US20110174629A1

US20110174629A1 - Method for functionalising the wall of a pore

Info

Publication number: US20110174629A1
Application number: US12/866,340
Authority: US
Inventors: Aurélie Bouchet; Emeline Descamps; Pascal Mailley; Thierry Livache; Vincent Haguet; Francois Chatelain
Original assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2008-02-05
Filing date: 2009-02-05
Publication date: 2011-07-21
Also published as: FR2927169B1; CA2714185A1; JP2011511167A; FR2927169A1; EP2250122A1; WO2009109727A1

Abstract

The invention relates to a method for functionalising at least a portion of a wall of a pore of a carrier material, characterised in that it comprises: a) contacting the pore with a solution of electrically activated entities and positioning two electrodes in said solution in order to create inside the pore, and when an electric signal is applied between the two electrodes, a voltage drop capable of generating a localised deposit on said wall; and b) applying an electric signal between the two electrodes in order to activate the electrically activated entities and carry out said functionalisation function.

Description

The present invention relates to a functionalization process and more particularly to a process for biofunctionalizing at least part of the wall of a pore.
The term “pore” (or channel or capillary) denotes any cavity emerging or not emerging from a material.
The three-dimensional structure of a pore or channel or capillary makes its functionalization difficult. Specifically, techniques commonly used for the functionalization of flat surfaces, such as spraying or “spotting”, for example, become difficult or even impossible to implement for pores, channels or capillaries, this being all the more true the smaller their size.
In general, the known manufacturing processes do not enable or have great difficulty in functionalizing pores in a localized manner.
In order to functionalize a pore, it is common practice to use standard surface functionalization techniques. The most common use self-assembly properties of molecules on a support.
First, silanization achieves the covalent grafting of organosilanes to the surface of materials such as glass or silicon. This process usually consists in first performing a functionalization with a reactive group that will then allow the immobilization of the molecule of interest (Iqbal, S. and coworkers, “Solid-State nanopore channels with DNA selectivity”, Nature Nanotechnology, 2007, 2: p. 243 et seq.; Karnik and coworkers, Nano-Letters, 2007, 7(3): p. 547 et seq.; Kim, Y.-R. and coworkers, Biosensors & Bioelectronics, 2007. 22: p. 2926 et seq.; Wanunu, M. and coworkers, Nano-Letters, 2007, 7(6): p. 1580 et seq.). Despite its common use, the silanization process still remains poorly controlled and requires control of the parameters of the material, which is critical for the reliability of the surface modification and the stability of the deposit (nature of the surface functions, absence of contamination, roughness of the surface, etc.).
The formation of self-assembled alkanethiol monolayers (Lee S. B. and Martin C. R., Chemistry of Materials, 2001, 13 (10); p. 3236 et seq.; Smuleac V. and coworkers, Chemistry of Materials, 2004, 16 (14): p. 2762 et seq.; Jagerski and coworkers, Nano-Letters, 2007, 7 (6): p. 1609 et seq.) is based on the chemisorption of thiol groups on various metal surfaces such as gold (most commonly used), silver, platinum, copper, etc. This strategy has been exploited to achieve the functionalization of nanopores with thiolated DNA strands (Harrell, C. C. and coworkers, Journal of the American Chemical Society, 2004, 126, p. 15646 et seq.).
One of the major drawbacks of the techniques mentioned previously is the fact that the functionalization usually concerns not only the pore but also the reactive flat surface surrounding it, wherever there is a deposit of solution containing the organosilane or the alkanethiol, without localization. In the case of alkanethiols, the support is necessarily metallic.
A recent article by Joakim Nilsson and coworkers, entitled “Localized Functionalization of Single Nanopores” (Advanced Materials, 2006, 18, p. 427 to 431) describes the use of a focused ion nanobeam or nanoFIB (FIB: Focused Ion Beam) for the creation of a pore in a silicon nitride surface. The etching of the pore, the deposition of a layer of silicon dioxide under the beam of the FIB beam and a silanization lead to the creation of surface reactive functions that enable the localized attachment of DNA strands. However, this process is multi-step and requires prior silanization of the support.
Other authors have described the immobilization of conductive polymers onto dielectric surfaces by means of silanization of the support with a pyrrole-functionalized organosilane. Pyrrole monomers are then added to the medium and the polymerization is initiated by means of an oxidizing agent (Simon and coworkers, Journal of the American Chemical Society, 1982, 104: p. 2031 et seq.; Faverolle and coworkers, Chemistry of Materials, 1998. 10: p. 740 et seq.).
It has also been described in the literature that it is possible to functionalize pores in polycarbonate membranes, for example, by involving polymerizable species. It is thus possible to obtain conductive polymer tubes by performing the polymerization in a confined framework, delimited by physical barriers (pore, channel, etc.) (Martin, C. R. and coworkers, Journal of the American Chemical Society, 1990, 112, p. 8976 et seq., Martin, C. R., Science, 1994, 266 (5193): p. 1961 et seq.) or in the presence of external agents that structure the polymerization medium so that it takes place in an oriented manner (Carswell and coworkers, Journal of the American Chemical Society, 2003, 125: p. 14793 et seq.; Qu and coworkers, Journal of Polymer Science: Part A: Polymer Chemistry, 2004, 42: p. 3170 et seq.). The application of these structures is usually linked to the connections, which leads the majority of authors, where appropriate, to dissolve the matrix after creation of the polymer tubes. In this case, the pore is only a “mold”, the creator of the cylindrical shape of the generated polymers, and is not intended to be used as an active support.
Schematically, two different processes are known and used: chemical polymerization and electro-polymerization.
1) Chemical polymerization (abovementioned article by Martin, C. R., Science, 1994, 266 (5193): p. 1961 et seq.; Martin C. R., Advanced Materials, 1991, 3: p. 457 et seq.).
One means for obtaining polymer nanotubes is to perform a “chemical” polymerization of a monomer such as pyrrole, which is often cited. The experimental technique consists in placing a porous membrane (polycarbonate, etc.) between two aqueous solutions: a solution containing the pyrrole monomer and the other solution containing an oxidizing agent (for instance FeCl₃), which leads to polymerization at the points where the two solutions meet, i.e. in the pores of the membrane.
2) Electropolymerization (Menon, V. P. and coworkers, Chemistry of Material, 1996, 8: p. 2382 et seq.; Demoustier-Champagne and coworkers, European Polymer Journal, 1998, 34 (12): p. 1767 et seq.).
It is a matter in this case of first depositing onto one side of a membrane an adhesion layer (for example chromium) and of then depositing thereon a metallic layer (gold). The electropolymerization of the pyrrole may then be performed on this surface by means of a three-electrode electrolytic cell.
These processes make it possible to obtain, during one of the steps, pores functionalized with a polymer to obtain organized structures using the pore as “mold”. Functionalizations with biotins have thus been performed by Sapp and coworkers (Chemistry of Materials, 1999, 11: p. 1183 et seq.) by performing electrochemical polymerization of thiophene and pyrrole monomers bearing an amine function, allowing the grafting of a biotin derivative.
It will moreover be noted that pyrrole monomers bearing biomolecules are known per se (especially French patent applications FR 2 703 359 and FR 2 720 832).
Patent applications FR 2 787 582 and FR 2 784 466 concern a standard electropolymerization technique according to which an electrode is placed at the bottom of a non-emerging frustoconical microcuvette and another electrode is placed in an electrolyte, in an unspecified position. In this case, there is no functionalization of the surface of the microcuvette, but only of the electrode located at the bottom thereof. In other words, this known technique makes it possible to achieve deposition only on one of the electrodes.
The invention relates to a process for performing functionalization of a pore located at its surface, while simplifying the process. The basic idea of the invention is that of generating in the pore an electrical voltage gradient that can allow deposition onto the walls of the pore.
The invention thus relates to a process for functionalizing at least part of the wall of at least one pore of a support material, characterized in that it involves:

- a) placing the pore in contact with a solution of electro-activatable species and positioning two electrodes in said solution, on either side of the pore, so as to create inside the pore, and when an electrical signal is applied between the two electrodes, a voltage drop, especially of greater than 1000 V/m, capable of generating a localized deposition onto said wall,
- b) applying an electrical signal, potential difference or current between the two electrodes to achieve said functionalization.

By generating a high voltage gradient between the electrodes inside the pore, deposition is obtained on the wall of the pore(s), and also concomitantly on the anodic polarization electrode as observed during a standard electropolymerization deposition.
In the case of a non-emerging pore, an electrode is arranged at the bottom of a non-emerging cavity or at the bottom of the pore. The other electrode is placed at the end of the pore (d=0) or at a distance from the end of the pore (d>0), the voltage drop in the pore being sufficient to enable deposition onto the walls.
In the case of an emerging pore, the electrodes are placed at the ends of the pore (d=0) and/or at a distance from this end (d>0), the voltage drop in the pore being sufficient to enable deposition onto its wall.
For example, the field may reach 10⁶V/m, or even more.
The electrical signal may be constant or modulated as a function of time (periodic or not, pulsed, amplitude-modulated or frequency-modulated, step, ramp, etc.).
The support is not necessarily conductive. There is no need to line the interior of the pores with a conductive layer as described for electropolymerization, which greatly simplifies the experimental process. The electropolymerization is performed “remotely” with electrodes located on either side of the surface to be functionalized. It will be understood that the term “on either side of the pore” includes the case where d=0. The support, formed from organic or inorganic material, may be of insulating, semiconductive or conductive nature.
The remote electropolymerization does not require the presence of an oxidizing chemical agent.
The remote electropolymerization process may be performed in a single operating step.
The preferential formation of the polymer on all or part of the wall of the pore may be explained by the fact that since an electrical signal is applied across the pore, the voltage drop that it produces is mainly localized inside the pore, resulting in a strong potential gradient that induces a preferential formation of the polymer.
The process may comprise at least one repetition of a and b with a second solution of electro-activatable species. These species may be the same or, advantageously, different species, which makes it possible especially to arrange layers deposited on each other or side by side.
According to a first variant, the pore is open at both ends and a solution is placed in two compartments, in each of which emerges one end of the pore, at least one of the two compartments containing said electro-activatable species.
According to a second variant, the pore has only one emerging end and one of the two electrodes is placed at the bottom of the pore, the other electrode being placed in a compartment in communication with the emerging end of the pore.
After b, rinsing may be envisioned.
The support material may be silicon-based.
The electro-activatable species may be electro-polymerizable monomers, especially pi-conjugated conductive monomers, preferably a pyrrole, or alternatively may be species bearing electro-graftable functions, especially diazonium groups, or alternatively may be chosen from metals, metal oxides, catalytic particles, salts and metal complexes or may be formed by an electrophoretic paint.
The solution of electro-activatable species may comprise ligands.
The solution of electro-activatable species may comprise a mixture of electro-activatable species, especially an electropolymerizable monomer and said electro-activatable species coupled to ligands, for example grafted with an oligonucleotide.
In particular, the solution may have an oligonucleotide (pyrrole-oligonucleotide) probe, or more generally pyrrole coupled to a biomolecule.
The solution of electro-activatable species may include doping ions of interest, especially heparin and/or chondroitin.
The support material may be silicon-based.
The electrical signal may be a voltage of between 10 mV and 500 V and preferably between 100 mV and 10 V. The criterion to be respected is that the field inside the pore be sufficient to generate a deposit on its wall. The voltage difference may be applied for a time of between 10 μs and 100 s and more particularly between 10 ms and 100 s, for example in the form of a pulse. The voltage application time determines the thickness of the deposit.
The concentration of electro-activatable species may extend over a wide range, namely between 1 nM and 500 mM.
The process may have a step of detaching the electro-activatable species from the support, for example by destroying the support or by the action of ultrasound. The support may have at least one flared functionalization region (optionally comprising stages) that extends the wall of a pore.
The electro-activatable species may comprise probe molecules, and the process may comprise a step of association by recognition, especially of hybridization with complementary target molecules.
The process may then comprise a step of denaturing said association by recognition, optionally followed by a step of new association by recognition, especially of rehybridization.
The process thus enables the association by affinity of a species of interest and allows the manufacture of molecular assemblies.

Other characteristics and advantages of the invention will emerge more clearly on reading the description below, in relation with the drawings, in which:

FIG. 1 is a schematic diagram illustrating the process according to the invention,

FIGS. 2 a to 2 c represent a cell for receiving a chip containing a pore (assembly a),

FIGS. 3 and 4 illustrate an assembly adapted to a multipore chip, FIG. 4 being a detail thereof relative to the pore P₁under the experimental conditions,

FIG. 5 illustrates the format of the fluorescence test used to validate the functionalization of the pores,

FIGS. 6, 7, 8 a and 8 b represent different profiles of pores used in the examples, and

FIGS. 9 a and 9 b represent two examples of profiles of non-emerging pores.

The present invention relates to a process for functionalizing the surface of a pore with an organic or inorganic species, in particular with a polymer, which has been generated electrically by means of applying an electrical signal, especially an electrical potential difference across the pore. It makes it possible to achieve functionalization of pores or channels irrespective of their size (for example with a diameter of between 1 nm and 5 mm), in particular of pores or channels of micrometric and/or nanometric size, by:

- active groups that allow low-energy interactions, for instance:
- surface charges,
- molecular or biomolecular recognition groups, for instance biomolecules, reactive chemical groups or ion chelators,
- organic or inorganic species, especially for the purpose of reducing the aperture diameter of the pore.

The term “pore” or “channel” or “capillary” means any cavity, emerging or non-emerging, which is in a material. Their spatial distribution on the support may be defined (for example in the case of a manufactured membrane) or statistical (in the typical case of a sinter). The invention concerns any size of pore.
A pore 1 (FIG. 1) is placed between two leaktight compartments 3 and 4 containing a solution 2 of electro-activatable species that also bathes the pore 1. A potential difference, for example 2 V, is applied by a voltage source 5 between two electrodes 6 and 7 arranged across the pore 1, a few millimeters away from each other.
The electro-activatable species may be chosen especially from:

- electropolymerizable monomers such as pyrroles, thiophenes, indoles, anilines, azines, phenylene-vinylenes, phenylenes, pyrenes, furans, selenophenes, pyridazines, carbazoles, acrylates or methacrylates, and derivatives thereof. Preferably, the electro-polymerizable unit is a pyrrole. This monomer is readily functionalizable with a species of interest. Furthermore, polypyrrole is a biocompatible polymer, which is stable in air and in solution at physiological pH, which is an advantage in the context of an application in the field of biosensors,
- derivatives bearing electrograftable functions such as diazonium groups,
- metals and metal oxides, for example iridium oxide, catalytic particles, salts and metal complexes,
- electrophoretic paints.

The porous support may be of organic and/or inorganic nature and, without preference, conductive, semi-conductive or electrically insulating. Semiconductive materials such as silicon or oxide and nitride derivatives thereof are preferably used.
In Example I below, the monomer used is pyrrole. Specifically, polypyrrole is a polymer that has the advantage of being biocompatible and is therefore very advantageous for producing biosensors. It also has the advantage of being stable under the operating conditions of biochemical tests (physiological pH, aqueous buffers, presence of oxygen, etc.). It is also a conductive polymer of hydrophilic nature allowing its use in biological systems. Furthermore, chemically, the synthesis of pyrrole-biomolecule conjugates is very well controlled and takes place in good yield.
Polypyrrole, polycarbazole, polyaniline, PEDOT, polyindole and polythiophene belong to the group of pi-conjugated conductive polymers.
It is known that the corresponding monomers are electropolymerizable, namely they lead to the formation of a polymer under the effect of application of an anodic potential to the surface of an electrode. These species thus behave in the same way taking into account solvent and oxidation conditions that are not identical from one species to another.

POLYPYRROLE—IMPLEMENTATION OF THE EXAMPLES

I.—Materials
A) Reagents and Consumables:
Pyrrole is divided into aliquots at a concentration of 1 M dissolved in acetonitrile and then stored at −20° C. Pyrrole bearing an oligonucleotide was prepared according to the protocol described in patent application FR 2 703 359.
The DNA sequences used are as follows:

- Py-probeZip6: Py^5′-(T)₁₀-GAC CGG TAT GCG ACC TGG TAT GCG^3′ (Py-SEQ ID No. 1)
- Target-Zip6-bio: biotin^5′ CGC ATA CCA GGT CGC ATA CCG GTC^3′ (biotin-SEQ ID No. 2)

The chips used are silicon oxide or silicon nitride membranes. They comprise nine pores of micrometric size distributed over an area of 2×2 cm².
B) Buffers Used (Given as a Guide):

- Electropolymerization buffer: 6 g/L Na₂HPO₄/NaH₂PO₄, 2.9 g/L NaCl, 10% v/v glycerol, 2% v/v acetonitrile (v/v=volume per unit volume).
- Hybridization buffer: 0.02 M Na₂HPO₄/NaH₂PO₄, 1.1 M NaCl, 5.4 mM KCl, 4% v/v 50× Denhardt, 0.2% v/v salmon sperm DNA, 0.3% v/v Tween 20 at pH 7.4.
- Rinsing buffer: PBS 5 tablets/L, NaCl 23.375 g/L, Tween 20 0.15% v/v.

C) Experimental Assemblies for the Remote Electro-Polymerization:
Two experimental assemblies were validated.
a) Electropolymerization Cell (see FIGS. 2 a to 2 c):
The material of this cell C is “Delrin” (registered trademark) polyoxymethylene known as “Delrin POM”. The cell is split into two leaktight compartments 3 and 4 by introducing into its rectangular receptacle 34 a support piece 8 comprising one or more pores 1. Platinum wires, for example, may be introduced into each of the compartments 3 and 4 through the apertures 9 ₁and 9 ₂of the lid 9 to form the electrodes. 9 ₃and 9 ₄denote the apertures for fixing the lid 9 to the cell C, and 9 ₅and 9 ₆the holes for fixing the screws to the cell C.
b) Assembly on a “Multipore” Chip:
If it is desired to functionalize differently each of the pores of a chip, it is appropriate to work in parallel with a multichannel voltage source or several sources of monochannel voltage, for example a multichannel potentiostat, or several monochannel potentiostats.
In the assembly described in relation to FIG. 3, the chip 10 has 9 pores P₁. . . P₉. Each pore is isolated between two leaktight compartments (3 ₁, 4 ₁; 3 ₂, 4 ₂; . . . 3 ₉, 4 ₉). In other words, it is possible to place each of the nine pores P₁, P₂. . . P₉which do or do not have the same diameter in contact with a different solution S₁, S₂. . . S₉of electroactivatable species. Each of these compartments is equipped with electrodes to which is applied a given electrical voltage difference and which are arranged a distance d from the end of each pore. The distance d may or may not be the same for the two electrodes of the same pore. It may be different from one pore to another according to the functionalization needs. There are thus nine working electrodes E_t1, E_t2, . . . E_t9which are or are not connected to the same potentiostat (or more generally to the same voltage source) and nine counterelectrodes that may be coupled to reference electrodes E_a1, E_a2, . . . E_a9. The solutions S₁, S₁, . . . S₉may or may not be the same. A “multi-channel” potentiostat PT allows these voltages to be applied simultaneously (even if the values thereof are different).
Each pore (P₁, . . . P₉) of a chip 10 is positioned between two leaktight compartments 3 ₁, 4 ₁, . . . ; 3 ₉, 4 ₉which have herein a volume of 10 μl.
The assembly comprises one or two printed circuits 21, (FIG. 4) having an integrated circular electrode 23, 28 that may be connected to an external potentiostat. Into at least one of the electrodes are pierced two holes 26, 27 for introducing and evacuating liquid via polytetrafluoroethylene capillaries. Two leaktight compartments 3 ₁and 4 ₁are created between the chip 10 and the printed circuits 21 and 22 by virtue of toric seals 24 and 25 (FIG. 4). In order to achieve the electropolymerization so as to obtain a localized deposit 30 on the walls of the pores, the following are used:

- either the electrodes integrated into the printed circuits,
- or electrodes (metal wires, not shown) introduced into capillaries on either side of the pore,
- or electrodes dipping directly into a compartment. In this case, a plastic card (not shown) is used instead of the printed circuit.

II) Implementation
A) Preparation of the Substrate:
a) Cleaning:
In a first stage, the chip undergoes cleaning (67% sulfuric acid, 33% hydrogen peroxide v/v) in a white room so as to remove any contaminant of organic nature. The chip is dipped for 10 minutes into the solution and then rinsed with a circulation of water until a resistivity of 9 MΩ.m is obtained. The chip is then dried in an oven at 180° C. for 10 minutes. It may then be stored at room temperature.
b) Increase in Hydrophilicity of the Surface by Applying an Oxygen Plasma:
This step makes it possible to make the surface hydrophilic, which is advantageous for the purpose of filling the pore, irrespective of its size, with a predominantly aqueous solution. The chip is thus placed for 45 seconds in an O₂plasma at a power of 100 W.
c) Polypyrrole Deposition:
A polymerization solution containing 20 mM of pyrrole and 5 μM of py-probeZip6 in electropolymerization buffer was used to perform polymer depositions in the pores.
The two assemblies (a and b above) were used.
In each case, the chip comprising emerging pores is introduced so as to be between two compartments. The polymerization solution is introduced into the two compartments. An electrode is inserted into each compartment and a voltage difference equal to 2 V is applied. It will be noted in practice that a voltage of between 10 mV and 500 V and preferably between 100 mV and 10 V may be used depending on the size of the pores, the pursued aim being to obtain a sufficiently high field inside the pore in order for the deposition to take place on its wall. Monitoring of the deposition process is performed by plotting the curve of the change in current intensity as a function of time: the shape of this curve (presence or absence of an electrical signal) makes it possible to see whether the liquid has penetrated into the pore (electrical contact) or not (absence of an electrical signal). The chip is then removed from the assembly and then rinsed with water, dried with compressed air and stored dry at 4° C.
d) Checking of the Functionalization by Fluorescence Microscopy
In order to check the formation of a polypyrrole deposit, fluorescence microscopy is used. The test format used is illustrated in FIG. 5. It is performed by placing drops of 15 μl of liquid onto a pore. The pore is first saturated with hybridization buffer (5 minutes at room temperature). Next, one drop of biotinylated target at 100 nM in hybridization buffer is added (15 minutes, room temperature). The chip is then rinsed thoroughly with the rinsing buffer. Each pore is then incubated in a streptavidin-phycoerythrin solution (SAPE) at 10% (v/v=volume per unit volume) in rinsing buffer (15 minutes at room temperature). The chip is then placed between a slide and watch glass to be observed by fluorescence microscopy at 530 nm, the emission wavelength of phycoerythrin.
Biofunctionalization of a Pore with Nucleic Acids

Example I

Assembly a

i) Creation of a Polypyrrole Deposit
A multipore chip comprising pores of variable shape ratio (shape ratio Rf=diameter of the pore/thickness of the membrane in which it is pierced) and which has undergone a plasma treatment is introduced into the “Delrin POM” two-compartment cell described above. The polymerization solution is introduced successively into the two compartments of the cell. It is formed from 20 mM of pyrrole and 5 μM of pyrrole-probe Zip6 (py-probe Zip 6) in electropolymerization buffer. Next, two platinum wires are introduced, one on either side of the chip. The first is connected to the counterelectrode coupled to the reference electrode of the potentiostat, and the second to the working electrode. A potential of 2 V is applied for a given time (between 100 ms and 1 s) between the two electrodes (working electrode and counterelectrode). The chip is then removed from the cell, rinsed thoroughly with water and then dried with compressed air and stored at 4° C.
The functionalization efficacy is checked by fluorescence microscopy according to the process described above.
ii) Results
The manipulation was performed with pores with different shape ratios:

- R_f=35: pore 70 μm in diameter in a membrane 2 μm thick and with a side length of 500 μm (see FIG. 6).

Fluorescence emission is observed in the form of a circle of light present on either side of the chip. Its dimensions correspond to those of the contour of the pore, which makes it possible to deduce that the functionalization technique is effective and allows a deposition of polymer located on the walls of a pore of micrometric size.
The scanning electron microscopy images show a fine layer of deposit—of about 30 nm—on the contour of a pore. This deposit is absent from a non-functionalized pore.

- R_f=1 with a circular pore 18 μm in diameter in a membrane 20 μm thick: in the case of a pore pierced into a square membrane with a side length of 50 μm, and 20 μm thick. The deposition is performed at a voltage of 2 V applied for 100 milliseconds (FIG. 7).

Performing the fluorescence test outlined above leads to the presence of rings of light on each side of the pore, which certifies the effective and localized functionalization of the pore with a polymer bearing oligonucleotides.

- R_f=0.25 pore with a diameter of 2 μm in a membrane 8 μm thick. The pore 2 μm in size is at the bottom of a cone with a largest diameter equal to 10 μm. The environment of the pore is said to be of the “funnel” type (FIGS. 8 a and 8 b).

The fluorescence microscopy images show that the wall of the pore has been functionalized in a localized manner, as has the contour of the top of the cone (of dimension 10 μm).
This constitutes a result that leaves the possibility of controlling the place of functionalization according to the morphology of the environment of the pore.

Example II

Assembly b

i) Creation of a Polypyrrole Deposit
A multipore chip comprising pores of variable shape ratios R_fwhich have undergone an O₂plasma treatment is placed in the assembly b described above (FIGS. 3 and 4). The polymerization solution is successively introduced into the two compartments. It is formed from 20 mM of pyrrole and 5 μm of pyrrole-probe-Zip6 in electropolymerization buffer. Next, two electrodes are positioned on either side of the chip. The first is connected to the auxiliary electrodes and reference electrodes of the potentiostat and the second to the working electrode. A potential of 2 V is applied for 100 ms between the two electrodes. The chip is then removed from the cell, rinsed thoroughly with water and then dried with compressed air and stored at 4° C.
ii) Results
The functionalization efficacy was checked by fluorescence microscopy according to the process described above. Each of the 9 pores of the multipore chip may be studied independently, optionally with a specific functionalization for each pore.

- R_f=1 pore 18 μm in diameter in a membrane 18 μm thick.

Fluorescence microscopy confirms that the surface functionalization process also functions for all the abovementioned values of R_fusing this assembly (presence of a fluorescent ring).
Controls were performed to establish the specificity of the biochemical interaction resulting in the functionalization characterized by fluorescence emission. These controls were performed on pores 18 μm in diameter (R_f=1) of a multipore chip:
a) Electrical Potential
To do this, 15 μl of a polymerization solution composed of 20 mM of pyrrole and 5 μM of pyrrole-probe-Zip6 in electropolymerization buffer are deposited on a pore and left in contact with the surface for 5 minutes. The chip is then rinsed with water and dried with compressed air (procedure identical to that performed after a remote electropolymerization). The chip is stored at 4° C. and then undergoes the fluorescence test procedure described previously. No fluorescence was observed, which is proof that the application of an electrical potential is necessary for functionalization of the pore.
b) Pyrrole-Oligonucleotide Conjugate Adsorption
Another control was performed for the purpose of studying whether the application of a potential promotes the adsorption of DNA onto the surface of the support. To do this, a solution of py-ProbeZip6 at 5 μM in electropolymerization buffer was used (no pyrrole in this case), and an electrical potential difference was then applied according to the same protocol as that used for the pyrrole/py-ProbeZip6 copolymer. The absence of fluorescence shows that, under the working conditions, the non-specific adsorption of pyrrole-oligonucleotide conjugate is negligible.
c) Non-Specific Adsorption During the Revelation Procedure
i) On a non-functionalized pore that has not been in contact with the polymerization solution, the hybridization and revelation procedure described previously is performed, the first step being saturation of the pore with hybridization buffer. Under the operating conditions, there is no spurious fluorescence associated with the non-specific adsorption of the biotinylated DNA target.
ii) On a non-functionalized pore that has not been in contact with the polymerization solution, the revelation procedure described previously is performed, the first step being saturation of the pore with hybridization buffer, followed by incubation for 15 minutes in hybridization buffer alone (without the corresponding target). SAPE diluted in rinsing buffer is then added according to the protocol described above (II, A, d). The fluorescence microscopy image confirms that, under the operating conditions tested, SAPE is not adsorbed onto the surface of the support.
d) Denaturing of the Hybridization
On a functionalized pore that has undergone the fluorescence revelation procedure described previously, rinsing is performed with 0.2 M NaOH solution for 2 s, followed by thorough rinsing with water and drying with compressed air. The pore is then observed by fluorescence microscopy at the usual wavelength and with the same camera sensitivity parameters (luminosity, contrast). Disappearance of the fluorescence after denaturing of the hybridization is observed. This shows the specificity of the fluorescence emission observed in the case of a complementary hybridization.
e) Fluorescence after Rehybridization
The fluorescence of a pore that has undergone denaturing via the addition of NaOH (d) reappears after rehybridization of the DNA probes with their complementary target. The experimental procedure followed for this second hybridization and its fluorescence revelation is exactly the same as that described previously for the hybridization.

Example III

Functionalization with Iridium Oxide

i) Creation of an Iridium Oxide Deposit
An iridium oxalate solution is prepared according to the following protocol (described in the article by A. M. Marsouk, Analytical Chemistry, 2003, 75: p. 1258 et seq.): 75 mg of IrCl₄monohydrate are dissolved in 50 mL of distilled water; 0.5 ml of 30% hydrogen peroxide, 365 mg of potassium oxalate hydrate and anhydrous potassium carbonate, to adjust the pH to 10.5, are then added. Stirring for 10 minutes is required between each addition of product. The solution is then heated at 90° C. for a few minutes, until a final dark blue color characteristic of the complexed form of iridium (IV) is obtained. The solution may then be stored for several months at 4° C.
A multipore chip comprising pores with a shape ratio Rf=1 and which has undergone an O₂plasma treatment is placed in the assembly b described above (FIGS. 3 and 4).
The iridium oxalate solution is successively introduced into the two compartments. Next, two electrodes are placed on either side of the chip. The first is connected to the auxiliary and reference electrodes of the potentiostat and the other to the working electrode. A potential of 0.80 V or 0.85 V or 0.90 V is applied for a time of 5 s or 10 s. In the same manner as for the polypyrrole deposits, the monitoring of the deposition is performed by chronoamperometry so as to check the correct electrical contact across the pore.
The chip is then removed from the cell, rinsed thoroughly with water and then dried with compressed air and stored at 4° C.
ii) Results
The functionalization efficacy was checked by scanning electron microscopy (SEM).
The images obtained show that a deposit is created on the walls of the pore and only inside the pore, the surrounding surface being totally clean. A control pore, which has not undergone functionalization, does not have any deposit on the inner walls of the pore. This shows that the functionalization process also functions for electro-activatable species such as these metal oxides.
The texture of the various deposits obtained appears to be different from one pore to another, which may possibly be explained by variable degrees of oxidation of the iridium. Thus, the electrochemical half-reactions involved in the case of iridium oxides are as follows:
Ir(OH)+H₂O<->Ir(OH)₂+H⁺+e⁻ (−0.1 V)
Ir(OH)₂+H₂O<->Ir(OH)₃+H⁺+e⁻ (0.3 V)
Ir(OH)₃+H₂O<->Ir(OH)₄+H⁺+e⁻ (0.8 V)

- or IrO₂+2H₂O+H⁺+e⁻ (divergence according to the publications).

Given the heterogeneous visual aspect of the deposits observed by SEM inside the pores, it is possible that, under the experimental conditions used, the same average degrees of oxidation of iridium are not obtained in the oxide(s) formed.

Example IV

Production of Structured Objects by the Remote Electrodeposition Technique

i) Creation of a Polypyrrole Deposit:
A polycarbonate membrane, comprising nanometric-sized pores, may be inserted either into the assemblies a or b. Deposits of a copolymer of pyrrole/pyrrole coupled with an oligonucleotide may then be obtained inside these pores according to the protocol described previously.
ii) Detachment of the Deposits Formed from Their Support:
The membrane is then rinsed with water and introduced into a dichloromethane bath in order to dissolve the polycarbonate and to release into solution the objects created inside the pores. The electro-activatable species may also be detached without dissolving the membrane, for example by the action of vibrations created by ultrasound. Via successive filtrations, the objects of interest are then isolated; these are pyrrole nanostructures bearing DNA probes having the shape of the pores of the membrane.

Example V

FIGS. 9 a and 9 b are two variants of non-emerging pore shapes with an electrode 61 that covers all or part of the bottom of the cavity 60. In the case of FIGS. 9 a and 9 b, the area of the pore in which the deposition takes place corresponds to the only necking region 62, 62′ that concentrates the field. Thus, the shape of the pore makes it possible to specifically localize the deposition onto only a part of its wall.
For the deposition of a polymer such as polypyrrole or a functional derivative, polarization of the electrode 61 at the bottom of the cavity 60 may be anodic or cathodic in order, respectively, to form or not to form a deposit of the same polymer onto the surface of the electrode 61, in addition to the deposit on the necking region 62, 62′.
Conclusion:
The functionalization technical process according to the invention is efficient and relatively easy to implement.
The reproducibility of the deposits is satisfactory and may be further improved by controlling the manipulation parameters more strictly:

- fixed inter-electrode distance
- temperature control
- hygrometry control.

This novel technique makes it possible efficiently to control the localization of functionalization of the surface of a pore with reactive groups. Specifically, this localization is essentially associated with the organization of the electrical field lines within the pore, which is itself dependent on the structure of the environment of the pore (namely its geometry).
The process according to the invention has the advantage of being inexpensive:

- in financial terms, since only limited equipment is necessary: potentiostats, electrodes, etc.,
- in terms of time, since the deposition procedure lasts only a few minutes.

The experimental device is furthermore of relatively small bulk and is easy to transport.
The strategy is adaptable to any type of porous support, of organic or inorganic nature, conductive, semiconductive or insulating, irrespective of the pore dimension.
To measure whether the liquid has penetrated into the pore, one means is to check whether the electrical contact between the two electrodes is effective, in which case the chronoamperogram measured during the deposition (for example of polypyrrole) has a signal of non-zero intensity.
To characterize the formation of the deposit, for example of polypyrrole, it is possible to use fluorescence microscopy, or even confocal fluorescence microscopy in order to have a three-dimensional view of the fluorescence inside the cavity. Scanning electron microscopy may also make it possible, for example in the case of deposition of iridium oxide, to characterize the deposit formed.
Since electro-activatable species, in particular electropolymerizable species (pyrroles, thiophenes, etc.), can be functionalized, this technique is entirely transposable to the immobilization within pores of active groups involved in low-energy interactions, for instance ionic groups, peptides, antibodies, enzymes or ion chelators, for example.
The polymer may also serve as a “starting layer” for a localized deposition, in particular using doping anions of interest such as polysaccharides (for example heparin, which promotes the adhesion of cells (Zhou et al. Reactive & Functional Polymers, 1999. 39: p. 19 et seq.)) or surfactants.
Stacks of “multilayer” type may be envisioned starting with the deposits obtained by “remote electro-deposition”. It is thus possible to prepare a localized deposit (first layer) having, for example, a certain surface charge or a reactive chemical group that promotes the binding of a given second layer of organic or inorganic species relative to the bare support.
The technique has experimentally enabled DNA immobilization, the latter being a biomolecule of modular aspect, i.e. which can be used as a biomolecular recognition element for the immobilization via hybridization of a molecule of interest functionalized with complementary DNA targets. The process also made it possible to immobilize a species of biological interest, biotin, by hybridization with immobilized DNA probes with a biotinylated complementary target, which underlines the modular aspect of the technique.
The experimental device may furthermore integrate thermal and optical devices allowing, for example, crosslinking experiments or visualization of the organization of the deposits produced.
Several applications may be envisioned in the field of ultrasensitive miniaturized biosensors. Bio-functionalized porous membranes may find applications in the health sector, in particular for detecting (bio)molecules present in small amount in biological samples. Many research teams have thus directed their studies toward the design of systems for detecting individual molecules. These molecular Coulter counters have given encouraging results with protein pores (Vercoutere, W. and coworkers, Nature Biotechnology, 2001. 19: p. 248; Bayley and Cremer, Nature, 2001. 413: p. 226 et seq.).
The considerable advantage of synthetic pores relative to the latter lies in the possibility of:

- modulating their properties by creating charges or reactive groups at the surface,
- controlling the geometry (pore diameter, membrane thickness, etc.),
- performing easier integration in a microfluidic device.

The process is also suitable for applications in the field of micro- or nanochromatography (ion exchange, steric exclusion, affinity chromatography or adsorption chromatography) for the purification of (bio)molecules.
Functionalized pores may also be useful for capturing bacteria or cells, especially by immobilizing heparin or chondroitin inside a pore.
Functionalized porous membranes also conventionally find applications in purification and filtration systems (for water, effluents, etc.), the presence of ion chelators or ion exchangers at the surface of the pores being able to allow the selective separation of certain components of the liquid passing through the membrane.
The immobilization of catalytic particles, for example containing metals such as palladium or platinum, in pores, via the described process, may allow the creation of micro- or even nanoreactors for performing chemical reactions, for instance hydrogenations. By being able to run these reactions in parallel using networks of pores distributed in a membrane, micro/nano-combinatorial chemistry becomes possible. The electrodeposition of metals via this technique may also find applications in the fields of catalytic exhausts (gas-phase catalysis).
Finally, the process is compatible with the use of molecular imprint techniques, which opens advantageous applications in the field of capillary electrophoresis, for example.
It will be understood that the solution of electro-activatable species for which the possible presence of DNA probes has been mentioned above, may more generally optionally comprise ligands, namely:

- molecular and/or biomolecular recognition elements, especially nucleotides, oligonucleotides, polynucleotides, DNA, RNA, PNA, peptides, polypeptides, antibodies, antigens, enzymes, proteins, amino acids, glycopeptides, biotins, haptens, sugars, oligosaccharides, polysaccharides, lipids, glycolipids, steroids, hormones or receptors,
- other affinity groups, especially ion chelators and ion exchangers,
- chemically active functions, especially amine, amide, oxyamine, active ester, alcohol, carboxylic acid, alkyne, thiol, epoxide, anhydride, acyl chloride or aldehyde functions, and derivatives thereof,
- single objects (in the context of an individual or collective immobilization of objects), especially microparticles and nanoparticles. The particles may be and/or may contain biological cells and/or cell components and/or products, especially cell lines and/or globules and/or liposomes and/or cell nuclei and/or chromosomes and/or DNA or RNA strands and/or nucleotides and/or ribosomes and/or enzymes and/or antibodies and/or protids and/or proteins and/or peptides and/or active principles and/or parasites and/or bacteria and/or viruses and/or pollens and/or polymers and/or biological factors and/or growth stimulants and/or inhibitors and/or beads suspended in a liquid and/or bioparticles suspended in a solution and/or molecules. The manipulated particles may be and/or may contain insoluble solid particles such as magnetic particles and/or dielectric particles, or conductive particles, or functionalized particles, or pigments, or dyes, or protein crystals, or powders, or polymer structures, or insoluble pharmaceutical substances, or fibers, or yarns, or carbon nanotubes, or aggregates (clusters) of small size formed by agglomeration of colloids,
- groups
- with particular surface features,
- in terms of pH, especially weak acid/base pairs and amphoteric compounds,
- and/or in terms of hydrophilicity and/or hydrophobicity and/or amphiphilicity,
- and/or in terms of polarity,
- and/or having low-energy interactions, especially
- hydrogen bonds,
- Van der Waals interactions,
- ionic interactions, especially proton exchange,
- electrostatic interactions,
- salt bridges, especially those formed by divalent ions such as calcium and magnesium ions between negatively charged groups,
- and/or being surfactants,
- surface modifications preparing a subsequent modification: the functionalization layer deposited onto the walls of the pore comprises, for example, means for interacting or reacting with molecules and/or biomolecules. These are especially modular groups such as DNA, photo-activatable groups such as benzophenone, electro-activatable groups such as the electro-activatable species mentioned above, or heat-activatable groups such as thermosetting polymers.

It will be noted that the ligand must be coupled to an electro-activatable species to enable the functionalization via the process according to the present invention.
It is not necessary for there to be in the solution both electro-activatable species and electro-activatable species coupled to a ligand: there may also be only electro-activatable species coupled to a ligand, or alternatively only electro-activatable species.

Claims

1. A process for functionalizing at least one part of the wall of at least one pore of a support material, the process comprising:

a) placing the pore in contact with a solution of electro-activatable species and positioning two electrodes in said solution, so as to create inside the pore, when an electrical signal is applied between the two electrodes, a voltage drop capable of generating a localized deposition onto the wall,

b) applying at least one electrical signal between the two electrodes to activate the electro-activatable species and generate the localized deposition, thereby achieving the functionalizing.

2. The process of claim 1, wherein the voltage drop inside the pore is greater than 1000 V/m.

3. The process of claim 1, further comprising:

at least one repeating the placing a) and the applying b), with a second solution of electro-activatable species.

4. The process of claim 1, wherein

the pore is open at both ends, and

a solution is placed in two compartments, in each of which emerges one end of the pore, at least one of the two solutions comprising the electro-activatable species.

5. The process of claim 1, wherein

the pore has only one emerging end,

one of the electrodes is placed at a bottom of a cavity or at a bottom of the pore, and

the other electrode is placed in a compartment in communication with the emerging end of the pore.

6. The process of claim 1, further comprising, after the applying b), c) rinsing.

7. The process of claim 1, wherein the electro-activatable species is at least one electropolymerizable monomer.

8. The process of claim 7, wherein the electro-activatable species is at least one selected from the group consisting of a pyrrole, a thiophene, an indole, an aniline, an azine, a phenylenevinylene, a phenylene, a pyrene, a furan, a selenophene, a pyridazine, a carbazole, an acrylate, a methacrylate, a derivative of a pyrrole, a derivative of a thiophene, an derivative of an indole, an derivative of an aniline, an derivative of an azine, a derivative of a phenylenevinylene, a derivative of a phenylene, a derivative of a pyrene, a derivative of a furan, a derivative of a selenophene, a derivative of a pyridazine, a derivative of a carbazole, an acrylate, and a derivative of a methacrylate.

9. The process as of claim 1, wherein the electro-activatable species bears at least one electro-graftable function.

10. The process of claim 1, wherein the electro-activatable species is at least one selected from the group consisting of a metal, a metal oxide, a catalytic particle, a salt, and a metal complex.

11. The process of claim 1, wherein the electro-activatable species are formed by an electrophoretic paint.

12. The process of claim 1, wherein the solution comprises the electro-activatable species coupled to at least one ligand.

13. The process of claim 12, wherein the solution of electro-activatable species comprises a mixture of electro-activatable species, and the electro-activatable species coupled to at least one ligand.

14. The process of claim 13, wherein the solution comprises pyrrole coupled to a biomolecule.

15. The process of claim 1, wherein the solution of electro-activatable species comprises doping ions of at least one selected from the group consisting of heparin and chondroitin.

16. The process of claim 1, wherein the support material comprises silicon.

17. The process of claim 1, wherein the electrical signal is an electrical voltage difference of between 10 mV and 500 V.

18. The process of claim 17, wherein the voltage difference is applied for a time of between 10 μs and 100 s.

19. The process of claim 1, wherein a concentration of electro-activatable species is between 1 nM and 500 mM in the solution.

20. The process of claim 1, further comprising d) detaching the electro-activatable species from the support.

21. The process of claim 1, wherein the support comprises at least one flared functionalization region that extends the wall of a pore.

22. The process of claim 1, wherein the pore comprises a necking region constituting a functionalization region.

23. The process of claim 1, further comprising:

f) associating by recognition, wherein the electro-activatable species comprises at least one probe molecule.

24. The process of claim 23, further comprising:

g) denaturing the associating by recognition; and

h) optionally, associating again by recognition.

25. The process of claim 7, wherein the electro-activatable species is at least one pi-conjugated conductive polymers.

26. The process as of claim 9, wherein the electro-activatable species bears at least one diazonium group.