was found optimal (Figure 5A). The electrophoretic coating operation (electroplating in a bath + annealing) must be repeated 2 or 3 times in order to obtain an effective insulating coating with a thickness of 3 μπι. During the first electroplating in the bath, a current control determines a plateau phase during the ramp where the deposition of the layer is finished (current I is equal to about 300 μΑ), followed by a decrease. zero current indicating the end of the process (see Figure 5B).

The insulation of the coating was tested directly by studying the tuning fork coated in a buffer solution (pH = 8.2, Tris = 10 mM, NaCl = 10 mM, MgCl ₂ = 10 mM) by performing resonance curves, on the one hand, to show that the excitation and the detection of the signal coming from the probe is possible without problem of current leakage between the electrodes and the conducting solution, and, on the other hand, to test the influence of the environment liquid on the mechanical performance of the probe.

Thus, the probe was tested, firstly, completely immersed in a drop of buffer solution, then, with only the tip glued to the end of one of the branches of the tuning fork immersed in the buffer solution. The tip is a Platinum / Iridium wire, diameter 50 μπι and about 4 mm long, glued with epoxy glue on a branch of tuning fork coated with electrophoretic paint. Three configurations are the tuning fork in the air, the entire tuning fork in the buffer solution and only the tip of the tuning fork in the same buffer solution were studied. As shown in FIG. 6, the resonance curves obtained show a shift of the resonance frequency towards the low frequencies when the tip or the complete probe have been immersed in the buffer solution (f0 = 31 '630 Hz in the air, fO = 31 '595 Hz with only the tip in the solution, fO = 31' 146.8 Hz with the entire probe immersed in the solution). At the same time, the hydrodynamic effects of the liquid (Maali et al., 2005, J. Appl Phys., Vol 97, pages 074907.1-074907.6) increase the energy dissipation decreasing the quality factor of the probe (Q = 2000 in air, Q = 1300 with only the tip in a solution, Q = 60 with the entire probe immersed in the solution).

This means that the sensitivity of the force detection, depending on the ratio between the quality factor and the resonator stiffness constant, will be the best with only the tip immersed in the solution, keeping the body of the piezoelectric resonator coated with an electrophoretic paint outside the liquid.

II.2. Parylene coating.

The method is faster than the previous one to obtain a conformal coating of the probes. This is a one-step process. The parylene deposit for the passivation coating is a deposit pyrolytic physics of monomers, with the sample to be coated at room temperature. This process by pyrolysis is known to those skilled in the art and reference may be made for example to the protocol of EL Heintz et al., 2001, IEEE-NANO 2001. Proceedings of the 2001 Ist IEEE Conference on Nanotechnology, pp. 346-351 (2001).

Parylene is well known to be hydrophobic and biocompatible, and widely used to coat surgical tools. There are several types of parylene. The most effective for a passive deposition are parylene N ([2, 2] paracyclophane) and parylene C (Dichloro [2, 2] paracyclophane). The tuning fork piezoelectric ^¬ electrical was coated in a vacuum at room temperature, after having been rinsed with ethanol and acetone. The parylene thickness was characterized at 545 nm and conforms to the shape of the resonator. Parylene N was used in this case.

Again, as shown in Figure 7, amplitude and phase resonance curves were performed in air (Figure 7A) and after complete immersion of the same tuning fork in the buffer solution (Figure 7B).

The thinnest parylene layer compared to the electrophoretic paint layer allows a higher quality factor and a higher resonant frequency in the air but also in the tuning liquid (fO = 32'724 Hz, Q = 10'131 in air and fO = 31,223 Hz, Q = 109 the entire probe immersed in the solution). This means that the sensitivity of the force detection, depending on the ratio of the quality factor to the constant of the spring of the resonator, will be better with a thin layer of parylene.

III. Manufacture and chemical functionalization of the tip.

III.1. Fabrication of the tip on the piezoelectric resonator.

As shown in Figure 8, the body of the tuning fork (1) holds, on one of its branches, a rigid tip (2a) of platinum / iridium, maintained with a point of conductive epoxy glue (16), such as Epotek H21D glue. An adhesive point (17) is also added to the opposite branch so as to maintain mass symmetry during oscillation and thus maintain a high quality factor.

The vibration mode of the tuning fork and the relationship between the mass symmetry of the branches with the quality factor are explained in detail in the article by Ng et al. (2009, Ultramicroscopy, 109, pages 291-295). At the end of the rigid tip (2a), the end of the tip used for the chemical functionalization (2b) is also attached to the end of the rigid tip (2a) with a conductive epoxy glue point (18).

The length of the rigid tip (2a) and also the length of the flexible tip (2b) are very critical to maintain good oscillation properties and to avoid artifacts in the measurements due to the deformation of the flexible tip (2b). ) while approaching the point towards the surface. Therefore, these lengths were controlled by etching the tip electrochemically.

The device for this etching is shown in FIG. 9. The tuning fork (1) is mounted on a Z-translator (19) to control the size of the tip (2) to be etched inside the electrochemical solution (20). The counter-electrode (21) connects the electrochemical solution to a connector of a frequency generator (22). The material of the counter-electrode (21) and the chemical composition of the electrochemical solution (20) depend on the material of the tip to be cut or etched.

To engrave and cut the tip of a platinum / iridium wire having a diameter of 50 μπι, fixed to a tuning fork, a platinum counter-electrode having a diameter of 500 μπι and a length of 8 mm was used with a solution Electrochemical CaCl ₂ (5g) diluted in Millipore ultrapure water (30ml) with acetone (2ml).

In order to etch and cut a 10 μm diameter graphite wire fixed on the rigid platinum / iridium tip, a carbon counter electrode having a diameter of 4 mm and a length of 8 mm was used with an electrochemical solution of NaHCC> 3 diluted in Millipore ultrapure water (0.07 g / ml). Then, the other frequency generator connector is connected (23) to a platinum electrode (24) attached to the XYZ transducer (25) to make electrical contact with the end of the tuning fork tip (2) or the base of this tip (2) glued with the conductive epoxy on the branch of the tuning fork (1). Thus, the waveform, the frequency, the amplitude and the duration of excitation of the electrical signal delivered by the frequency generator (22) makes it possible to control the fine etching or electrochemical cutting of the tip. An ammeter in the circuit makes it possible to follow the etching process and to stop it in order to obtain a fine engraving, or to wait for a measured current of zero, which means that the tip is cut off.

For example, to cut the tip of a platinum / iridium wire, a continuous sinusoidal waveform of 22 volts (peak-to-peak) with a frequency of 400 Hz was used. To burn the apex of the thin platinum / iridium tip, bursts of 250 ms, spaced 750 ms, a 22-volt sine wave (peak-to-peak) with a frequency of 400 Hz were used (see Figure 10).

To cut the graphite tip, a continuous sine waveform of 16 volts (peak-to-peak) with a frequency of 46 Hz was used. To etch the apex of the thin graphite tip, bursts of 250 ms, spaced 750 ms, an 8-volt sine wave (peak-to-peak) with a frequency of 46 Hz were used (see Figure 11) . III.2. Chemical functionalization of the tip of the probe.

Thus, the end of the tip of the graphite probe has been selectively grafted with φ-ΟΗ (hydroxyl), φ-Ν¾ (amino) or (p-CH ₃ (methyl) groups, as illustrated in FIG. 12. The grafting Direct protein on the tip tip can also be considered.

A. Functionalization by φ-ΝΗ ₂

Solution No. 1 (100 ml of 0.1 M amine):

1.8107 g of 1,4-phenylenediamine (for example Sigma-Aldrich) are dissolved in 100 ml of 0.5M HCl (21.64 ml of 37% HCl in 500 ml of H ₂ 0). The following product which can be used for approximately 2 hours at ambient temperature is obtained:

Ct-NH ₃ ^* ^^ --- NH ₃ -C | -

Solution No. 2 (100 ml of 0.1 M HN0 ₂ ): 0.69 g of sodium nitrite NaNO ₂ (for example Fluka) is dissolved in 100 ml of Millipore ultrapure water to carry out the following chemical reaction:

NaN0 ₂ + ¾0 → HN0 ₂ + NaOH

Mix of solutions n ° 2 and n ° 1:

A magnetic bar is placed in the reaction bath. Then, 10 ml of solution No. 2 are added dropwise to 10 ml of solution No. 1, this being the first diazotization such that:

HNO ₂ + NH ₂ -cp-H ₃ ⁺ NCl ^~ → 2H ₂ 0 + NH ₂ -cp-N ₂ ⁺ Cl ^~ Stirring is maintained for 5 min, the reaction bath then taking an orange-brown color with foam on the surface. Then, 1.5 g of iron powder is then added as a reducing agent, thus allowing:

H ₃ ⁺ N-cp-N ₂ ⁺ + Fe + 3H ⁺ → Η ₃ ⁺ Ν-φ · + Fe ²⁺ + N ₂ The mixture is then heated at 40 ° C for a period of time to polymerize a layer of several diazonium salts with a -N¾ terminus on the immersed surface in the bath and to functionalize. The thickness and thus the density of the grafted φ-Ν¾ can be controlled by adjusting the At. An At 15 min was used for the functionalization of the apex of the graphite tip.

Finally, the functionalized surface was rinsed with acetone and ethanol, before being dried under nitrogen.

B. Functionalization by φ-ΟΗβ

0, 294 g of a hemi (salt of zinc chloride) of p-diazo-N, N-diethyl-m-toluidine (formula below) is diluted in 20 ml of Millipore ultrapure water:

H _g C ^

The solution is stirred for 30 minutes, before the addition of 1.5 g of iron powder in the bath.

The surface to be functionalized (i.e., the tip of the graphite tip) in this bath is immersed for 15 minutes, then rinsed with acetone and ethanol and dried under nitrogen.

C. Functionalization by φ-ΟΗ

The protocol followed is identical to that for functionalization by φ-Ν¾.

The surface of the functionalized apex is put, for 2 min, in a 15 ml bath of HC1 with 15 ml NaNC> ₂ to diazoter the -N¾ terminal again and replace it with a -N ₂ ^{+ group} .

Immediately thereafter, in a light-protected area, a drop of Millipore ultrapure water is placed on the surface bearing -N ₂ ^{+ groups} and held for 60 min before rinsing the surface with ethanol and water. dry with nitrogen. Thus, the -N ₂ ^{+ group} is replaced by a hydroxyl group making it possible to obtain a stable phenol.

D. Functionalization of φ-Protein Golden Mica (BSA)

A mica previously gilded by spraying or thermal evaporation of a gold layer a few tens of nanometers thick, on which we apply the protocol similar to that for functionalization by φ-ΟΗ; except that in the last step, instead of adding a drop of Millipore ultrapure water, a protein solution is deposited and left for 15 minutes, before rinsing with a buffer solution (such as PBS or 10 mM Tris). The prepared protein solution is a solution of 25 mg of BSA (bovine serum albumin) in 4 ml of Millipore ultrapure water. The surface thus prepared is shown schematically in Figure 13.

IV. Experiments of "Molecular Harpoon".

IV.1. Principle and conditions.

It is well known that by playing with the pH of the solution, the chemical functions at the tip of the tip can bind the groups -COOH or -N¾ of certain amino acids of the proteins via hydrogen bonds.

Therefore, all "Molecular Harpoon" experiments were performed in a buffer solution of Tris = 10 mM, MgCl ₂ = 10 mM and NaCl = 10 mm with pH = 8.2 to activate hydrogen bonds with the grafted proteins on the test surface. The functionalized tip of the probe is immersed in this solution.

The test surface is a freshly cleaved mica sample coated with gold and covered with diazonium salts to graft BSA. Functionalization of the gold-coated mica surface by the diazonium salt and BSA was verified by Fourier transform infrared spectroscopy or "FTIR spectroscopy".

Since the tip size of the graphite tip is too small to be characterized by FTIR spectroscopy or X-ray photoelectron spectroscopy (XPS) analysis, only the spectroscopic force spectroscopy shown in the results below can be used to verify the functionalization of the tip.

Thus, a combination of different chemical force spectroscopy experiments was conducted with the piezoelectric probe prepared according to the protocols of points I, II and III above in AM-AFM mode using the approach curves. withdrawal to the surface. The signals acquired at the output are the amplitude of oscillation and the phase shift relative to the relative distance tip-surface. These two information (amplitude and phase versus distance) can then be used to recover the chemical interaction forces using the formalism proposed elsewhere (Sader et al., 2004, Katan et al., Uchihashi et al., 2004).

IV.2. Results on an unfunctional surface.

In order to verify the selectivity of the "molecular harpoons" grafted at the end of the tip, the tests were first performed on a surface of clean mica, coated with gold without chemistry, with three different tip ends (φ- ΟΗ, φ-ΝΗ ₂ and cp-CH ₃ ).

Figure 14 illustrates three approach / withdrawal curves obtained under these conditions. The amplitude and the phase signal are acquired simultaneously with, in thick lines, the curves of approach towards the surface and, in thin line, the curves of withdrawal, when the tip is withdrawn.

The decrease of the amplitude and the increase of the phase are synonymous with the fact that the tip comes into contact with the surface. The approach and withdrawal curves for each case are very similar. This means that no chemical grafting occurs between the tip of the tip and the surface.

IV.3. Results on a functionalized surface with BSA proteins.

Using the same points whose end is functionalized by φ-ΟΗ, φ-Ν¾ or (p-CH ₃ , the same approach / withdrawal curves have been realized on a mica surface, covered with gold and grafted with BSA proteins.

Comparing the results obtained in Figure 15 with those in Figure 14, sharp differences appear in the withdrawal curves (thin line) showing different steep steps, more visible on the phase curves. Each step corresponds to the detachment of one (or more) chemical link (s) created during the approach of the tip and during the residence time of the end of the tip on the surface, controlled by the force feedback given by the piezoelectric probe.

Therefore, a clear signature of some specific molecular links between tip chemistry and surface biochemistry can be obtained by means of chemical force curves with a piezoelectric integrated sensor probe working in a saline environment necessary for study the biological material.

Based on previous work (Wang et al.,

2004, Langmuir, vol. 20, pages 7753-7759; Ebner et al., 2005, ChemPhysChem, Vol. 6, pages 897-900), the sensitivity of the system is of the order of one hundred picoNewtons.

The graph of Figure 15D shows the curves obtained using a graphite tip whose end is non-functionalized and the same surface of gold-coated mica and functionalized with BSA. In the latter case, no specific chemical grafting appears on the withdrawal curves (the approach and withdrawal curves for each case are very similar) which demonstrates that chemical steepness curves with steep steps during tip removal are actually due to the presence of specific and controlled chemistry, both at the tip end and at the the surface of the sample.

Claims

1) Atomic force microscope probe comprising:

i) a piezoelectric resonator provided with two electrodes and coated with an insulating layer; and ii) a point fixed on said resonator coated and functionalized with at least one group or a molecule of interest.

2) Probe according to claim 1, characterized in that said piezoelectric resonator is made of a piezoelectric material especially selected from quartz, topaz, tourmaline, berlinite, aluminum nitride, zinc oxide , cadmium sulphide, lead titanate, lead titano-zirconate, lithium niobate, lithium tantalate, potassium niobate, langasite, gallium arsenide, sodium barium niobate, bismuth germanium oxide, indium arsenide, antimonide, a piezoelectric polymer and mixtures thereof.

3) probe according to claim 1 or 2, characterized in that said electrodes are metallic and in particular of a metal selected from gold, nickel, aluminum, copper, tin and silver.

4) Probe according to any one of claims 1 to 3, characterized in that said piezoelectric resonator provided with said two electrodes is in the form of a piezo tuning fork. electric (or "Tuning fork" in English) with an electrode disposed on each of the branches of the tuning fork, or in an interdigitation configuration of the electrodes arranged on the two branches of the tuning fork.

5) Probe according to any one of the preceding claims, characterized in that said insulating layer is:

- a cathode or anodic electrophoretic painting commercial or

in a material selected from silicon dioxide, parylene or another derivative of

[2, 2] paracyclophane, polyimidine, aluminum oxide, aluminum nitride, a vinyl polymer and a hydrophobic silane.

6) Probe according to any one of the preceding claims, characterized in that said tip has:

- a ^1st element attached to the resonator and

a 2 ^nd element attached to the 1 ^st element and functionalized by a group or a molecule of interest.

7) A probe according to claim 6, characterized in that said 1 ^st element is a platinum / iridium wire and said 2 ^nd element a platinum wire, a graphite fiber or a carbon nanotube. 8) Probe according to any one of the preceding claims, characterized in that said group of interest is chosen from -OH, -C (O) OH, -NH ₂ , -CH ₃ , -C (O) O ^~ , -NH ₃ ⁺ , -C (O) H, -N ₃ and a terminal alkene

9) Probe according to any one of claims 1 to 7, characterized in that said molecule of interest is chosen from peptides, proteins such as enzymes, antibodies or antibody fragments, cell or membrane receptors, polysaccharides, cells or cell parts such as organelles or cell membranes, nucleic acids such as DNA and RNA, and natural or synthetic molecules for therapeutic purposes.

10) Probe according to any one of the preceding claims, characterized in that the functionalization of the tip by the group or the molecule of interest is indirect and involves a bonding arm covalently bonded, on the one hand, to the tip and, secondly, the group or the molecule of interest.

11) Probe according to claim 10, characterized in that said linker comprises a (hetero) arylene group. 12) Probe according to claim 11, characterized in that said connecting arm comprises a polyethylene glycol (PEG) group. 13) A method for preparing an atomic force microscope probe according to one of the preceding claims, said method comprising the steps of:

a) depositing on a resonator provided with two electrodes an insulating layer;

b) fixing, on the resonator provided with two electrodes and coated with an insulating layer obtained in step (a), a tip;

c) optionally cut and / or engrave the tip set in step (b);

d) functionalizing the fixed and refined peak in steps (b) and (c) by a group or a molecule of interest.

14) Method according to claim 13 characterized in that the deposit during said step (a is a pyrolytic physical deposit or electrodeposition.

15) Method according to claim 13 or 14, characterized in that said step (c) is a step of electrochemical etching of the tip.

16) Method according to any one of claims 13 to 15, characterized in that said step (d) implements at least one element selected from an aryl diazonium salt bearing a grouping of interest or a precursor of such a group, an aryl diazonium salt bearing a molecule of interest or a precursor of such a molecule, an aryl diazonium salt bearing an amino function or a precursor of such salts, aryl diazonium.

17) Use of a probe as defined in any one of Claims 1 to 12 or capable of being prepared by a process as defined in any one of Claims 13 to 16, for chemical force spectroscopy in biological solution.

18) Use of a probe as defined in any one of Claims 1 to 12 or capable of being prepared by a process as defined in any one of Claims 13 to 16, for AFM imaging in solution organic. 19) Use according to claim 17 or

18, characterized in that said probe is used in dynamic mode at an excitation frequency close to or equal to its resonance frequency. 20) Use according to claim 17 or

18, characterized in that said probe is used in off-resonance dynamic mode at an excitation frequency remote from its resonance frequency, or at a frequency corresponding to a harmonic different from its fundamental resonance frequency.