Highly specific surfaces for biological reactions, method ofpreparation and utilization

~ 1 ~ 2 ~~ 6 i W095 / 22USG PCTIFR95 / OOIGJ 1 HIGHLY SPECIFIC SURFACES FOR BIOLOGICAL REACTIONS, METHOD FOR THEIR PREPARATION AND METHOD FOR THEIR USE.

The present invention relates in particular to very highly specific surfaces which can be used in biology, as well as their applications and processes for their preparation.

We know for a long time the very great specificity and the very great selectivity of certain biological reactions, in particular 1ü antigen-Atlantibody reactions, DNA or RNA hybridization reactions, interpratein reactions or of the avidin / streptavidin / biotin type, same as the reactions of ligands and their receptors.

We now know how to take advantage of these specificities, in particular 1i to demonstrate the presence or absence of one of the elements of the reaction couple in a sample or even possibly to separate one of the elements of the couple from a more medium. complex.

However, when it is desired to detect the presence of a molecule at very low concentration in a very complex medium, the currently known methods sometimes give very random results, in particular taking into account the problem of background noise occurring during the separation steps and. detection.

This is why, what will be called hereinafter "molecular fishing", that is to say the possibility of being able to demonstrate each of the molecules which are sought when they are at very low concentrations, n has so far not been liable.

By way of example, the analysis of a DNA sample involves the use of a so-called hybridization probe corresponding to the sequence complementary to the desired sequence.

Under these conditions, the problem posed is to isolate the hybrid from the medium and to detect with a low signal-to-noise (S / N) ratio the possibly reduced number of positive reactions.

CA 02182906 2004-12-16 7 This is why we now use, in most cases, an intermediate step intended to amplify the sequence to be detected, for example using the PCR method or methods of amplification leading to the same results, under these conditions the concentration of the sequence to be determined in the sample is increased and this detection is obviously much more convenient.

However, the amplification step is sensitive to contaminants and leads to errors which are specific to it.

It would therefore be preferable, as far as possible, to be able to detect the presence of the nucleic acid sequence without an amplification phase.

It has been proposed to use, in order to demonstrate the specific hybridization reaction, an intermediate step of anchoring the hybridization product on a solid surface exhibiting certain specificities.

For example, it is possible to use certain pretreated surfaces making it possible to fix certain proteins or DNA, whether or not it has been modified.

Such surfaces are commercially available (Covalink, Costar * Estapor, Bangs, Dynal * for example) in the form of beads or wells having on their surface COOH, NH2 or OH groups for example.

It has also been proposed to obtain such groups to use an intermediate step having a vinyl group which is then oxidized to present COOH or OH groups (USA 1,539,061 and EP X35,785).

The DNA can then be functionalized with a reactive group, for example amine, and a reaction can be carried out with these surfaces.

However, these methods require a particular functionalization of the DNA to be fixed.

A technique has also been described which allows anchoring without prior treatment of the DNA.

This process consists of reacting the free phosphate at the S 'end of the molecule with a secondary amine (NH Covalink surface).

* (trademarks). WQ 95! 22056 PCT / FR95 / OüIG4 3 The DNA can also be attached to a group or a Pp protein to make it react with a surface covered with a group or 'a protein I't, capable of reacting specifically with P0 . The Pp / Pt couple can be a couple of the biotin / streptavidin type or a digoxigenin / antibody directed against digoxigenin (anti-DIG) for example.

Such surfaces are, however, in most cases insufficiently specific (VV.

Lund et al., Nucl.

Acids Kes., 16, 1861 (1988)).

Thus, the presence of parasitic interactions, even weak ones, of the non-specific adsorption type leads to effective adsorptions for language molecules capable of contracting a large number of weak interaction points with the solid.

These surfaces lead to potential applications lacking in sensitivity and / or with a high shearing noise rate in the case of a small number of molecules to be fished.

In addition, some of these surfaces exhibit a high rate of parasitic fluorescence potentially troublesome during the detection phase.

As regards the detection proper, in particular for the detection of DNA, French patent 78 10975 describes a method coupling the probe to an enzyme which allows detection using a chromogenic substrate. I (is, moreover, possible to quantify the reaction by a colorimetric measurement.

However, such a technique is not suitable directly for the detection of traces, which is why, here too, it must be preceded in most cases by a step of amplification of the quantity of nucleic acid required. , for example by the PCft method.

This so-called "cold probe" detection method was developed to avoid the use of radioactive markers which give results which in sensitivity are similar but which obviously present handling problems, given the presence of radioactive products and problems. long revelation times if you are looking for great sensitivity.

W0 45! 22Q56 PCTIFRySltlti tl ~ q For certain particular applications, in particular methods derived from ex-vivo imaging, a direct method of observation of the reaction has been proposed by coupling the product of the hybridization to microbeads, in particular of PAfMIA, suitably chemically treated on their surface. (.a method is based on the direct identification under a scanning electron microscope of the presence of these microbeads with a typical diameter of 60 nm and moreover is based on solid anchoring techniques known but insufficiently specific, as well as has been described above.

The above techniques are of course not limited to the detection of nucleic acids.

In the mind, we have. proposed for example the detection of antibodies.

These are the EIdSA type tests which we will not redescribe here and which, to summarize, make it possible to couple the presence of an antibody 3 to an associated anchoring of a T5 antigen molecule on a solid. Again, the problems of specificity and parasitic reactions arise.

The detection phase can then be based on coupling to a chromogenic reaction having its own sensitivity problems.

In summary, the methods of the prior art or their ~ 3 combinations have a certain number of drawbacks, in particular - either to be potentially dangerous by using radioactive products, - or to require excessively long development times, ? 5 - either to be generated by specific problems at the level of the amplification phase, - or to go through insufficiently specific solid surfaces, or to be too insensitive, - or, finally, to require, in addition to the phase of attachment to a solid, the inconvenient use, of course, of an electron microscope.

Finally, in most cases, the known processes do not make it possible to recognize on a given molecule the specific position of the desired motif.

However, this type of recognition is important CA 02182906 2005-09-22 when one seeks to make a cartography, in particular within the framework of the mapping of the genome, one seeks to know first of all the approximate spatial position with respect to ûne e ~ rtr extremity of the molecule of a given gene on a DNA or an RNA.

The present invention, which proposes to remedy the drawbacks of the prior methods, is based on the use of very highly specific surfaces which, during their implementation, lead to excessively limited background noise, in particular due to the fact that they eliminate parasitic fixings. .

More particularly, the present invention relates to a highly specific surface for biological reactions, characterized in that it comprises a support having on the surface at least one monomolecular and compact layer of an organic compound of elongated structure:

- Having at least one fixing group exhibiting an affinity for the support, and an exposed group comprising an ethylenic double bond having little or no affinity for said support and said fixing group under the fixing conditions, but exhibiting a affinity for a type of biological molecule; and said surface exhibiting, on the outside of the layer, the exposed group comprising the ethylenic double bond having an affinity for the type of molecule with biological activity, said molecule being a chemical compound exhibiting biological activity and being capable of s' anchor directly by absorption on said surface for a pH greater than 5 and strictly less than 8.

The term “affinity” should be understood here to mean both chemical reactivity and adsorption of any type, this under the conditions for fixing said biological molecules.

The term “support” is understood to denote both a solid support and a support consisting of a non-solid element such as a liquid or gaseous particle having, in particular, a compact layer as described above.

CA 02182906 2004-12-16 5a The surface is “essentially compact”, that is to say it limits the access of the molecule with biological activity to the lower layers and / or to the support, it being understood that defects of surface coverage are tolerable. , ~ i ~~~ r ~~ W0 9512205 (, PC1'IFR95l001G4 6 These highly specific surfaces for biological reactions, comprise utx support having on the surface double bond groups, in particular vinyl (-Cl-I = CH2, hereinafter surfaces C = C) accessible to the solution.

They are capable of anchoring S directly molecules of biological interest (DNA, RNA, PNA, proteins, lipids, saccharidesy in certain copditions of pH or ionic content of the medium.

In particular, these surfaces do not require any particular chemical modification either of the surface or of the biologic molecules to be anchored.

There are no documents mentioning such use of a surface with v ~ nyl groups.

By "anchoring", it is meant here a fïxatïon by covalent bond resulting from a chemical reactivity, or non-covalent bond resulting from physicochemical interactions such as an adsorption of any type, this d.tns the conditions of pH or ionic content of the binding medium of said biological molecules.

The surfaces according to the present im = ention can be obtained by carrying out various methods.

Mention may be made, by way of example (A ~ a layer of carbonaceous polymer, optionally branched, of 2ü hands 1 nm thick, having groups comprising an ethylenic double bond,. The remainder of the layer consisting of hydroorfluorocarbon groups;

(F3) surfaces obtained by anchoring on a solid one or more molecular layers, these can be obtained by the formation of successive layers fixed by non-covalent bonds, of the Langmuir-131odgett film type, or by self- molecular assembly; this allows the formation of a layer fixed by 3p covalent bond.

In the first case, the surface can be obtained by polymerization of at least one monomer generating on the surface of the polymer said group comprising an ethylene double bond, or by partial depolymerization of the surface of the polymer to generate said group, or else by deposition of polymer.

~~ 8 ~ 'i WO 95122056 PCT / FR95JOD1G4 7 In this process, the polymer formed has vinyl bonds such as a polyene derivative, in particular synthetic rubber type surfaces, such as polybutadiene, poly ~ isogrene or natural rubber â In the second case, the highly specific surface for biological reactions according to the present invention comprises - on a support, a substantially monomolecular and compact layer of an organic compound of elongated structure having at least 1ü. a fixing group having an affinity for the support, and an exposed group comprising an ethylenic double bond, having little or no affinity for the said support and the said fixing group in the conditions of the fixing, but having an affinity for a type of biological molecule.

In order to obtain an essentially compact layer, the different organic compounds are preferably capable of reacting with each other outside the exposed group to create cross-links, thus obtaining an "essentially" compact monomolecular layer to which the support is obtained. becomes not or hardly accessible for side reactions.

Preferably, the organic compound has a binding moiety at one end and an exposed moiety at the other end.

It is of course possible to provide different embodiments in which, for example, the fixing group would be located in the middle of the molecule, the latter having at each of its ends an exposed group.

Surfaces can be analyzed according to:

30 a) the support, b) the molecule having an exposed group and a binding group on the support, c) the interaction between the support and said molecule ensuring the attachment.

WO 95J22U56 PCTlFR95J (I (it ti.i 8 The attachment can be first of all of the non-covalent type, in particular of the hydrophilelhydraphile and hydrophobic / hydrophobic type, as in the films of Langmuir-Blodgett {K.Ii. ülodgett, j .

Am.

Chem. 'Scx. 57, 1007 (1935) and US 5,102,798).

In this case, the fixing group will be either hydrophilic or hydrophobic, in particular alkyl or haloalkyl groups such as Cl-I3, CF3, CHF3, CllyF.

The binding can also be of the covalent type, the binding group will then react chemically on the support.

i0 Certain surfaces of similar structure have already been mentioned in the electronic field, in particular when the fasteners are coveted, L.

Netzer and j.

Sagiv, j.

Ant.

Chem.

Soc. 105, 674 (1983) and IlS-A- ~ 539,061.

Those skilled in the art have a very wide range of groups available. By way of nonlimiting example, mention will be made of groups of metal alkoxide type, such as silane, silane chloride, ethoxysilane, methoxysilane.

The fixing group is obviously chosen according to the support used.

The support according to the invention can be constituted at least on the surface, by a polymer, a metal, a metal oxide, a semiconductor element or an oxide of semiconductor element such as an oxide of silicon or one of their co: mbinaisnn.

Mention is made in particular of glass and silicon oxidized on the surface.

Among the attachment groups, mention should be made more particularly of groups of the metal alkoxide type such as silane, chlorosilane, chlorosilane, silanoI, methoxysilane, ethoxysilane, silazane, phosphate, hydroxy, hydrazide, hydrazine, amine, amide, diazonium, pyridine, sulfate. , sulfonic, carboxylic, boronic, halogen, acid halide, aldehyde.

30 In particular, as the oxidation group, it will be preferred to use groups capable of reacting transversely with an equivalent, neighboring group, to provide the transverse bonds, for example it will be a question of silance-type drives, in particular dictxlorosilane, trichlorosilane, dimethoxysilane, trimethoxysilane. , diethoxysilane and triethoxysilane.

21 ~ 2 ~~ WO 95! 22056 FCTIFIt95 / OOlG4 9 These cross links can also be made at any point in the thickness of the monolayer. by polymerizing it using reactive groups optionally present on the drain between the binding site and the exposed group. Thus, the p diacetylenic groups are known to allow one-dimensional or two-dimensional polymerization of the monolayer.

The choice of the Rxatian group will obviously depend on the nature of the support, groups of silane type are well suited for covalent attachment to glass and silica.

Preferably, the chains connecting the group e:, posed to the group of I'matïon are chains comprising at least 1 carbon atom, preferably more than 6 and in general from 3 to 30 carbon atoms.

When there is formation of a lateral coupling inside the same layer, whether this coupling is ionic, of coardinance or cavalier, one 1 ~ obtains highly ordered layers obtained by self-assembly, even if the initial surface exhibits only a reduced number of active anchoring sites compared to the number of molecules obtained in a compact manolayer.

One can advantageously use, in the case of glass or silica, the known techniques of surface functiannalisation using silane derivatives, for example: Si-OI-I + CL3-Si-R-CH = CI-IZ gives Si- O-Si-R C1-f = CHZ, R consisting for example of (CH 2) .y.

Such a reaction is known in the literature, with the use of ultra-pure solvents.

The reaction results in a carpet of molecules having their C = C end on the surface exposed to the outside.

In the context of obtaining a very high specificity surface, the present invention also relates, for such C = C double bond molecule grafting reactions, to the use of a gas phase, making it possible to avoid the use of solvent.

30 In the case of gold, the latter optionally being in the form of a thin layer on a substrate, the known techniques of t '~' O! T5122t156 PCT1FR94 / Ot) 16.1 surface functionalization use thittl derivatives, for example example: Au + HS-R-CH = CH2 gives Au-SR-CH = C1I2, lt being for example (CH2) .t.

Such a reaction is described in a liquid medium and leads, like the previous trichlorosilane-silica reaction, to a carpet of molecules having their C = C end at the surface exposed to the outside.

Of course the terminology of "support" encompasses both a single surface such as a blade, also particles, whether silica powder or polymer beads, and also any shapes such as bar, fiber. or structured support, which can moreover be made magnetic, fluorescent or colored, as is known in various assay technologies.

Preferably, the support will be chosen to be not or weakly fluorescent when the detection is carried out by fluorescence.

The surfaces obtained according to modes (A) or (B) above have a high specificity thanks to the presence of specific reactive sites from either exposed groups, coitus of the fixed molecule.

In addition, the surfaces obtained according to modes (A) to (B) exhibit the following unexpected and remarkable characteristics (i) a specific anchoring strongly pH dependent on the DNA by its ends without the need for a particular functionalization of the molecule, accompanied by a very low rate of non-specific interactions;

(ü) lot possibility of anchoring proteins and other molecules of biological interest therein, without particular chemical modification;

(iii) the possibility of preparing specific surfaces for an antigen (eg digoxigenin) or a ligand (eg biotin);

(iv) a very low level of intrinsic fluorescence, when required, a background fluorescence (with a typical area of ​​100 x 100 tam) lower than the fluorescence signal of a single molecule to be detected;

(v) the possibility of detecting isolated molecules with an S / N ratio independent of the number of molecules, which is susceptible by means of various high S / N ratio techniques described below and based on the identification of the presence. a macroscopic marker exhibiting a weak non-specific interaction with the surface.

The surfaces thus obtained are preferably coated with a molecule with biological activity chosen from - proteins, - nucleic acids, - lipids, - polysaccharides and their derivatives, Among the proteins, mention should be made of antigens and antibodies, ligands, receptors, but also products of the avidin or streptavidin type as well as derivatives of these compounds.

Mention should also be made, among RNAs and DNAs, of ha and p derivatives as well as thio derivatives and mixed compounds such as PNAs.

It is also possible to attach mixed compounds such as glycopeptides and lipopolysaccharides for example, or other elements such as viruses, cells in particular, or chemical compounds such as biotin.

2U The binding of biological molecules can be covalent or non-covalent, for example by adsorption, hydrogen bonds, hydrophobic or ionic interactions, for example, in which case one can advantageously proceed with a bridging ("cross-linking") between the grafted molecules. by known methods ("Chemîstry of Protein 25 Conjugation anl Cross-linking ~, SC

Wong, CRC Press (1991jj and this in order to strengthen their cohesion.

With an exposed group comprising a -CH = Cflz radical which will be called hereinafter "surface C = C" or "surface with ethylene liaïsori ~, direct anchoring, in particular of DNA or proteins, is possible.

30 In the context of the present invention, it has been demonstrated that these surfaces exhibit a very strongly gH dependent reactivity.

This feature allows nucleic acids or proteins to be anchored, in particular by their) excrete (s), using a determined pH region and often with a reaction rate which can be controlled by the pH.

~~ ~ '~~~~ WQ ~ JS12205f, PCTIFI295fOk1ld] 7 Thus, for DNA at pH 5.5, the anchoring reaction is complete in one hour (if not limited by diffusion) and occurs by eW remit.

At ply 8, on the other hand, the attachment is very weak (reaction speed of 5 to 6 orders of magnitude lower).

This pH-dependent and specific hooking effect of the ends, presents an improvement compared to other surfaces which require DNA functionalization (biotin, DIG, NHS, ...) or specific reagents (carbodümide, dirnéthy'le pïmélidate ) which make a peptide or phosphorimide bond between Nliz and -COOH or -POOII.

The surfaces according to the invention can anchor proteins directly (protein A, anti-DIG, antibody, streptavidin, etc.).

It has been observed that (i) the activity of the molecule can be preserved and (ü) that the reactivity of the prepared surface (initially C = C) is completely hidden to make room for the only reactivity of the molecule of interest. . It is therefore possible, from a relatively large initial reactivity, to pass a surface having a very highly specific reactivity, for example that of specific sites on a protein.

By anchoring a specific antibody on the surface (for example anti-DIG), a surface is created whose reactivity is limited to the antigen (for example ie the DIG group).

This indicates that the original chemical groups have all been obscured by the anchored antibodies.

It is also possible to anchor on the reactive surfaces (chemically or biochemically) other molecules with biological activity, in particular viruses or other components: membranes, membrane receptors, polysaccharides, PNA, in particular.

It is also possible to fix the product of a reaction of biological interest (eg PCit) on the prepared surfaces.

The present invention also relates to the surfaces obtained by carrying out the methods according to the present invention and all the methods using this type of surface, whether these are methods allowing the detection and / or the quantification of molecules. biological, but also the separation of certain biological molecules, in particular a sampling by implementing antigen / antibody and / or DNA, DNA / Al2N coupling techniques.

WO 95122056 PCTIFR95 / 001 (ia 13 The present invention also relates to processes for preparing highly specific surfaces for biological reactions as described above for obtaining the layers according to (A) and (B) and, in particular, the process characterized in that - is fixed on a support a substantially monomolecular and compact layer of an organic compound of elongated structure having at least one fixing group having an affinity for the support, and. an expanded group comprising an ethylenic double bond, having little or no affinity for said support and the fixing group under the fixing conditions, but having an affinity for a type of biological molecule.

The present invention also relates to the applications of the treated surfaces for the detection of isolated molecules using specific reagents and detection methods with an S / N ratio independent of the number of molecules detected.

Aia = generally, the present invention relates to a method for demonstrating and / or assaying a molecule with biological activity in a sample, characterized in that a surface such as described above is used, on which is located fixed a molecule with biological activity capable of recognizing the molecule of the sample, and in that the detection or the assay are carried out by means of a fluorescent reagent or not detecting the presence of the fixed molecule.

Among the reagents, a distinction is made between fluorescent reagents and non-fluorescent reagents.

Fluorescent reagents contain fluorescent molecules, advantageously chosen to be molecules greater than 0.1 wm long and specifically reacting directly or indirectly with the pretreated surfaces.

For example, but not limited to, a double-stranded DNA molecule stained with fluorescent probes (ethidium bromide, YOYO, fluorescent nucleotides, etc.) that can anchor directly to a C = C surface. , by modifying the molecule (DIG, biotin, etc.) on a surface presenting complementary proteins (anti-DIG, streptavidin, etc.).

CA 02182906 2004-12-16 1 ~ The non-fluorescent reagents consist, in particular, of beads anchored by the intermediary of a molecule fixed in a manner.

specific directly or indirectly to a pre-treated surface.

Thanks to the surface treatment, these beads exhibit a low non-specific S interaction with the surface.

For example, but not limited to, Dyna ~ beads coated with streptavidin and anchored via biotinylated DNA to a surface according to the present invention, exhibiting sites capable of reacting with the other end of the DNA molecule.

Depending on whether the desired molecule is detected directly by fluorescence or indirectly using the above reagents, we will speak of "direct detection" or "by flag".

In order to limit the problems associated with prohibitively slow reaction times, the times for diffusion of reagents to the surface can advantageously be reduced by using small reaction volumes.

For example, but not limited to carrying out the reaction in a volume of a few microliters determined by the spacing between two surfaces, one of which is treated to present reactive sites according to the present invention and the other is inert or treated. so as not to present reactive sites.

The detection of the number of specific reactions having occurred can be carried out on a small number of molecules (typically 1 to 1000), by a low noise macroscopic physical test requiring neither electron microscope nor radioactivity nor 2 ~ necessarily PCR.

The detection methods are likely to be implemented. by people with little laboratory experience.

Depending on the reagent, two implementations of the present invention (X mode and Y mode) can be used for the low noise macroscopic detection of a small number of reagent anchoring reactions.

* (trademark) CA 02182906 2005-09-22 In the so-called mode X implementation of the present invention, a test of the number of specific reactions having occurred is.

obtained directly by a fluorescence technique, allowing for certain embodiments of the present invention to individually identify the number of sites having reacted.

In this case, the highly specific surface is advantageously taken to have a very low level of fluorescence, in particular the support must have a low fluorescence.

After anchoring of the fluorescent reagent, the detection and counting of the possibly small number of anchoring reactions can advantageously be done with the aid of a fluorescence optical microscope using a large numerical aperture objective, making it possible to identify either directly at the eye, or after signal acquisition, the number of anchored fluorescent molecules.

It is advantageously possible to carry out a scanning of the observation field in order to explore a larger area than the only fixed field.

In the so-called Y-mode implementation of the present invention, a macroscopic bead-like reagent (fluorescent, magnetic, colored, for example) is detected.

The technique as described above allows the reaction to be revealed by the presence or absence of microbeads.

In one embodiment, a new method comprises (i) the use of beads with specific reactivity, (ü) the use of beads of sizes not nanoscopic but lying in the range 0.1 ~ m-200 wm , detectable by a macroscopic technique, and (iii) the absence of non-specific reaction between beads and surface due to the use of the product according to the present invention.

The number of these macroscopic beads each characterizing an anchoring reaction is then determined by a macroscopic physical method, including, but not limited to, the diffusion of light on the beads, optical microscopy and the fluorescence of the beads. balls.

WO 95! 22054 PCTIFR95101) lba 16 The specificity of some biological reactions may be limited.

Thus, in the context of hybridization, the hybrids may be imperfect (reactions with other sites) while exhibiting a reduced number of pairings and therefore a lower binding quality. I ~ present invention also covers the possible use of a step of testing the quality of the bonds obtained.

This test makes it possible to dissociate the paired products in a weak non-specific way, by adsorption, hybrophobic forces, imperfect hydrogen bonds, imperfect hybridization, in particular.

ï0 Therefore, the invention also relates in a method of detection or assay as described above, a method where the reaction product is subjected between the molecule with biological activity and the molecule of the sample. â a constraint in order to destroy the mismatches before detection.

This process offers, in addition to the possibility of destroying the mismatched couples, the possibility of orienting the products of the coupling, which facilitates the measurements or the observations ~ ations.

It is thus possible to apply to the surfaces, after fixing of the complementary elements, a stress which can be constituted by? 0 the combined use of - cenirifugation, - magnetic field gradient applied to the fluorescent reagents then taken to include magnetizable or magnetic microbeads. , - agitation, liquid flow, - passage of meniscus, - electrophoresis - temperature variation, and / seen temperature gradient.

The low noise detection techniques described below are then determined to determine the number of systems which have remained integrated or have been destroyed.

~ W09512205G ~ PCTIFR951001Gd 1r It should be noted that thanks to the surfaces according to the present invention, it is possible to orient the molecules after their attachment by at least one point by passing the airJeau meniscus, in particular on DNA.

Thus, an observed that the passage of menicus air / water over DNA in solution and anchored to the surface, resulted in a regular expansion of the anchored molecules.

They then appear in the open air in the form of elongated fluorescent sticks.

These elongated molecules are stable in the open air and can be obsen ~ ed even after several weeks, without showing any apparent degradation.

These remarkable and unexpected observations suggest a possibility of counting the number of DNA molecules anchored to the surface on the one hand, the surfaces being very little fluorescent, the signal-to-noise ratio (S / N} is good, on the other hand, looking for a very correlated object (shape of rods), it is very easy to increase the S / N ratio. That is to say, to ignore the dust, the inhomogeneities, which do not present any particular spatial correlation.

It should be noted that in solution, the molecules in balls fluctuate thermally, which causes very significant variations in their fluorescence signal collected, in general, with a shallow depth of field and limits their observation.

1.a present invention also covers this technique of alignment and immobilization which therefore allows the ohsen ~ ation of isolated molecules with a very large S / N ratio.

It is remarkable that this ratio is independent of the number of anchoring reactions.

The S / N ratio posed by the detection of one? S molecule is the same as per 10,000.

In addition, this stretching technique makes it possible to easily discriminate between molecules of various lengths.

The following steps can advantageously be carried out to further improve the S / N ratio. The molecule being stationary, its fluorescence signal can be integrated.

- The observation under the microscope presents a reduced field (typically 100 wm x 100 wm with an objective x 100 with immersion, NA = 1.25).

For a sample of 1 cmz, one can either carry out a scanning, or consider the use of objectives of smaller magnifications (x 10 or x 20) but high numerical aperture.

~~~~ i wo 9srzzas6 pcr> rx ~ rsrmrtsa 1s - The rods being always parallel, we can consider an optical spatial filtering method to further increase the SlN ratio.

- Other global fluorescence methods are possible (~ P »10342f ~).

- Ia linearization of the molecules is also obsen ~ e within the framework of a chemical grafting (C = C) as in the case of immunological type bonds (DIClanti-DIG).

- Once on the surface in the open air, the DNA molecules are stable (remain intact, even after several weeks) and fluorescent.

This property can advantageously be used to defer the anchoring step from the step of locating / counting the anchored molecules, if this detection takes place for example, but without limiting itself to it, by fluorescence microscopy.

Such use is covered by the present invention.

- A doubla (or multi) fluorescence technique can possibly help to improve the SIN report or to detect a double or multi functionality.

- It is possible to extend the airlcau meniscus used here in order to stretch 2 () the molecule to other systems such as oil-water or water-surfactant / air, in particular.

It is also possible to use a dynamic orientation of the molecules in solution anchored at one end, by electrophoresis or flow in one or more successive directions, such a technique thus being able to lead to a synchronous detection of the presence of molecules in a given direction, eg. , r analysis of the temporal variations of the fluorescence signal corresponding to a given direction {for example, but without being limited thereto, by use of a suitably arranged optical spatial liter, making it possible to obtain a preferential signal for certain orientations of the molecules observed).

Nevertheless, the results observed show that this technique, in its simplest version (stretching in a single direction, without synchronous detection) is much less efficient than the use of the meniscus.

~~ ~~~~~ 3 R'O 95122056 PCTIFR95 / OO1G4 29 The surfaces and / or reagents and / or detection techniques described in Ia present inventian can be used paur many applications including, but without restricting the identification of one or more DNA or RNA sequencing elements that can be used with advantage for pathogen diagnosis or genetic mapping;

- measuring the size of DNA fragments that can be used with advantage for genetic mapping;

- improving the sensitivity of LISA techniques with the possibility of detecting a small number (possibly less than 1000) of immunological reactions.

The identification of DNA / RNA sequences can be done first by reaction in the volume of the solution of the DNA / RNA molecules with complementary probes (for example by hybridization or using specific proteins of the desired segment. ).

L? Had operating modes are then possible.

The descriptions which will follow will, for some, be made with reference to the appended figures in which: FIG. 1 shows diagrammatically the detection of a pathogen in a fluorescent DNA molecule by hybridization with an anchor molecule;

- the figure ? schematize your genetic mapping by extension of DNA and the use of a DNA marker;

- Ix Figure 3 shows schematically the detection of an immunological reaction?> (ELISA) using a "flag" molecule: a fluorescent DNA used as a reaction marker;

- Figure ~ i is a fluorescence photomicrograph showing the DNA tension of phxge 5, by the advancement of the meniscus, on the left we see DNA molecules in themselves stretched by the parallel evaporation flow at the meniscus, to the right of the DNA molecules in the open air after their stretching perpendicular to the meniscus;

~~ ~~~~~ a wa ssrz ~ ass rcTrlix ~ sloot6.s - Figures 5 (a) and 5 (b) are fluorescence microphotagrap.hies showing, respectively; DNA labeled with digogixenin (DIG) on a surface coated with anti-DIG and stretched by the meniscus, and, as a control, unlabelled DNA on an anti-DIG surface, will notice very high specificity. surfaces and the absence of non-specific anchoring;

- 1a figure 6 is a fluorescence photomicrograph showing a classic eam, mercial surface such as NUNC, one will notice the very large fluorescence inhamogenitis which makes these surfaces impossible to use for the fluorescent detection of a single molecule.

In the "diagnostic" mode, the probes (the "anchors") have a reactive group (DIG, butine, etc.) capable of being anchored in a specific manner to a surface according to the present invention (having for example as a site of anchoring an anti-D1G antibody or streptavidin ~).

The detection of the anchoring reaction can be done directly by detecting the fluorescence of the DNA molecule stained by fluorescent molecules (ethidium brornide, YOYO, fluorescent nucleotides) (Figure I).

It can also be done indirectly by detection of a; "flag molecule": a reagent according to the present invention capable of binding to the DNAIAItN molecule (for example by hybridization, DNA practicein interaction, etc.), but not exhibiting any affinity for the anchoring sites of the probe.

In the "mapping" mode, the complementary probes ~ 5 can be directly coupled to a fluorescent reagent according to the present invention.

It can be for example a single strand of complementary DNA having bases modified to be fluorescent or of a long double strand of DNA stained with a fluorophore A and ending with a single stranded segment complementary to the sequence. sought.

30 For different probes, fluorophores of different colors can be used.

It is also advantageously possible to dye the DNA molecule on which the probes hybridize with a fluoraphor of another color. The DNA-probe hybrid is anchored at one of its ends and stretched by one of the methods described above.

The 3S distance from the anchor point to the hybridization points, or between the hybridization points, is determined by detecting the fluorescence of the probe, according to the methods described previously (FIG. 2).

CA 02182906 2004-12-16 21 For example, without being limited thereto, a DNA marker, of approximately 3000 base pairs and having. At one of its ends a single-stranded segment complementary to the desired gene, is colored. with a fluorophore A (for example YOY01).

This DNA is hybridized and then ligated with the single-stranded DNA to be mapped, then the latter is stained with a second fluorophore B (POPO1) (after reaction by "random primming", to transform it into double-stranded DNA).

The molecule is then anchored by one of its e ~. ~ Hoppers (for example by DIG / anti-DIG bond) and stretched by the action of the meniscus.

The distance between the end of the molecule and the postion of the labeled gene, observable in double fluorescence microscopy (2 colors A and B) makes it possible to establish the position of the desired gene with a precision of the order of 1000 pairs of basics (0.3 gym).

The identification of DNA / RNA sequences can also be done by reaction between the desired sequence and the reactive sites of a surface according to the present invention (for example complementary oligonucleotides or the reaction site of a specific protein of the segment. research).

The detection of the anchoring reaction can then be done directly or indirectly (using a “flag molecule”) as described previously.

It is understood that the identification of DNA / RNA sequences according to the present invention can equally well be used for diagnostic purposes (for example the detection of the presence or absence of a viral or chromosomal pathogen) as well as for genetic mapping purposes.

It can be preceded by an amplification step by any method, in particular PCR.

Furthermore, genetic mapping can proceed by measuring the size of DNA fragments.

However, the coupling between the surfaces according to the present invention and the original techniques for stretching the molecules described above (in particular and with advantage the stretching by the meniscus) allows a measurement of the length of the stretched molecules and this over a very small sample (a few thousand molecules).

One can, for example, but without restriction, to proceed in the following manner wo ~ snzosc rcz'rFxvsromsa zt A DNA sample is fragmented (using restriction enzymes), stained with a fluoroghore guis anchored on a surface with reactive groups (for example C = C surfaces).

The molecules are then stretched by the meniscus and the size of the stretched S fragments determined by fluorescence light microscopy with a resolution and a size limit of the order of 1000 bp (0.3 gym).

The surfaces according to the present invention can be used for carrying out known methods allowing the detection and quantification of an antigen or of an antibody, in particular the ELISA methods using enzymatic systems or methods of the type. RIA using radioactive markers. These are technologies which will not be described in detail.

One can also and advantageously use the surfaces according to the present invention as a support for the immunological reactions of an EL1SA process having a step of anchoring a reagent according to the present invention ("flag") on one of the reagents of the. ELISA (Figure 3).

The detection can naturally be done globally by measuring fluorescence.

It is also possible to proceed to the counting of the number of reactions, this can advantageously be carried out according to the detection methods described in the present invention, in particular the tension by menic, and this thanks to the low taus of fluorescence and of. non-specific interaction of the product of this statement.

This allows the detection of a small number of reactions, possibly less than 1000) with an excellent S / N ratio.

It is therefore geut, by a minor modification of the sandwich ELISA methods (antibody-antigen-modified antibody, for example biotinylated), to graft onto the surface a reagent according to the present invention, for example fluorescent DNA anchored to streptavidin.

All variants of the EI1SA technique are applied with much better sensitivity.

Global fluorescence measurement techniques have already been used to determine the quality of FLLISA reactions.

However, the method according to the invention allows a much better detection because it is sensitive to the fluorescence signal of a single molecule.

WO 9512056 PCTlFR95I0016 ~ t 23 Of course, it is possible to use these surfaces as binding surfaces of at least one product of a reaction of biological interest, by way of example without being limited thereto, amplification reaction products, whether PCI2 or a related S method and, enim, it is possible to use this type of surfaces as a support for affinity chromatography, either preparative or detection.

In this case, it is possible, for example, to graft a given population of oligonucleotides onto sïlice beads which will constitute the stationary phase of a chromatography column.

The chromatography step must allow a particular specificity of the column with respect to eluents, for example a mixture of DNA, some of which have sequences complementary to or very close to the grafted oligonucleotide.

1 ~ The present invention finally relates to the use of surfaces according to the present invention in diagnostic or separation kits.

Other characteristics and advantages of the present invention will become apparent on reading the examples below.

~ (materials and methods DNA-> 'and the monoclonal antibody (Anti-DIG) come from Boehringer-Mannheim.

The trichlorosilanes come from Roth Sachiel.

The fluorescent nucleic acid probes (YOYO1, YOYO3 and POPO1) come from Molecular Probes.

Ultra clean glass coverslips are from Erie Scientific (Coverslips (ESCO).

The magnetic particles come from Dynal.

The màcroscope is a Diaphot inverted microscope from NIKKON, equipped with a Xenon lamp for epifluorescence and a Hamamatsu intensified CCD camera for visualization.

Surface treatment Blast glass coverslips cleaned for one hour by UV irradiation under an oxygen atmosphere (by formation of ozone).

They are then immediately placed in a desiccator previously purged of traces of water with a stream of argon.

A volume of about 100 to 3S 50O w1 of the appropriate trichlorosilane (HZC = CH- (Cli2) xr-SiCl3 is introduced into the desiccator, from which the surfaces are removed after about 12 hours (n = 6) or 1 hour ( n = 1).

When leaving, the surfaces are clean and non-wetting.

WO 95122051 PCTlFit9Slüü764 2 = t the lamellae thus functionalized can react with proteins.

A volume of 300 Ixl of an aqueous solution (2O ~ g / ml) of proteins (protein A, streptavidin, etc.) is deposited on a coverslip functionalized as a group (112C = CH-).

This coverslip is incubated for about two hours ~ room temperature, then rinsed in very pure water ~.

The treated surfaces are clean and wetting.

The surfaces treated with protein A can then react with an antibody, for example âttti-DIG, by incubation in a solution of 20 ~ g / ml of antibody.

Native DNA anchor on double linker surface A drop of 2 ~ 1 of a solution of fluorescently labeled DNA-> ,, (YOYO1, POPOI to YOY03, but without particular termination) of varying concentration and in different buffers (number total malecules <107) is deposited on a pretreated strip (HIC = CH-) and covered with a coverslip of untreated glass (diameter 15 mm).

The preparation is incubated for about 1 hour at room temperature in an atmosphere saturated with water vapor.

In a 0.05 M MES buffer (pH = 5.5), an almost general anchoring of the DNA molecules is observed.

On the other hand, in a 0.01 hl Tris buffer (pH = &) there is almost no anchored molecule (ratio> 10E '.

This dependence may allow control of the activation of surfaces (vis-à-vis DNA) via pH.

I îétewicm of a ~ tcra2e oar the action of the meniscus By transferring the previous preparation in a Z; dry atmosphere :, the solution, while evaporating, will stretch the d: DNA molecules, anchored to the surface, perpendicular to the meniscus.

The capillary force on the rIDN molecule (a few tens of pïcoNewtons) is indeed sufficient to completely stretch the molecule (greater than the forces of entropic elasticity), but too weak to break the bond between the end of the molecule. and the surface treated. The DNA being labeled by fluorescence, an obsen ~ e individually and easily stretched molecules (total length approximately? Z gym). As the anchoring between the surface and the DNA was limited to the ends, it was possible to stretch both phage x, YAC and E coli DNAs (greater total length ~ 400 µm).

This preparation of stretched, fluorescent and free DNA WO 95/22056 PCTfFFt95I0016d is stable for several days and can be observed non-destructively, by epilluorescence (Nikkon Diaphot inverted microscope with x100 objective, ON: 1.25).

Detection of anchorage nar electroohoresis An electrophoretic cell is formed by a paraffin ring (thickness approximately 100 g) caught between a treated coverslip and an untreated glass coverslip between which two platinum electrodes are inserted. The whole is made integral by briefly melting the paraffin ring.

By means of two openings left in the paraffin ring the DNA solution is introduced into this cell by capillary action, then the two openings are sealed with paraffin.

Incubation, as before, at room temperature.

By applying a low voltage (a few volts) between the two platinum electrodes, one observes by fluorescence a movement of the free DNA molecules (a few tens of microns per second) and an extension in the direction of the flow of the molecules. anchored which can thus be easily and individually identified by epifluorescence microscopy.

Specific Anchoring and Detection By treating the surfaces as described above with a specific monoclonal antibody, their specificity can be controlled very precisely.

Thus, we tested the specificity of anti-DIG treated surfaces vis-à-vis DNA-hybridized with an oligonucleotide complementary to one of the Cos ends and having a digoxigenin group (DIG) and vis-à-vis unhybridized A17N. .

In the first case, an almost general extension was observed by the action of the meniscus of the anchored molecules.

In the second case, only a few DNA molecules (<10) were observed anchored in the whole sample.

It is therefore estimated that the specificity of the method according to the invention is better than 10 ~.

Scope of detection In order to determine the sensitivity of the meniscus extension detection method, 2.5 w1 drops of a DNA solution in 0.05 were deposited on double bond surfaces. M MES (pI-i = 5.5) containing a total of 105, 10a and 1000 molecules. The anchoring is carried out as described previously.

The coverslips are then observed by epifluorescence microscopy to determine the density of anchored molecules ~~ B ~~ C wo asraaass rcr ~ i ~ srooma 26- This corresponds well to that estimated: about 4-6 molecules of DNA per eham, g of vision (100 wm t 100 ~ .m) for a total of 105 DNA molecules.

For the lowest concentration, about ten molecules extended by the action of the meniscus were observed.

This number is essentially limited by the large number of fields of view necessary to cover the whole sample (approximately? 5,000), which makes manual research difficult, but can be advantageously carried out automatically and with a weaker objective, but at larger field.

In conclusion, the sensitivity of the method according to the invention allows detection and individual counting of less than 1000 DNA molecules.