Process for forming a polymer layer with a uniform thickness on the surface of a solid support, solid support obtained and its applications

Abstract

The present invention relates to a process for forming a polymer layer with a uniform thickness on the surface of a solid support, to the solid support obtained using this process, and to its applications.

Description

[0001] The present invention relates to a process for forming a polymer layer with a uniform thickness on the surface of a solid support, to the solid support obtained using this process, and to its applications.

[0002] At the present time, there is a great interest in the multiparameter analytical tools known as DNA chips. These tools are dedicated to the analysis of biological macromolecules such as, for example, nucleic acid molecules and proteins.

[0003] They make it possible, by means of a polymer layer bonded to at least one surface of a solid support, to fix these biological macromolecules by means of specific functional groups. The molecules thus immobilized can then be subjected to various qualitative and quantitative analytical techniques or can also serve as probes, for the purpose of fixing other biological molecules of interest, especially in the case of the hybridization of complementary DNA strands.

[0004] At the present time, the deposition of polymers, and in particular of conducting polymers combined with specific (biological or chemical) functional groups for the purpose of fixing biological macromolecules, is carried out in various ways, including chemical or electrochemical deposition.

[0005] The polymer layers of the systems currently available on the market are of greater or lesser thickness depending on the desired applications.

[0006] The deposited polymer layers associated with the fixing of biological elements are described as a succession of links of a chain formed by one or more different monomers, some of these different monomers themselves carrying biological elements.

[0007] As an illustration, this kind of construction may be shown schematically by the following structure: 1

[0008] in which A and B represent the monomers of the polymer and BE represents a biological element carried directly by the monomers A and/or B.

[0009] In the particular case of conducting polymers, the polymerization is carried out electrochemically. During the polymerization process, the conducting polymer chain grows by successive insertion of the various monomers. Statistically, the monomers carrying biological elements are randomly fixed to the chain as it grows in length. Since the length of the chain is not controlled, it continues to grow until the polymerization process is stopped, this usually being caused, over time, by reducing the deposition potential or else by the exhaustion of the monomers. This technique does not allow the length of the chains to be easily controlled and consequently results in polymer layers being obtained which are nonuniform in terms of thickness.

[0010] Moreover, when the polymerization is carried out chemically, a chemical oxidizing agent is, for example, mixed with the monomers, at the time of use, in order to initiate the polymerization process (oxidative polymerization). In this case, it is only the time for which the various monomers and the chemical oxidizing agent are left in contact with each other, or the amounts of monomers initially present, which allows the final length of the polymer chains to be varied. This polymerization technique therefore allows easy control neither of the final thickness of the polymer layer formed, nor of the amounts of any biological elements fixed to the chains thus formed.

[0011] Moreover, since the biological elements are more accessible at the surface of the polymer layers than within this layer, many workers have sought to reduce the thicknesses of the polymer layers by depositing them rapidly (short deposition time, generally less than 1 second).

[0012] Such rapid deposition requires great control of the parameters involved during preparation of the polymer layer (polymerization time, deposition potential in the particular case of electrochemical deposition, surface cleaning, etc.). Rapid deposition, although allowing thin polymer layers to be obtained, is therefore difficult to implement and requires very precise operating conditions.

[0013] Moreover, when it is desired to polymerize several sites simultaneously, the deposition conditions mean that it is extremely difficult to achieve uniform deposition thicknesses at all the sites. This is because the physico-chemical conditions of the surface of the site to be polymerized affect the start of deposition, with the consequence that the thickness of the various polymer layers obtained on the various sites to be polymerized is nonuniform. This is typically the case when producing biochips of the MICAM® type, such as those described for example in patent application FR-A-2 781 886 and in the article by M. P. Caillat, “Sensors and Actuators”, B-Chimie, 1999, 61, 154-162.

[0014] It is therefore to remedy all these drawbacks and to provide a process for obtaining solid supports, at least one of the surfaces of which is covered with a uniform polymeric layer of small thickness, that the inventors have developed what forms the subject matter of the present invention.

[0015] The inventors have in particular demonstrated, surprisingly and unexpectedly, that it is possible to prepare such supports using a mixture of various monomers, the said mixture consisting, to at least 50%, of monomers having a chemical functional group capable of blocking the polymerization reaction.

[0016] The first subject of the present invention is therefore a process for forming a polymeric layer with a uniform thickness on at least part of one of the surfaces of a solid support, wherein said polymeric layer is obtained by copolymerization of a monomer solution containing monomers carrying a functional group capable of stopping the copolymerization reaction, and in which solution the amount of monomers carrying said functional group represents at least 50% of the total amount of monomers present within said solution.

[0017] The inventors have in fact demonstrated that the process according to the invention makes it possible for the deposition of the monomers, and thereby the growth of the polymer chains, to be self-regulated by successively stopping the growth of the chains undergoing polymerization. This makes it possible to greatly reduce the rate of chain growth and to obtain more flexible conditions for deposition at the sites that it is desired to cover. Surprisingly, this process then makes it possible to obtain remarkably uniform thin polymer layers.

[0018] In addition, when it is desired for the polymerization to be carried out at several sites simultaneously, for example in the case of biochips possessing several thousands of different sites, the initiation of polymerization may differ between two sites because of the physico-chemical conditions of the surfaces. In this case, the process according to the invention makes it possible to retard the growth of the layer by successive stopping of the growing chains and to reduce the difference owing to delayed initiation between two sites as the initial deposit continues to grow. In the end, the difference in thickness between the various sites is reduced and the biochip has uniform polymer thicknesses at each of its various sites.

[0019] The process according to the invention also allows the amounts of biological elements fixed to a polymer chain to be limited and controlled.

[0020] According to an advantageous embodiment of the invention, the amount of monomers carrying said functional group preferably represents at least 60%, and even more preferably 60 to 95%, of the total amount of monomers present in said solution.

[0021] The process according to the invention makes it possible to control the growth of the polymer layer, the thickness of which is therefore more uniform. More specifically, the process according to the invention makes it possible to reduce the rate of formation of the polymer layer by a factor of about 10. The standard deviation (E) of the thickness measurement (made on polymer layers whose thickness may vary between a few nanometers and a few tens of nanometers) may be calculated from the following equation: 1$E=\sqrt{\frac{1}{n}\sum {\left(X_i-X_m\right)}^2}$

[0022] in which n represents the amount of measurements, X1 represents the thickness value in ångstroms at the measurement point and Xm represents the mean thickness value.

[0023] This standard deviation E goes from 90 for a polymeric layer obtained by using only unfunctionalized monomers to 9 only if one uses a monomer mixture containing about 85% of monomers functionalized by a functional group capable of stopping the copolymerization reaction with respect to the total amount of monomers.

[0024] According to the invention, the actual nature of the monomers is not critical.

[0025] The monomers that can be used in the process according to the invention are in fact chosen depending on the type of application envisioned and especially on the types of macromolecules that it is desired to fix.

[0026] These monomers may be especially chosen from pyrroles, anilines, thiophenes and azulenes.

[0027] The copolymerization reaction may be initiated, depending on the nature of the monomers, either by a chemical oxidizing agent or by an electrical signal (in electropolymerization) in the case of conducting monomers.

[0028] Among chemical oxidizing agents, mention may especially he made of ferric chloride, cupric chloride, molybdenum (V) chloride and ruthenium trichloride.

[0029] According to a preferred embodiment of the invention, said monomers are chosen from pyrrole monomers and the copolymerization reaction is an electropolymerization reaction.

[0030] Among functional groups capable of stopping the copolymerization reaction, mention may especially be made of aldehyde, nitrile, ester, alkyl and aryl functional groups.

[0031] Advantageously, said functional group is preferably chosen from those allowing attachment of a biological or biochemical element containing, for example, an amine functional group, such as for example a DNA strand, a biotin, a streptavidin or an antibody.

[0032] As an example, and when the monomer is a pyrrole carrying an aldehyde functional group, the attachment of the biological element will be able to take place directly on the functional group capable of stopping the polymerization reaction, according to the following reaction: 2

[0033] in which R represents a biological or biochemical element.

[0034] Thus, a solid support will be obtained which has a polymeric layer in which the biological or biochemical element is fixed to the end of a chain according to the following formula: 3

[0035] in which R represents a biological or biochemical element and S corresponds to the surface of a solid support.

[0036] This process therefore also allows solid supports to be obtained in which the polymer layer is particularly uniform and whose solid supports offer the further advantage of having biological or biochemical elements that are very accessible. This accessibility is particularly beneficial if it is desired to carry out hybridization reactions on DNA strands.

[0037] The solid supports that can be used according to the invention are preferably chosen from metal supports, such as gold supports, or glass or plastic supports.

[0038] The subject of the invention is also the solid support obtained by implementing the process described above. This solid support includes, on at least part of one of its surfaces, a polymer layer whose thickness is preferably between about a few nanometers and a few tens of nanometers.

[0039] The subject of the invention is also the use of at least one solid support according to the invention, for isolating, separating and/or analyzing biological and biochemical elements. Thus, one application of the invention is in the production of improved MICAM®-type biological chips, but also biological chips whose solid support is made of glass or plastic.

[0040] Apart from the above arrangements, the invention also comprises other arrangements which will emerge from the description which follows, this referring to an example of a measurement of the rate of formation of a polypyrrole layer on the surface of a solid support as a function of the pyrrole/(pyrrole carrying an aldehyde functional group) ratio, to an example of the measurement of the thickness and the uniformity of a polymeric layer on the surface of a solid support as a function of the pyrrole/(pyrrole carrying an aldehyde functional group) ratio, and to the appended figures in which:

[0041] FIG. 1 shows the rate of formation of the polymer layer (as a percentage of the initial rate) as a function of the percentage of pyrrole-2-carboxaldehyde monomer; and

[0042] FIG. 2 shows the standard deviation E of the thickness measurement as a function of the decreasing percentage of pyrrole-2-carboxaldehyde present in each monomer solution.

EXAMPLE 1

Measurement of the Rate of Formation of a Polypyrrole-Type Polymer Layer as a Function of the Percentage of pyrrole-2-carboxaldehyde Monomers Present Within a pyrrole/pyrrole-2-carboxaldehyde Monomer Mixture

[0043] 1) Equipment and Method

[0044] Nine pyrrole/pyrrole-2-carboxaldehyde (Aldrich) monomer solutions comprising from 10 to 90% of pyrrole-2-carboxaldehyde monomer relative to the total amount of pyrrole/pyrrole-2-carboxaldehyde monomers (with a 1 molar concentration in acetonitrile) were prepared.

[0045] Two monomer solutions (with a 1 molar concentration in acetonitrile) consisting, respectively, of only pyrrole monomers and of only pyrrole-2-carboxaldehyde monomers were also prepared.

[0046] The various monomer solutions thus prepared were deposited on gold terminals all having the same size. The deposition potential was set at 1.1 V (saturated calomel electrode). For each solution, deposition was carried out until a charge of 5 mC was obtained. The time needed to reach this charge was recorded. The mean deposition current was calculated by dividing the total charge by the deposition time. The polymerization reaction was initiated electrochemically. The support electrolyte used was 0.1 M aqueous LiClO4. The rate of formation of the polymeric layer is given by the mean deposition current.

[0047] 2) Results

[0048] By comparing the various mean currents obtained with one another, and by setting the maximum rate obtained at 100%, a curve showing the rate of formation of the polymer layer as a function of the percentage of pyrrole-2-carboxaldehyde monomer present in each monomer solution was obtained (FIG. 1).

[0049] These results show that a solution of pyrrole monomers containing at least 50% in number of pyrrole-2-carboxaldehyde monomer results in a reduction of about 10% in the rate of formation of the polymer layer. When the percentage of pyrrole-2-carboxaldehyde monomer is between 60 and 70%, the rate of formation of the polymer is reduced of about 50%.

EXAMPLE 2

Measurement of the Uniformity of a Polypyrrole-Type Polymer Layer as a Function of the Percentage of pyrrole-2-carboxaldehyde Monomer Present Within a pyrrole/pyrrole-2-carboxaldehyde Monomer Mixture

[0050] 1) Equipment and Method

[0051] Four pyrrole/pyrrole-2-carboxaldehyde (Aldrich) monomer solutions comprising from 46 to 86% (46%; 66%; 78% and 86%) of pyrrole-2-carboxaldehyde monomer relative to the total amount of pyrrole/pyrrole-2-carboxaldehyde monomers (with a 1 molar concentration in acetonitrile) were prepared.

[0052] Two monomer solutions (with a 1 molar concentration in acetonitrile) consisting, respectively, of only pyrrole monomers and of only pyrrole-2-carboxaldehyde monomers were also prepared.

[0053] The various monomer solutions thus prepared were deposited according to the method described above in Example 1.

[0054] For each solution, deposition was carried out until a charge of 15 mC was obtained.

[0055] The thickness and the uniformity of the deposited layer were then measured by means of an optical interferometer of the NANOSPEC® brand. The refractive index of the layer was set arbitrarily at 1.7.

[0056] Each deposited layer was measured at five points.

[0057] The uniformity of the deposited layer is characterized by the standard deviation (E) of the thickness measurements obtained.

[0058] The results obtained are plotted in FIG. 2, which shows the standard deviation E of the thickness measurement as a function of the decreasing percentage of pyrrole-2-carboxaldehyde present in each monomer solution. These results show that the standard deviation E is decreased by a factor of about 2 when the monomer mixture used contains, in accordance with the invention, 66% of monomers carrying a function capable of stopping the copolymerization reaction. These results also show that the standard deviation E goes from about 95 to only about 9 when the percentage of pyrrole-2-carboxaldehyde monomer goes from 0% to 85%.

[0059] These results are indicative of the very great uniformity of the polymeric layer obtained by implementing the process according to the present invention.

Claims

1. A process for forming a polymeric layer with a uniform thickness on at least part of one of the surfaces of a solid support, wherein said polymeric layer is obtained by copolymerization of a monomer solution containing monomers carrying a functional group capable of stopping the copolymerization reaction, and in which solution the amount of monomers carrying said functional group represents at least 50% of the total amount of monomers present within said solution.

2. The process as claimed in claim 1, wherein the amount of monomers carrying said functional group represents at least 60% of the total amount of monomers present in said solution.

3. The process as claimed in claim 2, wherein the amount of monomers carrying said functional group represents from 60 to 95% of the total amount of monomers present in said solution.

4. The process as claimed in any one of the preceding claims, wherein the monomers are chosen from pyrroles, anilines, thiophenes and azulenes.

5. The process as claimed in claim 4, wherein the monomers are chosen from pyrroles and the copolymerization reaction is an electropolymerization reaction.

6. The process as claimed in any one of the preceding claims, wherein the functional groups capable of stopping the copolymerization reaction are chosen from aldehyde, nitrile, ester, alkyl and aryl functional groups.

7. A solid support, which is obtained using the process as defined in any one of claims 1 to 6.

8. The use of at least one solid support as defined in claim 7 for isolating, separating and/or analyzing biological and biochemical elements.

9. The use of at least one solid support as defined in claim 7 for producing biological chips.