U.S. patent application number 12/866340 was filed with the patent office on 2011-07-21 for method for functionalising the wall of a pore.
This patent application is currently assigned to Comm. A L'Energie Atom. Et Aux Energies Alterna. Invention is credited to Aurelie Bouchet, Francois Chatelain, Emeline Descamps, Vincent Haguet, Thierry Livache, Pascal Mailley.
Application Number | 20110174629 12/866340 |
Document ID | / |
Family ID | 39767207 |
Filed Date | 2011-07-21 |
United States Patent
Application |
20110174629 |
Kind Code |
A1 |
Bouchet; Aurelie ; et
al. |
July 21, 2011 |
METHOD FOR FUNCTIONALISING THE WALL OF A PORE
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.
Inventors: |
Bouchet; Aurelie;
(Seyssinet- Pariset, FR) ; Descamps; Emeline;
(Sourcieux Les Mines, FR) ; Mailley; Pascal; (Le
Pin, FR) ; Livache; Thierry; (Jarrie, FR) ;
Haguet; Vincent; (Saint-Partin-Le-Vinoux, FR) ;
Chatelain; Francois; (Beaulieu, FR) |
Assignee: |
Comm. A L'Energie Atom. Et Aux
Energies Alterna
Paris
FR
|
Family ID: |
39767207 |
Appl. No.: |
12/866340 |
Filed: |
February 5, 2009 |
PCT Filed: |
February 5, 2009 |
PCT NO: |
PCT/FR09/00133 |
371 Date: |
February 8, 2011 |
Current U.S.
Class: |
205/131 |
Current CPC
Class: |
B81C 1/00206 20130101;
G01N 33/48721 20130101; B81B 2203/0353 20130101 |
Class at
Publication: |
205/131 |
International
Class: |
C25D 5/02 20060101
C25D005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 5, 2008 |
FR |
08 00601 |
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 .mu.s 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.
Description
[0001] 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.
[0002] The term "pore" (or channel or capillary) denotes any cavity
emerging or not emerging from a material.
[0003] 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.
[0004] In general, the known manufacturing processes do not enable
or have great difficulty in functionalizing pores in a localized
manner.
[0005] 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.
[0006] 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.).
[0007] 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.).
[0008] 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.
[0009] 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.
[0010] 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.).
[0011] 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.
[0012] Schematically, two different processes are known and used:
chemical polymerization and electro-polymerization.
[0013] 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.).
[0014] 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 FeCl.sub.3),
which leads to polymerization at the points where the two solutions
meet, i.e. in the pores of the membrane.
[0015] 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.).
[0016] 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.
[0017] 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.
[0018] 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).
[0019] 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.
[0020] 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.
[0021] 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: [0022] 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, [0023] b) applying an
electrical signal, potential difference or current between the two
electrodes to achieve said functionalization.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] For example, the field may reach 10.sup.6 V/m, or even
more.
[0028] 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.).
[0029] 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.
[0030] The remote electropolymerization does not require the
presence of an oxidizing chemical agent.
[0031] The remote electropolymerization process may be performed in
a single operating step.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] After b, rinsing may be envisioned.
[0037] The support material may be silicon-based.
[0038] 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.
[0039] The solution of electro-activatable species may comprise
ligands.
[0040] 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.
[0041] In particular, the solution may have an oligonucleotide
(pyrrole-oligonucleotide) probe, or more generally pyrrole coupled
to a biomolecule.
[0042] The solution of electro-activatable species may include
doping ions of interest, especially heparin and/or chondroitin.
[0043] The support material may be silicon-based.
[0044] 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 .mu.s 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.
[0045] The concentration of electro-activatable species may extend
over a wide range, namely between 1 nM and 500 mM.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] The process thus enables the association by affinity of a
species of interest and allows the manufacture of molecular
assemblies.
[0050] Other characteristics and advantages of the invention will
emerge more clearly on reading the description below, in relation
with the drawings, in which:
[0051] FIG. 1 is a schematic diagram illustrating the process
according to the invention,
[0052] FIGS. 2a to 2c represent a cell for receiving a chip
containing a pore (assembly a),
[0053] FIGS. 3 and 4 illustrate an assembly adapted to a multipore
chip, FIG. 4 being a detail thereof relative to the pore P.sub.1
under the experimental conditions,
[0054] FIG. 5 illustrates the format of the fluorescence test used
to validate the functionalization of the pores,
[0055] FIGS. 6, 7, 8a and 8b represent different profiles of pores
used in the examples, and
[0056] FIGS. 9a and 9b represent two examples of profiles of
non-emerging pores.
[0057] 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: [0058] active groups that allow
low-energy interactions, for instance: [0059] surface charges,
[0060] molecular or biomolecular recognition groups, for instance
biomolecules, reactive chemical groups or ion chelators, [0061]
organic or inorganic species, especially for the purpose of
reducing the aperture diameter of the pore.
[0062] 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.
[0063] 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.
[0064] The electro-activatable species may be chosen especially
from: [0065] 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, [0066]
derivatives bearing electrograftable functions such as diazonium
groups, [0067] metals and metal oxides, for example iridium oxide,
catalytic particles, salts and metal complexes, [0068]
electrophoretic paints.
[0069] 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.
[0070] 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.
[0071] Polypyrrole, polycarbazole, polyaniline, PEDOT, polyindole
and polythiophene belong to the group of pi-conjugated conductive
polymers.
[0072] 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
[0073] I.--Materials
[0074] A) Reagents and Consumables:
[0075] Pyrrole is divided into aliquots at a concentration of 1 M
dissolved in acetonitrile and then stored at -20.degree. C. Pyrrole
bearing an oligonucleotide was prepared according to the protocol
described in patent application FR 2 703 359.
[0076] The DNA sequences used are as follows: [0077] Py-probeZip6:
Py.sup.5'-(T).sub.10-GAC CGG TAT GCG ACC TGG TAT GCG.sup.3' (Py-SEQ
ID No. 1) [0078] Target-Zip6-bio: biotin.sup.5' CGC ATA CCA GGT CGC
ATA CCG GTC.sup.3' (biotin-SEQ ID No. 2)
[0079] The chips used are silicon oxide or silicon nitride
membranes. They comprise nine pores of micrometric size distributed
over an area of 2.times.2 cm.sup.2.
[0080] B) Buffers Used (Given as a Guide): [0081]
Electropolymerization buffer: 6 g/L
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4, 2.9 g/L NaCl, 10% v/v
glycerol, 2% v/v acetonitrile (v/v=volume per unit volume). [0082]
Hybridization buffer: 0.02 M Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4,
1.1 M NaCl, 5.4 mM KCl, 4% v/v 50.times. Denhardt, 0.2% v/v salmon
sperm DNA, 0.3% v/v Tween 20 at pH 7.4. [0083] Rinsing buffer: PBS
5 tablets/L, NaCl 23.375 g/L, Tween 20 0.15% v/v.
[0084] C) Experimental Assemblies for the Remote
Electro-Polymerization:
[0085] Two experimental assemblies were validated.
[0086] a) Electropolymerization Cell (see FIGS. 2a to 2c):
[0087] 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.sub.1 and
9.sub.2 of the lid 9 to form the electrodes. 9.sub.3 and 9.sub.4
denote the apertures for fixing the lid 9 to the cell C, and
9.sub.5 and 9.sub.6 the holes for fixing the screws to the cell
C.
[0088] b) Assembly on a "Multipore" Chip:
[0089] 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.
[0090] In the assembly described in relation to FIG. 3, the chip 10
has 9 pores P.sub.1 . . . P.sub.9. Each pore is isolated between
two leaktight compartments (3.sub.1, 4.sub.1; 3.sub.2, 4.sub.2; . .
. 3.sub.9, 4.sub.9). In other words, it is possible to place each
of the nine pores P.sub.1, P.sub.2 . . . P.sub.9 which do or do not
have the same diameter in contact with a different solution
S.sub.1, S.sub.2 . . . S.sub.9 of 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.sub.t1, E.sub.t2, . . . E.sub.t9 which 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.sub.a1, E.sub.a2, . . . E.sub.a9. The solutions
S.sub.1, S.sub.1, . . . S.sub.9 may 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).
[0091] Each pore (P.sub.1, . . . P.sub.9) of a chip 10 is
positioned between two leaktight compartments 3.sub.1, 4.sub.1, . .
. ; 3.sub.9, 4.sub.9 which have herein a volume of 10 .mu.l.
[0092] 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.sub.1 and 4.sub.1 are 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: [0093] either the electrodes integrated
into the printed circuits, [0094] or electrodes (metal wires, not
shown) introduced into capillaries on either side of the pore,
[0095] or electrodes dipping directly into a compartment. In this
case, a plastic card (not shown) is used instead of the printed
circuit.
[0096] II) Implementation
[0097] A) Preparation of the Substrate:
[0098] a) Cleaning:
[0099] 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 M.OMEGA..m is obtained. The chip is
then dried in an oven at 180.degree. C. for 10 minutes. It may then
be stored at room temperature.
[0100] b) Increase in Hydrophilicity of the Surface by Applying an
Oxygen Plasma:
[0101] 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 O.sub.2 plasma at a
power of 100 W.
[0102] c) Polypyrrole Deposition:
[0103] A polymerization solution containing 20 mM of pyrrole and 5
.mu.M of py-probeZip6 in electropolymerization buffer was used to
perform polymer depositions in the pores.
[0104] The two assemblies (a and b above) were used.
[0105] 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.degree. C.
[0106] d) Checking of the Functionalization by Fluorescence
Microscopy
[0107] 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 .mu.l
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.
[0108] Biofunctionalization of a Pore with Nucleic Acids
Example I
Assembly a
[0109] i) Creation of a Polypyrrole Deposit
[0110] 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 .mu.M 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.degree. C.
[0111] The functionalization efficacy is checked by fluorescence
microscopy according to the process described above.
[0112] ii) Results
[0113] The manipulation was performed with pores with different
shape ratios: [0114] R.sub.f=35: pore 70 .mu.m in diameter in a
membrane 2 .mu.m thick and with a side length of 500 .mu.m (see
FIG. 6).
[0115] 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.
[0116] 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. [0117] R.sub.f=1 with a
circular pore 18 .mu.m in diameter in a membrane 20 .mu.m thick: in
the case of a pore pierced into a square membrane with a side
length of 50 .mu.m, and 20 .mu.m thick. The deposition is performed
at a voltage of 2 V applied for 100 milliseconds (FIG. 7).
[0118] 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. [0119] R.sub.f=0.25 pore
with a diameter of 2 .mu.m in a membrane 8 .mu.m thick. The pore 2
.mu.m in size is at the bottom of a cone with a largest diameter
equal to 10 .mu.m. The environment of the pore is said to be of the
"funnel" type (FIGS. 8a and 8b).
[0120] 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 .mu.m).
[0121] 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
[0122] i) Creation of a Polypyrrole Deposit
[0123] A multipore chip comprising pores of variable shape ratios
R.sub.f which have undergone an O.sub.2 plasma 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 .mu.m 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.degree.
C.
[0124] ii) Results
[0125] 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. [0126]
R.sub.f=1 pore 18 .mu.m in diameter in a membrane 18 .mu.m
thick.
[0127] Fluorescence microscopy confirms that the surface
functionalization process also functions for all the abovementioned
values of R.sub.f using this assembly (presence of a fluorescent
ring).
[0128] 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 .mu.m in diameter (R.sub.f=1) of a multipore
chip:
[0129] a) Electrical Potential
[0130] To do this, 15 .mu.l of a polymerization solution composed
of 20 mM of pyrrole and 5 .mu.M 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.degree. 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.
[0131] b) Pyrrole-Oligonucleotide Conjugate Adsorption
[0132] 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 .mu.M 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.
[0133] c) Non-Specific Adsorption During the Revelation
Procedure
[0134] 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.
[0135] 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.
[0136] d) Denaturing of the Hybridization
[0137] 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.
[0138] e) Fluorescence after Rehybridization
[0139] 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
[0140] i) Creation of an Iridium Oxide Deposit
[0141] 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
IrCl.sub.4 monohydrate 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.degree.
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.degree. C.
[0142] A multipore chip comprising pores with a shape ratio Rf=1
and which has undergone an O.sub.2 plasma treatment is placed in
the assembly b described above (FIGS. 3 and 4).
[0143] 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.
[0144] The chip is then removed from the cell, rinsed thoroughly
with water and then dried with compressed air and stored at
4.degree. C.
[0145] ii) Results
[0146] The functionalization efficacy was checked by scanning
electron microscopy (SEM).
[0147] 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.
[0148] 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:
[0149] Ir(OH)+H.sub.2O<->Ir(OH).sub.2+H.sup.++e.sup.- (-0.1
V)
[0150] Ir(OH).sub.2+H.sub.2O<->Ir(OH).sub.3+H.sup.++e.sup.-
(0.3 V)
[0151] Ir(OH).sub.3+H.sub.2O<->Ir(OH).sub.4+H.sup.++e.sup.-
(0.8 V) [0152] or IrO.sub.2+2H.sub.2O+H.sup.++e.sup.- (divergence
according to the publications).
[0153] 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
[0154] i) Creation of a Polypyrrole Deposit:
[0155] 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.
[0156] ii) Detachment of the Deposits Formed from Their
Support:
[0157] 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
[0158] FIGS. 9a and 9b 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. 9a and 9b, 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.
[0159] 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'.
[0160] Conclusion:
[0161] The functionalization technical process according to the
invention is efficient and relatively easy to implement.
[0162] The reproducibility of the deposits is satisfactory and may
be further improved by controlling the manipulation parameters more
strictly: [0163] fixed inter-electrode distance [0164] temperature
control [0165] hygrometry control.
[0166] 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).
[0167] The process according to the invention has the advantage of
being inexpensive: [0168] in financial terms, since only limited
equipment is necessary: potentiostats, electrodes, etc., [0169] in
terms of time, since the deposition procedure lasts only a few
minutes.
[0170] The experimental device is furthermore of relatively small
bulk and is easy to transport.
[0171] The strategy is adaptable to any type of porous support, of
organic or inorganic nature, conductive, semiconductive or
insulating, irrespective of the pore dimension.
[0172] 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.
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] The experimental device may furthermore integrate thermal
and optical devices allowing, for example, crosslinking experiments
or visualization of the organization of the deposits produced.
[0179] 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.).
[0180] The considerable advantage of synthetic pores relative to
the latter lies in the possibility of: [0181] modulating their
properties by creating charges or reactive groups at the surface,
[0182] controlling the geometry (pore diameter, membrane thickness,
etc.), [0183] performing easier integration in a microfluidic
device.
[0184] 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.
[0185] Functionalized pores may also be useful for capturing
bacteria or cells, especially by immobilizing heparin or
chondroitin inside a pore.
[0186] 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.
[0187] 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).
[0188] Finally, the process is compatible with the use of molecular
imprint techniques, which opens advantageous applications in the
field of capillary electrophoresis, for example.
[0189] 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: [0190] 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, [0191] other affinity
groups, especially ion chelators and ion exchangers, [0192]
chemically active functions, especially amine, amide, oxyamine,
active ester, alcohol, carboxylic acid, alkyne, thiol, epoxide,
anhydride, acyl chloride or aldehyde functions, and derivatives
thereof, [0193] 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, [0194] groups [0195] with particular
surface features, [0196] in terms of pH, especially weak acid/base
pairs and amphoteric compounds, [0197] and/or in terms of
hydrophilicity and/or hydrophobicity and/or amphiphilicity, [0198]
and/or in terms of polarity, [0199] and/or having low-energy
interactions, especially [0200] hydrogen bonds, [0201] Van der
Waals interactions, [0202] ionic interactions, especially proton
exchange, [0203] electrostatic interactions, [0204] salt bridges,
especially those formed by divalent ions such as calcium and
magnesium ions between negatively charged groups, [0205] and/or
being surfactants, [0206] 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.
[0207] 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.
[0208] 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.
Sequence CWU 1
1
2134DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1tttttttttt gaccggtatg cgacctggta tgcg
34224DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2cgcataccag gtcgcatacc ggtc 24
* * * * *