U.S. patent application number 11/155773 was filed with the patent office on 2006-02-16 for method for producing matrices of addressed ligands on a carrier.
This patent application is currently assigned to COMMISSARIAT A L'ENERGIE ATOMIQUE. Invention is credited to Frederic Lesbre, Thierry Livache.
Application Number | 20060032750 11/155773 |
Document ID | / |
Family ID | 9541715 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060032750 |
Kind Code |
A1 |
Livache; Thierry ; et
al. |
February 16, 2006 |
Method for producing matrices of addressed ligands on a carrier
Abstract
The invention concerns a method for fabricating matrices of
addressed ligands on a carrier. According to this method, an
element is used such as a reservoir (1) filled with ligand and
containing an electrode (3) to deposit and electrochemically fix
the ligand to the conductive carrier (7). The ligand may be an
oligonucleotide or a peptide, and fixing may be obtained by
electropolymerisation of this oligonucleotide or peptide carrying a
pyrrole group at 5' with pyrrole.
Inventors: |
Livache; Thierry; (Haute
Jarrie, FR) ; Lesbre; Frederic; (St Egiene,
FR) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
COMMISSARIAT A L'ENERGIE
ATOMIQUE
Paris
FR
|
Family ID: |
9541715 |
Appl. No.: |
11/155773 |
Filed: |
June 20, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09890261 |
Aug 7, 2001 |
6962782 |
|
|
PCT/FR00/00289 |
Feb 8, 2000 |
|
|
|
11155773 |
Jun 20, 2005 |
|
|
|
Current U.S.
Class: |
204/601 |
Current CPC
Class: |
B01J 2219/00527
20130101; B01J 2219/00605 20130101; B01J 2219/00369 20130101; B01J
2219/00612 20130101; B01J 2219/00722 20130101; B01J 2219/00713
20130101; B01J 2219/00596 20130101; B01J 2219/00585 20130101; B01J
2219/00351 20130101; B01J 2219/0061 20130101; C40B 40/06 20130101;
B01J 2219/00628 20130101; B01J 19/0046 20130101; C40B 60/14
20130101; B01J 2219/00637 20130101; B01J 2219/00659 20130101; C40B
40/10 20130101; B01J 2219/00725 20130101; B01J 2219/0059 20130101;
C09D 5/4476 20130101 |
Class at
Publication: |
204/601 |
International
Class: |
C02F 1/40 20060101
C02F001/40 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 8, 1999 |
FR |
99 01438 |
Claims
1-15. (canceled)
16. Device for producing a matrix of ligands on a conductive
carrier or on conductive zones of a carrier, comprising: at least
one ligand dispensing means (1) provided with a conductive part
(3), means for connecting firstly the conductive carrier (7) or
conductive zones (13) of the carrier, and secondly the conductive
part (3) of the dispensing means to an electric generator, and
means for positioning and/or moving the carrier and/or the
dispenser means relative to one another and to place them in
contact such as to carry out several ligand deposits on the carrier
at different sites.
17. Device according to claim 16, in which said dispensing means
comprises a reservoir (1) containing the ligand and at least one
electrode (3, 5) arranged in said reservoir and forming the
conductive part of said means.
18. Device according to claim 17, which comprises several ligand
dispensing means assembled in the form of a print head.
19. Device for producing a matrix of ligands on a conductive
carrier or on conductive zones of a carrier, comprising: an
electrode (15) in wire or needle form able to be charged externally
with said ligand, means for connecting firstly the conductive
carrier (7) or conductive zones (13) of the carrier, and secondly
the electrode (15) to an electric generator, and means for
positioning and/or moving the carrier and/or electrode (15)
relative to one another such as to carry out several ligand
deposits on the carrier at different sites.
Description
TECHNICAL FIELD
[0001] The subject of the present invention is a method for
producing matrices of addressed ligands on a carrier.
[0002] The ligands may be natural or synthetic products having
biological activity or an affinity for biological or other
molecules, for example peptides, oligonucleotides, receptors or
other molecules of biological interest. Matrices of this type may
find numerous applications, in particular for the detection and
identification of constituents in biological samples and for
screening molecule libraries. Such matrices may in particular be
matrices of oligonucleotide probes.
PRIOR ART
[0003] In the past few years several methods have been developed
for producing matrices of this type. Three methodologies are known
in which addressing is made either by photochemical route, or by
mechanical route, or by electrochemical route.
[0004] In the document by Fodor S. et al, Science, 1991, 251, pages
767-773 [1] a method is described for making a matrix of
oligonucleotides by photochemical addressing. According to this
method, a carrier is used functionalised by functional groups
protected by photolabile protector groups; these protector groups
are then removed by radiation through a mask on the sites which are
to be coupled to the molecules of biological interest, then these
molecules are coupled to the de-protected functional groups.
[0005] This mode of photochemical addressing has the disadvantage
of requiring a large number of different masks to carry out all the
coupling operations.
[0006] The documents: Khrapko K. R. et al, DNA Sequence--I.DNA
Sequencing and Mapping, 1991, volume 1, pages 375 to 388 [2] and
GB-A-2 319 838 [3] describe a method for producing matrices by
mechanical addressing. In document [2] a carrier is used which is
coated with a polyacrylamide gel that is activated by substituting
certain amide groups by hydrazide groups. The oligonucleotides
activated in aldehyde form are then fixed to the hydrazide groups
by micropipetting the oligonucleotide solutions onto the sites to
which they are to be coupled.
[0007] In document [3] a carrier is used which is functionalised by
reagent groups and coupled to identical biological molecules. The
carrier is then cut into individual plaques each one corresponding
to the coupling of a molecule and then several plaques carrying
different molecules at desired sites are subsequently assembled on
a plate.
[0008] The use of these mechanical addressing techniques has the
disadvantage of having to bring the molecule to be fixed directly
to the site to be addressed. Therefore the size of the site cannot
be smaller than the size of the drop of dispensed reagent. Also,
the process requires two phases which are respectively a dispensing
phase and then a covalent attachment phase. Also the carrier has to
be modified such that a covalent bond may be formed between the
carrier and the molecule to be fixed.
[0009] The documents: Livache T. et al, Nucleic Acids Res., 1994,
22, 15, pages 2915-2921 [4] and WO-A-94/22889 [5] describe
electrochemical addressing techniques to produce matrices of
biological products.
[0010] In this case a carrier is used which comprises several
electrodes and these electrodes are used to fix the biological
molecules by electrochemical route. For this purpose, the carrier
fitted with its electrode is immersed in a solution containing the
molecule to be fixed, and by activation of the desired electrodes
they are coated with the molecule by electrochemical route. On this
account, the deposits of molecules can only be made in successive
manner. Moreover, it is necessary to use a carrier carrying
electrodes that can be individually addressed, therefore complex
systems that are possibly multiplexed.
[0011] The subject of the present invention is precisely a method
for producing matrices of biological products on a carrier, which
remedies the disadvantages of the above-mentioned methods and with
which it is possible in addition to conduct the addressing and
fixing of the biological molecule in a single step, without
requiring prior functionalisation of the carrier.
DESCRIPTION OF THE INVENTION
[0012] For this purpose, the invention puts forward a method for
producing a matrix containing at least one ligand fixed by
electrochemical route to a conductive carrier or to conductive
zones of a carrier, in which at least one element is used able to
dispense the ligand or ligands coupled to an electropolymerisable
monomer serving as electrode to achieve electrically assisted
synthesis of a polymer carrying the ligand or ligands on the
conductive carrier or on the conductive zones of the carrier.
[0013] According to the invention, an element is therefore used as
electrode which is able to dispense the ligand or ligands. This
element may be made up of a reservoir containing the ligand coupled
to the electropolymerisable monomer and comprising a conductive
part, or it may simply be formed of an electrode in the form of a
wire or needle which, after immersion in a container containing the
ligand to be fixed coupled to the electropolymerisable monomer, is
charged with this ligand by capillarity.
[0014] By using an electrode formed of said element according to
the invention, it is possible to place the ligand in contact with
the conductive carrier or the conductive zones of the carrier, then
to fix it directly to the conductive carrier (or the conductive
zone) by electrochemical activation, for example by setting up a
potential difference or by generating a current between the
conductive carrier (or the conductive zone) and the element acting
as electrode.
[0015] Therefore the dispensing and fixing of the ligand to the
carrier is conducted in a single step.
[0016] According to a first embodiment of the invention, said
element comprises a reservoir filled with the ligand and comprising
an insulating dispenser nozzle and at least one electrode arranged
in said reservoir, said nozzle being in direct contact with the
conductive carrier or at least one conductive zone of the carrier,
during the fixing operation.
[0017] The nozzle may in particular be a capillary tube which is
directly placed on the conductive surface.
[0018] According to a second embodiment of the invention, said
element comprises a reservoir filled with ligand, and comprising a
conductive dispenser nozzle, the contact between the conductive
nozzle and the conductive carrier or at least one conductive zone
of the carrier being assured via a drop of ligand leaving the
nozzle during the fixing operation.
[0019] In this case, the conductive nozzle is not in contact with
the conductive carrier or the conductive zone. As previously, the
conductive nozzle may be formed of a capillary tube.
[0020] According to a third embodiment of the invention, said
element is formed of an electrode in wire or needle form, charged
externally with ligand coupled to the electropolymerisable monomer,
the contact between the electrode and the conductive carrier or a
conductive zone of the carrier being assured during the fixing
operation by a drop of ligand withheld by the electrode.
[0021] In the different embodiments described above, the reservoir
generally contains a solution of ligand to be fixed and reagent(s)
that may optionally be needed to ensure fixing of the ligand by
electrochemical route.
[0022] According to the invention, the electrochemical fixing of
the ligand is made in particular by coupling it to an
electropolymerisable monomer. In this case, the solution may
contain the ligand coupled to the electropolymerisable monomer, the
electropolymerisable monomer and optionally a doping agent.
[0023] The elctropolymerisable monomer may in particular be one of
those described by Emr S. and Yacynych A., Electroanalysis, 1995,
7, pp. 913-923 [7]. They may belong to two categories, those
leading to conductive polymers such as pyrrole, aniline, thiophene,
and their derivatives, and those leading to insulating polymers
such as derivatives of phenol or benzene.
[0024] In this case, fixing of the ligand is achieved by
electrocopolymerisation of the monomer and of the ligand coupled to
the monomer.
[0025] The ligand may for example be an oligonucleotide, a
nucleotide, an amino acid or a peptide.
[0026] Said method of electrochemical fixing is described in
document [5] for ligands which are an oligonucleotide or a
nucleotide.
[0027] In this latter case, after conducting fixation, the chain of
the fixed oligonucleotide or nucleotide can be lengthened through
application of conventional synthesis methods for oligonucleotides
by successive coupling of the desired nucleotides, but by
conducting electrochemical de-protection of the last nucleotide
fixed.
[0028] In respect of peptides, it is possible to use the same
technique to lengthen the chain of the peptide by coupling the
desired amino acids.
[0029] The use of the electrodes described above to achieve the
depositing and fixing of a ligand by electrochemical route has the
following advantages: [0030] The depositing and fixing procedure is
carried out in a single step and it is very rapid. [0031] This
technique is easy to implement since it simply uses a mechanical
depositing technique, for example transfer using a mlcropipette,
but it is coupled to the space resolution possibilities of
electrochemistry. [0032] With this technique it is possible to
carry out several deposits in parallel mode. [0033] Also, this
method does not require the use of modified carriers or which carry
individually addressable electrodes.
[0034] For carriers in conductive material, these may be made
entirely in an electrically conductive material or they may be made
of an insulating material coated with a layer of conductive
material.
[0035] The conductive materials which can be used may be of
different types, they may for example be metals such as gold,
silver and platinum, or conductive oxides such as indium and tin
oxide (ITO), carbon or conductive organic polymers.
[0036] If the carrier comprises conductive zones, these may be made
in the conductive materials cited above and arranged on an
insulating carrier.
[0037] The insulating carrier may for example be in glass, silicon
or plastic material. It is also possible to use a carrier in a
conductive material whose conductive zones are delimited by
depositing an insulating material on the surface of the conductive
material.
[0038] According to the invention, the conductive zones may be
electrically interconnected or electrically addressable either
individually or in groups so that they can be activated
separately.
[0039] The method of the invention may be implemented such as to
fix identical or different ligands on different conductive sites of
the carrier.
[0040] In this case, simultaneous or successive fixing of identical
or different ligands may be made using several elements
respectively dispensing identical or different ligands. In this
case, at least two of the elements may be grouped together to form
a print head.
[0041] According to one variant of the invention, successive fixing
is made of at least two different ligands to different sites of the
carrier using a single element but by changing the ligand dispensed
by this element at least once.
[0042] In all the embodiments described above, the main advantage
lies in the ligand dispensing-coupling process which enables the
production of carriers carrying addressed molecules in extremely
fast manner.
[0043] A further subject of the invention is a device for producing
a matrix of ligands on a conductive carrier or on conductive zones
of a carrier, comprising: [0044] at least one ligand dispensing
means provided with a conductive part, [0045] means for connecting
firstly the conductive carrier or the conductive zones of the
carrier, and secondly the conductive part of the dispensing means
to an electric generator, and [0046] means for positioning and/or
moving the carrier and/or the dispensing means, relative to one
another and to place them in contact such as to make several
deposits of ligands on the carrier at different sites.
[0047] According to the invention, the dispensing means may
comprise a reservoir containing the ligand and at least one
electrode arranged in said reservoir and forming the conductive
part of said means.
[0048] According to one particular arrangement, the device
comprises several ligand dispensing means assembled in the form of
a print head.
[0049] According to one variant of embodiment, the device for
producing a matrix of ligands on a conductive carrier or conductive
zones of a carrier, comprises: [0050] an electrode in the form of a
wire or needle able to be charged externally with said ligand,
[0051] means for connecting firstly the conductive carrier or the
conductive zones of a carrier, and secondly the electrode to an
electric generator, and [0052] means for positioning and/or moving
the carrier and/or the electrode relative to one another such as to
make several deposits of ligands on the carrier at different
sites.
[0053] Other characteristics and advantages of the invention will
become clearer on reading the following description which is
evidently given for illustrative purposes and is non-restrictive,
with reference to the appended drawings.
SHORT DESCRIPTION OF THE DRAWINGS
[0054] FIG. 1 is a diagram of an element comprising a ligand
dispensing reservoir and at least one electrode to fix the ligand
to a conductive carrier.
[0055] FIG. 2 shows an element similar to the one in FIG. 1 to
achieve fixing of a ligand to a conductive carrier provided with
conductive zones that are electrically interconnected.
[0056] FIG. 3, on an enlarged scale, shows the nozzle of the
dispensing reservoir in FIG. 1, to carry out fixing of the ligand
on a carrier comprising multiplexed conductive zones.
[0057] FIGS. 4A and 4B illustrate the necessary steps to achieve
fixing of a ligand to a conductive carrier using an electrode in
wire form.
[0058] FIG. 5 shows a dispensing element, fitted with a fluid inlet
and outlet to ensure its filling and draining, between two
different ligand fixing operations.
[0059] FIG. 6 is a diagram of a print head comprising several
reservoirs for dispensing identical or different ligands
DETAILED DISCLOSURE OF THE EMBODIMENTS
[0060] FIG. 1 shows the first embodiment of the invention in which
as electrode an element is used comprising a reservoir 1 filled
with the ligand to be fixed and comprising a dispensing nozzle 1a.
Inside reservoir 1 are arranged a counter-electrode 3 made in
platinum or gold for example, and a control electrode 5.
[0061] The reservoir may contain a sufficient volume of reagent to
carry out a certain number of deposits, which may for example reach
one thousand.
[0062] In this first embodiment shown in FIG. 1, a conductive
carrier 7 is used which may comprise a glass substrate coated with
a gold layer.
[0063] This figure shows the deposits 9 made with said reservoir by
moving the carrier along directions x and y for example between two
deposits. If the nozzle 1a of the reservoir, in the form of a
capillary tube for example, is made in an insulating material it
can be placed on the conductive carrier 7 and, by setting up a
difference in potential or current between conductive surface 7 and
the counter-electrode 3, it is possible to obtain deposits 9 which
are fixed to the conductive surface 7 by electric impulse. In this
case, the size of the deposits 9 is determined by the size of the
reservoir/carrier interface located in the lines of the electric
field between the electrode and the conductive surface. This
interface must be as small as possible to reduce the size of the
deposit obtained.
[0064] The reservoir in FIG. 1 may also comprise a nozzle 1a in
conductive material. In this case, fixing of the ligand present in
the reservoir is made by contacting the conductive surface 7 with
the electrode formed by nozzle 1a by means of a drop leaving nozzle
1a. In this case the size of the deposits is also adjusted by the
interface between the liquid and the conductive surface located in
the lines of the electrical field.
[0065] The resolution of the deposits 9 may be improved by using a
carrier as shown in FIG. 2 formed of interconnected conductive
zones. In FIG. 2 the same references have been used as in FIG. 1 to
designate the reservoir 1 fitted with its nozzle 1A, a control
electrode 5 and a counter-electrode 3. In this case the carrier is
formed of an insulating carrier provided with conductive zones 13
insulated from each other but electrically interconnected. These
conductive zones may be made in gold on a glass or silicon
substrate for example. In this case, deposits 9 are obtained by
dispensing the ligand above the conductive zones, but only the
conductive zones in contact with the ligand can be coated with the
latter. Therefore the size of the deposits is adjusted by the size
of the conductive zones 13.
[0066] In this case, the conductive carrier used in fact only
comprises a single electrode; this immensely simplifies its
production and the costs involved may be very low since simple
sheets of plastic material coated with conductive material may be
used.
[0067] The use of a network of conductive zones makes it possible
to reduce the size of the deposits 9, but not to increase the
density of the matrix. This density is directly dependent upon the
size of the interface between the capillary nozzle 1a and the
carrier and it is limited by the size of the nozzle.
[0068] It is nonetheless possible to increase the density of the
matrix by using a carrier comprising conductive zones forming
multiplexed electrodes, as shown in FIG. 3.
[0069] FIG. 3 illustrates the nozzle 1a of the reservoir 1 in FIGS.
1 and 2 on an enlarged scale and part of an insulating conductive
carrier 11 provided with conductive zones 13 which are separately
connected to means for applying a potential or current so that they
can be activated separately. In this case, the size of the deposits
is determined by the size of the activated conductive zones 12 as
shown in the case in FIG. 3. The other conductive zones which are
in contact with the ligand cannot lead to fixing of the ligand
since they are not electroactivated. In this manner, it possible to
simultaneously achieve high space resolution and strong matrix
density.
[0070] In FIGS. 4A and 4B another embodiment of the invention is
shown in which the element able to dispense the ligand is formed by
an electrode 15 in wire form.
[0071] In this case, a conductive carrier 7 can be used as shown in
FIG. 4A. To make a ligand deposit, the electrode 15 is firstly
immersed in a container 17 containing the ligand to be fixed and
the electrode withholds a drop 19 of this ligand. The electrode
containing the drop 19 of ligand is then brought above conductive
carrier 7 as shown in FIG. 4B making electric contact by means of
drop 19. By applying an electric impulse between electrode 15 and
the conductive carrier 7 the formation of deposits of ligand is
obtained.
[0072] After this operation, the electrode 15 is rinsed in a
rinsing tank 21 so that it can be used again to make another
deposit 9 either with the same ligand or with another ligand.
[0073] When this type of electrode is used, the resolution of the
deposits may be lower but in this case the possible rinsing of
electrode 15 is a determinant advantage.
[0074] The method of the invention is of great interest since it
provides the possibility firstly of using a very small volume of
reaction medium and therefore of economising the molecules of
biological interest to be coupled. Also, the size of the deposits
made on the carrier may be adjusted whereas in mechanical
addressing methods involving conventional chemical activation
methods the size of the deposits could not be less than 50, even
100 .mu.m.
[0075] According to the invention, the size of the deposits can be
very easily reduced not by reducing the size of the drop which is
difficult in practice, but by reducing the surface of the zone that
can be electroactivated. The resolution of the deposits is
optimised through the fact that only the electrode/carrier
interface located in the lines of the electric field can be
activated; that is to say that if a drop spills outside this zone,
its content will not be fixed to the conductive surface.
[0076] Therefore if the diameter of the interface between the
nozzle 1a and the conductive carrier is 200 .mu.m, and if a
conductive zone is used whose side measurement is only 10 .mu.m,
only this conductive zone may be coated with the molecules of
biological interest.
[0077] According to the invention, it is possible to make deposits
9 of different ligands on a carrier. This may be achieved by
successively fixing at least two different ligands to different
sites of the carrier using a single element and by changing the
ligand dispensed by said element. In this case, the deposits may be
made successively, either by changing the content of reservoir 1 of
the elements shown in FIGS. 1 and 2, or by using the electrode in
FIG. 4 which is immersed in different reagents. It is also possible
to use a fixed reservoir provided with ligand adding and evacuation
means, that is to say comprising a fluid inlet and outlet system
for the ligand so as to change the content of the reservoir without
having to move it.
[0078] FIG. 5 illustrates said embodiment of reservoir 1 provided
with a fluid inlet 1b and an outlet 1c.
[0079] Evidently, it is also possible in order to make deposits 9
of identical or different ligands, to use several elements such as
those shown in FIGS. 1 and 4. These elements may optionally be
assembled to form a print head as shown in FIG. 6.
[0080] In FIG. 6 it can be seen that the print head contains a
first reservoir R1 filled with a ligand P1, a second reservoir R2
filled with a ligand P2 and a third reservoir R3 filled with a
ligand P3. With a multiple head of this type it is possible to make
three simultaneous deposits 9 of ligands P1, P2 and P3 respectively
on the conductive surface 7.
[0081] It is specified that the deposits may be made in an inert
atmosphere or in an electrochemically neutral liquid medium which,
if possible, is non-miscible with the reaction medium contained in
the reservoir.
[0082] After the depositing phase, the carrier may be rinsed and
used in conventional manner.
[0083] The following examples illustrate the production of matrices
of oligonucleotides or peptides using oligonucleotides or peptides
carrying a pyrrole group which are fixed to a conductive carrier by
copolymerising them with pyrrole by electrochemical route using the
method described in document [5]: WO-A-94/22889.
EXAMPLE 1
1--Production of Carriers Carrying Oligonucleotides
[0084] The conductive carriers used are glass plates coated with a
layer of chromium (for adherence) and a continuous layer of gold of
0.5 .mu.m. This layer is connected to the "working electrode"
outlet of an EGG 283 potentiostat.
[0085] Two different oligonucleotides carrying a pyrrole group at
5' are copolymerised on these carriers. Their sequences are as
follows: TABLE-US-00001 pyrM5: 5' pyr (T).sub.10 GGAGCTGCTGGCGT 3'
pyrCP: 5' pyr (T).sub.10 GCCTTGACGATACAGC 3'
[0086] They were synthesized using the method described by Livache
et al in [5].
[0087] To fix these oligonucleotides to the carrier, a reaction
medium is used containing 0.1M LiClO4, 20 mM pyrrole and 1 .mu.M
oligonucleotide carrying a pyrrole group at 5'.
[0088] This solution is added to a reservoir in polypropylene of
cone shape which contains a platinum counter-electrode (CE)
connected to the potentiostat. This reservoir is easily filled
using a micropipette whose volume may vary from 50 to 1000 .mu.l
reaction medium. The tip of this cone has a diameter of
approximately 0.8 mm. Finer or larger cones can be used for other
volumes of reagent.
[0089] The tip of the cone is placed in contact with the conductive
surface and the copolymer is made by cyclic voltametry (from -0.35
to +0.85V/CE at the rate of 100 mV/s). The charge recorded is used
to determine the thickness of the polymer formed. After the
formation of this first deposit, the cone is emptied, rinsed then
filled with a new reaction medium containing another
oligonucleotide. The conductive plate is moved (table x/y/z) and
the same copolymerisation operation is conducted on another area of
the conductive surface enabling the production of a deposit
carrying another oligonucleotide sequence.
[0090] In this manner two matrices are prepared solely comprising
pyrM5 oligonucleotides and two matrices solely comprising pyrCP
oligonucleotides.
[0091] It is checked that the matrices of oligonucleotides so
obtained have the desired properties for detecting complementary
oligonucleotides by hybridisation.
2--Hybridisation of Oligonucleotides and Detection.
[0092] The complementary oligonucleotides tested are the following:
[0093] biotinylated complementary M5: bio.sub.compM5; [0094]
biotinylated complementary CP: bio.sub.compCP.
[0095] The hybridisation of the complementary oligonucleotides is
conducted in PBS buffer (Sigma containing 0.5M NaCl, 100 .mu.g/ml
salmon sperm DNA (Sigma), 10 mM EDTA and 10 nM of complementary
biotinylated oligonucleotide. Hybridisation is conducted at
45.degree. C. in a volume of 20 mm for 15 min. Quick rinsing in
PBS/NaCl is made. Detection of the hybrids is then carried out
after incubation in PBS/NaCl solution containing 0.1 mg/ml R
phycoerythrine (Molecular Probe). Fluorescence is detected using a
cold camera (Hamamatsu) mounted on an epifluoresence microscope.
The results are expressed as shades of grey.
[0096] A spot of polypyrrole approximately 0.8 mm in diameter is
observed whose fluorescent intensity is reported below: [0097]
oligonucleotide on pyrM5 carrier hybridised with bio.sub.compM5:
110 [0098] oligonucleotide on pyrM5 carrier hybridised with
bio.sub.compCP: 5 [0099] oligonucloetide on pyrCP carrier
hybridised with bio.sub.compM5: 7 [0100] oligonucloetide on pyrCP
carrier hybridised with bio.sub.compCP: 84
[0101] Good hybridisation specificity is observed with a high
signal/noise ratio.
EXAMPLE 2
[0102] The same operating method is followed as in example 1 to
prepare matrices of pyrM5 and pyrCP oligonucleotides but using as
conductive carrier a carrier in plastic material coated with indium
and tin oxide (ITO).
[0103] The results obtained with these matrices for the detection
of biotinylated complementary oligonucleotides are the following:
[0104] oligonucleotide on pyrM5 carrier hybridised with
bio.sub.compM5: 95 [0105] oligonucleotide on pyrM5 carrier
hybridised with bio.sub.compCP: 5 [0106] oligonucleotide on pyrCP
carrier hybridised with bio.sub.compM5: 7 [0107] oligonucleotide on
pyrCP carrier hybridised with bio.sub.compCP: 105
EXAMPLE 3
[0108] In this example, the same operating method as in example 1
is followed to prepare a matrix of pyrM5 oligonucleotides on a
carrier in gold supported by glass but as counter-electrode a
platinum wire is used charged with reaction medium instead of the
reservoir fitted on the inside with a platinum electrode.
[0109] As shown in FIG. 4A, the platinum wire 15 is charged with
reaction medium by immersion in a reservoir 17 containing this
medium. The wire carrying the drop 19 is then brought to the
carrier until contact is made with the drop. The electrochemical
impulse is then made. The wire is lifted away and rinsed in water.
Other deposits are made in the same manner. In this way deposits of
approximately 1 mm in diameter are obtained and intense
fluorescence is visible when the matrix is used to conduct
hybridisation of the complementary oligonucleotide. The results
obtained are the following: [0110] oligonucleotide on pyrM5 carrier
hybridised with bio.sub.compM5: 400 [0111] oligonucleotide on pyrCP
carrier hybridised with bio.sub.compCP: 10
EXAMPLE 4
[0112] In the same manner, peptides may be deposited.
Pyrrole-peptides are synthesised using the procedure described by
T. Livache et al, Biosensor and Bioelectronics 13, (1998) 629-634
[6]. They are deposited following the usual procedure ( ). The two
peptides ACTH (18-39) and ACTH (11-24) are then detected by the
biotinylated antibodies Mab (34-39) and Mab (18-24)
respectively.
[0113] Fluorescence results after incubation with streptavidin
phycoerythrine are the following: TABLE-US-00002 Peptide ACTH 18-39
with Mab 34-39 640 Peptide ACTH 18-39 with Mab 18-24 510 Peptide
ACTH 11-24 with Mab 34-39 10 Peptide ACTH 11-24 with Mab 18-24
470
CITED REFERENCES
[0114] [1] Fodor S. et al, Science, 1991, 251, pp. 767-773. [0115]
[2] Khrapko K. R. et al, DNA Sequence--I.DNA Sequencing and
Mapping, 1991, vol. 1, pp. 375-388. [0116] [3] GB-A-2 319 838.
[0117] [4] Livache T. et al, Nucleic Acids Res., 1994, 22, 15,
pages 2915-2921 [0118] [5] WO-A-94/22889. [0119] [6] T. Livache et
al, Biosensors and Bioelectronics 13, (1998), pages 629-634. [0120]
[7] Emr S. and Yacynych A, Electroanalysis, 1995, 7, pp.
913-323.
* * * * *