JP2002530519A - Conductive electroactive functionalized binding polymers and uses thereof - Google Patents

Abstract

SUMMARY OF THE INVENTION The present invention is a conductive, electroactive functionalized binding polymer of formula (I '), wherein: Wherein n is an integer or zero and each R can be the same or different from each other and can be H, or an electrical bond to COON-hydroxyphthalimide, COO pentafluorophenol, an electrochemical probe and an activated ester. A functional group capable of covalently binding to a first biological molecule or an anti-ligand selected from the group consisting of chemical probes, provided that (a) at least one of said Rs in formula (I ') represents said functional group Or (b) when each YpR in formula (I ') is the same, they are different from CH2-COOH, each Yp may be the same or different, and p is zero or an integer And having a conductivity and electroactivity of substantially the same order of magnitude as the conductivity and electroactivity of the corresponding polymer of formula (I ′) when R represents H. About Rimmer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION AND PRIOR ART

Binding polymers such as polypyrrole, polythiophene, polyaniline, polyphenylene and derivatives thereof are known for their electroactive properties,
“Handbook of Organic Conducting Polymers” (TJSkotheim Editor, Marcel D
ekker, New York, 1986). These polymers are obtained in the form of a film on the electrode, in the form of a self-supporting film, or in the form of a composite when they behave like organic electrodes in combination with a polycationic or polyanionic polymer; Is charged according to the anodic oxidation treatment by the insertion of ions from the anode. This electrochemical treatment is reversible, and the reduction expels ions from the bound polymer or electroactive composite.

[0002] The second generation of binding polymers is described in the publication and is obtained by covalent grafting of monomeric units of the polymer with functional groups that can impart additional functions to the electroactive binding polymer. As an example, an electrocatalytic metal complex can be grafted onto a monomeric unit of polypyrrole to form a specific complexing macrocycle.
cle) is grafted to the polypyrrole or polythiophene chain to recognize the cation of the electrolytic medium, and a chiral group is grafted to the polythiophene to recognize the optically active anion. All of these functionalization routes also constitute the subject of color processing described in detail in the publication (F. Garnier, Angew. Chemie, 1989, 101, 529).
; A. Deronzier, JCMoutet, Acc. Chem. Res. 1989, 22, 249; J. Roncali, Chem Rev., 1
992,92,711).

[0003] During the last few years, researchers have been interested in the use of conductive polymers functionalized for the development of analyte scavenger color, especially for diagnostic purposes. However, as described in patent application EP0314009, especially when a functional group is introduced into a heteroatom ring, the conductivity of the polymer is impaired,
It has been generally accepted by the scientific community that pyrrole polymers substituted at either the nitrogen atom of the pyrrole ring or directly at the carbon atom are not good candidates for the color development of analyte scavengers. In order to solve such a problem, the present applicant has considered the use of a 2,5-di (2-thienylpyrrole) polymer grafted at the 3-position of a pyrrole ring at a reactive site where an organic molecule is covalently bonded. Was. However, due to the hydrophobic nature of the thiophene ring, the polymers are neither conductive nor electroactive in aqueous media and detect and / or detect analytes in biological samples.
Or did not appear to be suitable for characterization (J. Roncali et al., Chem. Co.
mm., 1986, p. 783 and G. Tourillon et al., Electronal. Chem., 161, 407, 1984).

[0004]

[Problems to be solved by the invention]

Quite surprisingly, and contrary to what has been accepted by those skilled in the art, the functional group can be converted to the 3- or 4- of the pyrrole ring using a functionalizing agent that moves the desired functional group away from the pyrrole ring. It has been found that the conductivity and electrical activity of polypyrrole is maintained if it is grafted to the position. The antiligand is covalently attached to the free end of the functional group without altering the above properties of the polymer. Such functionalized polymers have not been described before and have never been shown to be perfectly suitable as scavengers for biological ligands. In addition, polypyrrole has been shown to be an advantageous polymer because of its biocompatibility. Finally, the polypyrrole thus functionalized makes it possible to prepare electroactive and conductive polymers of significant thickness (up to a few millimeters), achieve a high density of functional sites and improve the sensitivity in proportion .

[0005]

Means for Solving the Problems and Embodiments of the Invention

Thus, the subject of the present invention is a conductive, electroactive functionalized polymer corresponding to formula (I),

Embedded image Wherein n is an integer other than zero, i is an integer ranging from 2 to n-1, R1, Ri and Rn may be the same or different from each other, and Is a functional group capable of covalently binding to a biological molecule or an anti-ligand, wherein said polymer is a corresponding non-functionalized binding polymer, ie, the conductivity of a corresponding polymer of Formula I, wherein R1, Ri and Rn each represent H. And a polymer having substantially the same order of conductivity and electroactivity as electroactivity.

In particular, the functional groups are independently selected from the following functional groups: Yp—C—X, where X is H, OH, a substituted or unsubstituted lower O-alkyl group, or a halogen, especially Cl. Yp-NHZ, Z represents H or an alkyl group; Yp-NH-CO-CF3; Yp-X, X correspond to the above definition, and p is preferably an integer equal to 0, 1 or 2. Activated ester groups such as -Si (alkyl) 3, -Si (alkoxyl) 3 or COON-hydroxysuccinimide;

Y is an alkyl group having 1 to 5 carbon atoms, an alkoxyl group having 1 to 5 carbon atoms, and m is an integer in the range of 1 to 3, and m ′ is 1 or 2. Indicate the polyether corresponding to the formula (CH2-CH2-O) m (CH2) m 'which is an equal integer.

The present invention also provides a conductive, electroactive functionalized binding polymer of formula (I ′),

Embedded image Wherein n is an integer or zero, and each R is the same or different and is H, or a functional group capable of covalently binding to a first biological molecule or anti-ligand; (A) Formula (I ′)
Wherein at least one of said Rs represents said functional group, or (b) each YpR of formula (I ')
Are the same as CH2-COOH, each Yp may be the same or different from each other, and p is zero or an integer, and R is H It also relates to polymers having conductivity and electroactivity of substantially the same order of magnitude as the conductivity and electroactivity of the corresponding polymer of formula (III) when indicated.

Preferably, p is 0, 1 or 2.

Preferably, the polymer of formula (I ′) is: —R is COX (X is H, OH, substituted or unsubstituted lower O-alkyl or halogen); Activated ester; NHZ, Z is H, Alkyl group or CO-CF3
Si (alkyl) 3; Si (alkoxyl) 3; an electrochemical probe; an electrochemical probe bound to an activated ester; the electrochemical probe is preferably selected from the group consisting of ferrocene and quinone, and / or Activated esters are COON-hydroxysuccinimide, COON-hydroxyphthalimide, C
A polymer selected from OO pentafluorophenol, wherein the halogen is preferably chlorine; a polymer wherein -p is at least 1 and R is selected from the group consisting of H, OH, substituted lower O-alkyl and halogen; Is an alkylene group having 1 to 5 carbon atoms; an oxyalkylene group having 1 to 5 carbon atoms; a formula wherein m is an integer in the range of 1 to 3 and m ′ is an integer equal to 1 or 2 A polyether having [(CH2-CH2-O) m (CH2) m ']; (CH2) mCONH (CH2) wherein m and m''are the same or different and are an integer in the range of 1 to 3; And (CH2) mCON (m) wherein m is an integer in the range of 1 to 3, and m ′ '' is 2 or 3.
CH2) a polymer selected from the group consisting of m ′ ″.

The anti-ligand can form an anti-ligand / target molecule complex. Preferably, the complex is a peptide / antibody, antibody / hapten, hormone /
Selected from the group consisting of receptors, polynucleotide hybrids / polynucleotides and polynucleotide / nucleic acid pairs.

[0012] The target molecule can include a histidine tag.

[0013] More preferred polymers are: -Polymer (A), p is 1 and Y is CH2, CH2-CH2 and CH2-
CH2-CH2, and at least one of said R is CO
OH; the substituent YpR can be reactive towards the amino function of a biological probe (eg, oligonucleotide, oligonucleotide, amino acid, peptide derived with an amino function); It allows for further grafting of biological probes on homopolymers or copolymers; such substituents are very beneficial in increasing the hydrophilicity of the homopolymer or copolymer, but this is due to the electrochemical behavior in aqueous solutions. -Polymer (B), p is 1 and Y is CH2, CH2-CH2 and CH2-
Selected from the group consisting of CH2-CH2, and wherein at least one of said R is an activated ester; preferred activated esters are selected from the group consisting of COON-hydroxysuccinimide, COON-hydroxyphthalimide and COO pentafluorophenol. This substituent YpR has a high reactivity with the amino function of the amino acid or peptide and allows further grafting of such biological probes on homopolymers or copolymers;-polymer (C), where p is 1 And Y is (CH2) mCONH (CH2) wherein m and m ″ are the same or different and are an integer in the range of 1 to 3.
m '', wherein at least one of said R is an electrochemical probe; a preferred electrochemical probe is selected from the group consisting of ferrocene and quinone; the electrochemical probe is selected for a sharp electrochemical signal; Improve the electrochemical detection sensitivity of the biological sensor;-polymer (D), p is 1, Y is the same or different m and m '' and is in the range of 1-3 (CH2) mCONH (CH2) which is an integer
m '', wherein at least one of said R is an electrochemical probe linked to an activated ester; preferred electrochemical probes and preferred activated esters are as described above; the purpose of this substituent YpR is To allow further grafting of the biological probe via the activated ester; polymer (E), where p is 1 and Y is the same or different m and m ″ (CH2) mCONH (CH2) which is an integer in the range of 1 to 3
m ″, wherein at least one said R is NH 2 or any substituent having a terminal amino function NH 2; amino functions are present on biological probes (eg, amino acids, peptides, oligonucleotides, antigens) Acidic functional groups (COO
Allowing further grafting of the biological probe via H or P (OH) n); polymer (F), p is 1 and Y is an integer in the range 1 to 3 And (CH2) mCON (CH2) m ′ ″ wherein m ″ ″ is 2 or 3,
At least one said R is COOH; formula (CH2) mCON (CH2CO
This substituent YpR having OH) m ′ ″ is a transition metal (Ni, Co, Fe, Cu
,. . . ), And the complexing properties of some derivatives such as histidine with these transition metal ions allow for further conjugation of biological probes; for example, histidine tags Attached to the biological probe, allowing the probe to bind to the metal ion and thus to be immobilized on the functionalized poly (pyrrole).

[0014] Preferred mono- or co-polymers are selected from a variety of solvents such as acetonitrile or propylene carbonate in the presence of an electrolyte such as LiClO4
At a concentration of about 0.1 M / l, at a constant voltage of about 0.9 V / SCE, or at a constant mA /
It is obtained at a constant current density of cm 2 or by cyclic voltammetry by electropolymerization of the corresponding monomer units. Typical concentrations of monomer units are
It is in the range of 10-2 to 10-1 M / l. These are polymerized alone to give homopolymers. The mixture of monomers results in a copolymer and can be associated with various properties. For example, copolymers of poly (A, B, C) may have the hydrophilic properties of A required for detection in aqueous solution, the sharp signal of electrochemical probe C, and the biological target in solution. B with the recognition properties exerted by the biological probe.

Another subject of the present invention is a conductive electroactive binding polymer comprising at least one functional group covalently linked to a first biological molecule or an antiligand corresponding to the following formula (II): ,

Embedded image Wherein n is an integer other than zero, i is an integer ranging from 2 to n-1, and R′1, R′i and R′n may be the same or different from each other , H, or a functional group capable of covalently binding to a first biological molecule or anti-ligand.

The functional groups attached to the biological molecule or ligand are advantageously selected from the following functional groups before reaction with the latter: Yp-CX [X is H, OH, a substituted or unsubstituted lower O-alkyl group, or halogen, especially Cl]; Yp-NHZ, Z represents H or an alkyl group; Yp-NH- CO-CF3; Yp-X, X corresponds to the above definition, p is preferably an integer equal to 0, 1 or 2; -Si (alkyl) 3, -Si (alkoxyl) 3 or COON-hydroxysuccinimide Such activated ester groups.

In particular, the functional group attached to the biological molecule is identical and consists of-(CH2) -COOH before the reaction with the first biological molecule, The molecule or anti-ligand may be a peptide or peptide derivative, especially Gly-Phe, Phe-
Pro and Phe-HEA-Pro, and polynucleotides such as CC
It is selected from the oligonucleotide sequence of TAAGAGGGAGTG.

Further, the present invention provides a conductive, electroactive functionalized binding polymer of formula (II ′),

Embedded image Wherein n is an integer or zero, each R ′ can be the same or different from each other, H, and can or are covalently bound to the first biological molecule or anti-ligand Selected from the group consisting of functional groups, with the proviso that at least one of the R ′ of formula (II ′) represents the functional group covalently linked to the first biological molecule or anti-ligand; A binding arm, which may be the same or different and p is zero or an integer, wherein the first biological molecule comprises a polynucleotide or peptide sequence.

Preferred polymers of the formula (II ′) are: —p is 1, Y is selected from the group consisting of CH 2, CH 2 —CH 2 and CH 2 —CH 2 —CH 2, and at least one of said functional groups is A polymer which is COOH prior to binding to the first biological molecule or anti-ligand; -p is 1, Y is selected from the group consisting of CH2, CH2-CH2 and CH2-CH2-CH2, and A polymer wherein at least one of said functional groups is an activated ester before binding to a first biological molecule or anti-ligand; preferred activated esters are COON-hydroxysuccinimide, COON-hydroxyphthalimide and COO pentafluoro Selected from the group consisting of phenols; -p is 1 and Y is the same or different m and m '' Te, 1
(CH2) mCONH (CH2) m '', which is an integer in the range from 1 to 3, and wherein at least one of said functional groups is electrochemically attached to the first biological molecule or antiligand. A polymer which is a probe; a preferred electrochemical probe is selected from the group consisting of ferrocene and quinone; -p is 1, Y is m and m '' are the same or different and 1
(CH2) mCONH (CH2) m "which is an integer in the range from 1 to 3 and wherein at least one of said functional groups is activated ester before binding to the first biological molecule or antiligand. A preferred electrochemical probe and a preferred activated ester are as described above;-p is 1, Y is m and m '' are the same or different. There is one
(CH2) mCONH (CH2) m "which is an integer in the range from 1 to 3 and wherein at least one of said functional groups is NH2 or terminal prior to binding to the first biological molecule or antiligand. A polymer which is any substituent having an amino function NH2; -p is 1, Y is m an integer in the range from 1 to 3, and m '"is 2 or 3 (CH2) mCON (CH2) m ''', and wherein at least one of said functional groups is bound to a first biological molecule or anti-ligand.
A polymer that is COOH.

[0020] The first biological molecule or anti-ligand is preferably an amino acid, a peptide,
The oligonucleotide is selected from the group consisting of an antigen.

Another subject of the invention is the in vitro or in vivo binding of said binding polymer to a second biological molecule or ligand which is different from and specifically interacts with said anti-ligand. Use for detection or assay. The ligand is detected and / or assayed by observing and / or measuring a potential difference or change in current between the binding polymer that is not bound to the ligand and the binding polymer that is bound to the ligand.

In particular, the polymers of the present invention can be used in enzymes, such as proteolytic enzymes, and especially,
To detect and / or assay for carboxypeptidase A, or a polynucleotide, or to extract a second biological molecule that interacts specifically with and differs from an antiligand in vitro or in vivo Used for

In one embodiment of the present invention, the bound polymer is deposited on a conductive substrate, such as a metal or carbon derivative, or is in the form of a self-supporting film.

Finally, the present invention relates to a conductive substrate such as a metal or carbon derivative and a self-supporting film and an electrode comprising the above polymer.

[0025] In one embodiment, the anti-ligand is specific for the ligand or target molecule. The anti-ligand is selected to form an anti-ligand / target molecule complex. By way of example, the conjugate is represented, inter alia, by peptides / antibodies, antibodies / haptens, hormones / receptors, polynucleotide hybrids / polynucleotides, polynucleotide / nucleic acid pairs and the like.

The term “polynucleotide” as used in the present invention includes, for example, inosine,
Nucleotides containing modified bases, such as 5-methyldeoxycytidine, 5-dimethylamino-deoxyuridine, deoxyuridine, 2,6-diaminopurine, 5-bromodeoxyuridine, or any other modified base that causes hybridization. 5 shows at least 5 deoxyribonucleotide or ribonucleotide sequences, optionally comprising at least one modified nucleotide. The polynucleotide can also be used at internucleotide linkages (eg, phosphorothioates,
H-phosphonate and alkyl phosphonate linkages), or backbones such as alpha-oligonucleotides (FR2,607,507) or PNAs (M. Egho
lm et al., J. Am. Chem. Soc., (1992), 114, 1895-1897). Each of these modifications may be used in combination.

The term “peptide” as used in the present invention refers in particular to any peptide of at least two amino acids, in particular proteins, protein fragments or oligopeptides, which are extracted, separated or substantially isolated Or any peptide in which one or more amino acids from the L-series have been replaced by amino acids from the D-series, or vice versa, in the sequence. At least one of the CO-NH bonds of the peptide chain, preferably CO-
Any peptide in which all of the NH bonds are replaced by at least one (one or more) NH-CO bond; at least one of the CO-NH bonds, preferably all of the CO-NH bonds are one or more NH-CO In any peptide substituted by a bond, the chirality of each aminoacyl residue may or may not be included in one or more of the above-described CO-NH bonds, whether or not the aminoacyl residue constituting the reference peptide, mimotope These compounds are also known as immunoretroids.

Many classes of peptides may be grafted, as shown in the following non-exhaustive list: adrenocorticotropic hormone or a fragment thereof; angiotensin analog or an inhibitor thereof (regulates renal hypertension) Components of the renin-angiotensin system); natriuretic peptides; bradykinin and its peptide derivatives; chemotactic peptides; dinorphin and its derivatives; endorphins and the like; encephalin and its derivatives; Derivatives; gastrointestinal peptides, peptides related to the release of growth hormone; neurotensin and the like; opioid peptides; oxytocin, vasopressin, vasotocin and derivatives; kinase proteins.

[0029] Peptides and polynucleotides have high biological activity and are known to control many biological functions (AS Dutta, Advances in Drug Research, B. Tes).
ta Editor, Academic Press, New York, 1991, 21, 145). For example, peptides have very important therapeutic potential as agonist or antagonist receptors and as very potent inhibitors that bind strongly to enzymes, which is the principle on which affinity chromatography is based. Furthermore, upon selective hybridization reactions with other target nucleic acid fragments or nucleotides, the polynucleotide undergoes advantageous recognition phenomena, generating particularly novel gene scavengers. Therefore, the target nucleic acid or nucleic acid fragment is detected and / or
Or contacting the functionalized polymer, at least partially conjugated to the anti-ligand polynucleotide, with a sample suspected of containing the target to assay.
If it occurs, the hybridization reaction can be by directly measuring the potential difference, or the diversity of currents between the unbound and bound polymer that has reacted with the target, or as described above, but reacting with the target. Can be detected indirectly by measurement using an additional detection polynucleotide, said additional polynucleotide preferably being adjacent to the antiligand polynucleotide and labeled with an electroactive molecule.

The term “antibody” as used herein is obtained by any monoclonal or polyclonal antibody, any fragment of said antibody, such as a Fab, Fab′2 or Fc fragment, as well as by genetic modification or recombination. Any antibody is meant.

The functionalization of the polypyrrole at the 3 or 4 position of the pyrrole ring may be carried out on monomer units, but with a subsequent polymerization step, or on the monomer units of a pre-synthesized polymer. Any suitable functionalizing agent can be used, provided that it contains at least one reactive functional group capable of reacting with atoms 3 and / or 4 of the pyrrole ring. Thus, the functionalizing agent may be a monofunctional agent, but after the step of grafting onto the pyrrole ring, a new reactive functional group
Provided that it is introduced for a subsequent reaction with the antiligand and that this reactive functional group is polyfunctional, for example a bifunctional agent, in particular a homo- or heterobifunctional agent. And By way of example, the functionalizing agent is selected from alkyl or alkoxyl or polyether chains, which are substituted or unsubstituted, groups which carry a reactive functional group. Reactive functional groups are, in particular, carboxyl, hydrazine,
It is represented by functional groups such as amine, nitrile, aldehyde, thiol, disulfide, iodoacetyl, ester, anhydride, tosyl, mesyl, trityl and silyl groups.

The formation of a complex resulting from the covalent coupling of an antiligand, for example a polynucleotide, with the functionalized polypyrrole according to the invention can be carried out according to the well-known, so-called direct or indirect methods. For example, in the case of a polynucleotide, a direct method such as the 5′-end or 3
A polynucleotide having a reactive functional group is synthesized at any site of the nucleotide chain, such as at the terminus, at the base or internucleotide phosphate, or at the 2 'position of the sugar. The polynucleotide is then prepared in advance from a polymer containing reactive functional groups that complements the above, in other words, one of the two complementary reactive functional groups carried on the polynucleotide and the other on the functionalized polymer. Coupling with a polymer that forms a covalent bond by the reaction between. For example, primary amines may be coupled to activated carboxylic acids or aldehydes, or thiol functions may be coupled to haloalkyls, in well-known fashion. Preferably,
The reactive functional group of the polynucleotide to be coupled with the polymer is at the 5 'or 3' end.

In the indirect coupling method, each of the polynucleotide and the polymer carries a reactive functional group, and these reactive functional groups may be the same or different from each other, and these two functional groups are complementary. However, it can react with an intermediate coupling agent that is a bifunctional reagent (homobifunctional if the two functional groups are the same, or heterobifunctional if the two functional groups are different). Among the homobifunctional coupling agents, mention may be made of DITC (1,4-phenylenediisothiocyanate), DSS (disuccinimidyl suberate) or analogs thereof. Among the heterobifunctional coupling agents, SMCC (succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate) or SMPB capable of reacting on the one hand with primary amines and on the other hand with thiols. (Succinimidyl
-4- (p-maleimidophenyl) butyrate).

The invention will be better understood on reading the following detailed description, with reference to the accompanying drawings, in which:

The polymers of the present invention can be used, inter alia, for the detection of biologically active species that may be present in a sample and that can react with an antiligand or a grafted antiligand. Indeed, as described above, conjugated polymers functionalized at the 3-position of their heterocycle and wherein one or more antiligands are grafted after reaction with one or more ligands Exhibiting an altered electrochemical reaction relative to a control polymer that did not react with the ligand or ligand of the biological medium is visually observed by a change in oxidation potential. This change in the redox of the polymer in the electrochemical voltammogram gives a scavenger-type function,
Thus, it can be used for quantitative measurement of biologically active species by changing the potential at a constant current, or by changing the current at a constant potential, and by producing field effect microelectrochemical transistors.

Additionally, the polymers of the present invention can be used for the extraction of biologically active species in solution. In many cases, the biologically active species in solution will bind strongly to antiligands, such as bioactive peptides or polynucleotides, grafted to the polymer chain, thereby selectively extracting the biologically active species from the medium. It is possible to do.
This type of extraction can be performed in vitro or in vivo when the supporting polymer is biocompatible, for example, pyrrole.

Finally, the polymers of the present invention can be used to transfer biologically active species (from one medium to another)
Enzymes, etc.). Functionalization of the electroactive conductive polymer of the present invention with a group that exhibits recognition for a compound of biological interest, such as polypyrrole, can be extended to the recognition of nucleic acids (NAs). For example, grafting of a polynucleotide or oligonucleotide, ODN, along the chain of a conjugated polymer should allow for the distinction of the corresponding NAs or NA fragments in a biological medium. This recognition is based on the selective hybridization of the ODN grafted to the polymer and the corresponding NA present in an external medium in which a film of the functionalized polymer is immersed, such as the “peptide / enzyme” recognition described below. Will be done by “ODN / NA” conjugation results in a change in the physicochemical properties of the conjugated polymer, which will allow the presence of the desired NA to be confirmed.

An essential point relates to the nature of the physicochemical properties of the polymer that have been determined for changes during “ODN / NA” recognition. Indeed, in order to develop a rapid, sensitive and quantitative method for measuring the presence of NA, it is an object of the present invention that the electrochemical reaction is altered after `` ODN / NA '' hybridization. Related to the development of wax electrochemical materials.
The change may relate to a change in the potentiometric type, such as a change in the oxidation potential of the polymer, or a change in the observed oxidation (or reduction) current at a given potential. These changes in the electrochemical reaction can be measured quantitatively, and the functionalized polymer film can be grafted onto a polypyrrole as an amperometric or potentiometric electrochemical scavenger, or as described above. Used in field effect microelectrochemical transistor structures in the case of peptide-initiated enzyme recognition. The advantages of this type of measurement are speed, sensitivity, and the possibility to easily create a matrix card of 2n measurement elements, including n-target and non-target ODNs, which easily allows the presence and absence of genes in the medium. The existence can be distinguished.

As in the case of enzyme recognition described above, a second essential point is the fact that functionalization of the 3-position of the hetero (pyrrole) ring is essential in order to obtain an electrochemical reaction to the recognition phenomenon. About.

In order to confirm the exact electrochemical type of reaction for these polymers,
It is necessary that the functionalization of the conjugated chain be compatible with the considerable electroactivity of the functional polymer. The need for such electroactivity, in the case of hydrophilic polyheterocycles such as polypyrrole, requires functionalization to be performed at the 3-position of the pyrrole ring. The polymers of the present invention may have all monomer units functionalized with an antiligand, such as an oligonucleotide, or only some monomer units may be so functionalized.
It is an electrochemical polymer. It is clearly understood that the monomer units can be functionalized with the same or different anti-ligands, in which case the polymers of the invention can be used for the detection of several target ligands in the same sample. Is done. The polymers of the present invention can be prepared by different routes as follows.

A) Fully Functionalized Polymer In this route, the first step involves the functionalization of a monomer such as pyrrole with an antiligand such as a given oligonucleotide. The second step then involves the polymerization of this monomer, resulting in the formation of a polymer film with all monomer units functionalized. b) Partially Functionalized Copolymer In this particular case of the target nucleic acid sequence, noting that they are generally of large size, all functionalization of the small-sized, monomeric units in the polymer Is not necessary. And one of the routes relates to the preparation of copolymers which on the one hand comprise the functionalized monomer units described under a) and which are not functionalized with antiligand oligonucleotides and also comprise pyrrole units. c) Functionalization of the precursor polymer

The partial functionalization of a polymer film can be accomplished by conjugated polymer films into which chemical groups compatible with grafting of anti-ligands such as oligonucleotides have been introduced.
You can do it. In this route, a monomer containing a synthon to be grafted, such as [N-3-hydroxysuccinimidopyrrole], is first made. Synthons [N-hydroxysuccinimide] are known to allow subsequent grafting of oligonucleotides. This monomer is then polymerized or copolymerized with other pyrrole derivatives. The obtained polymer film is then immersed in a reaction medium containing the oligonucleotide, and then a reaction of grafting the oligonucleotide to the pyrrole monomer is performed. In fact, this grafting involves only some of the pyrrole monomer units that make up the polymer.

[0043]

【Example】

Example 1 Synthesis of Monomer In the following example, polypyrrole (1) was chosen as the conjugated polymer support because of its biocompatibility (H. Narrmann, personal communication). The acetyl spacer arm A is grafted between carbon atom 3 of the pyrrole ring and the peptide substituent in order to keep the conductivity and the electrochemical properties of the corresponding functionalized polypyrrole. Various peptides in which the carboxylic acid terminal functionality was unprotected or protected in the methyl ester form were selected for their biological relevance and grafted onto the pyrrole acetate monomer, PyA (1). . Some mono- and dipeptides were grafted to give the following pyrrole derivatives as shown in FIG. 2: pyrroleacetic acid, PyA (1),
Pyrrole (glycine-d-phenylalanine), Py (Gly-DPhe) (2)
And its ability to complex with proteolytic enzymes such as carboxypeptidase A (Sigma) and trypsin (Sigma) (JR Uren, Biochim. Acta, 1971,
236, 67), pyrrole (valine), Py (Val) (3), pyrrole (phenylalanine), Py (Phe) (4), pyrrole (phenylalanine-proline)
, Py (Phe-Pro) (5). Bulkier dipeptide derivatives can also be grafted, such as phenylalanine-hydroxyethylamine-proline, Py (Phe [HEA] Pro) (6), and AIDS HIV.
-1 virus is known as a potential advantageous inhibitor for proteases. These monomers have been described (D. Delabouglise, F. Garnier,
Synth. Met., 1990, 39, 117). These monomers were purified and characterized by NMR, microanalysis, and mass spectrum.

Example 2 Polymerization These monomers were prepared in a propylene carbonate medium containing 0.5 M NaCl
. With a 7 cm 2 platinum electrode and a 10 cm 2 surface area platinum grid, a constant potential of 0.8 V
/ SCE electrochemically polymerized. A thick polymer film with a thickness of up to 10 μm was obtained. As shown in FIG. 3, at neutral pH 7, the electroactivity was increased to 0.
Confirmed by cyclic voltammetry in 5M H2O-NaCl medium. Oxidation potential values on the order of 0.30 V / SCE close to unsubstituted polypyrrole confirmed the electroactivity of these polypyrroles functionalized with dipeptides.

Example 3: Recognition of Carboxypeptidase A The specific properties of complexing these polypyrroles to proteolytic enzymes are known to form stable complexes with (Gly-DPhe) at neutral pH. The analysis was performed using carboxypeptidase A. 0.5cm H2O-N of 5cm3
Solutions in which the concentration of carboxypeptidase A in aCl was increased from 1 mg to 5 mg were analyzed. When a non-specific electrode such as unsubstituted polypyrrole or poly (3,4,5 or 6) is immersed in this solution, the same voltammogram as obtained in Example 2 without change is observed. However, poly (pyrrole (Gly)
-DPhe)), when using poly (2), the voltammogram shows a shift to higher potential, from an initial zero amount of carboxypeptide A of 0.340 V / SCE to a 0.5 mg amount of 5 mg of carboxypeptidase A in solution. / SCE up to the limit value. The results are shown in FIG. 4 and show that a potential shift contributing to steric hindrance and stiffening of the polypyrrole chain is formed during the complexation of the enzyme with the dipeptide borne by the polymer chain. The formation of this complex between the enzyme and the poly (pyrrole-dipeptide) is conventionally performed by affinity chromatography (IM Chaiken, M. Wilchek, I. Parkih, Affinity Choromatography and B
iological Recognition, Academic Press, New York, 1983), enzyme pH = 3
Was confirmed by release into the acidic medium. The released enzyme is a conventional Bradford
Characterized by a test, which involves measuring the enzyme activity with Coomassie Brilliant Blue and using standard bovine serum albumin. Poly (Gly-Dphe) containing 5 × 10 −6 monomer units corresponding to a polymerization charge of 1 coulomb
2.) When a film is used, a large amount of 400 mg of carboxypeptidase A
Was obtained after release into the acidic medium. Considering the size of this enzyme, which is 307 amino acid units, this amount of released enzyme is 2% of (pyrrole-dipeptide)
It shows that about one molecule of enzyme per 00 monomer units forms a complex, which is likely to be a reasonably given size difference (or factor of 100). The results also indicate that the enzyme must partition into the polymer film, thereby indicating the permeability of the film to the enzyme. Comparative results were obtained when trypsin was used as the enzyme.

As shown in FIG. 4, at a given electrode potential, a change in current is observed as a function of enzyme concentration. This result corresponds to an amperometric response of the electrode, one of the advantageous properties of which relates to its linearity with respect to its enzyme concentration as shown in FIG.
Such a linear relationship suggests that one proposes a quantitative assay for biological species in solution, either by using an electrochemical type scavenger or by developing a field effect transistor, as shown in the scheme in FIG. enable. The principle of operation is as follows. The polymer consists of source and drain electrodes and is arranged on a substrate. The assembly is immersed in the solution to be analyzed and the opposite electrode in the same medium represents the grid of the transistor. The grid electrode is at the potential where the change in voltammogram as a function of enzyme concentration is at a maximum, in the case shown in FIG.
, The conductivity of the polymer varies significantly with the concentration of the enzyme. The applied potential between the source and drain electrodes then makes it possible to obtain an amplified signal, which operates according to the field-effect transistor principle.

Example 4 Controlled Release of Enzymes A further advantageous feature of these electrodes is that, in the literature, poly (pyrrole-acetic acid) or poly (1) is used in an electrolyte medium when subjected to electrochemical oxidation. Have been shown to release protons (P. Baerle et al., Adv. Mater., 199
0, 2, 490). Large changes in pH can be observed with small electrolytic volumes up to values on the order of three. This proton release is also observed when the carboxylic acid function is released from the pyrrole ring, as is the case for peptides conjugated to unprotected amino acids or pyrrole. For the controlled release of protons, there are two routes; use of peptides in which the carboxylic acid terminal functionality is unprotected, ie having COOH,
Or the use of a copolymer between pyrrole-acetic acid and a pyrrole-protected peptide, respectively. An example of this second path is given. Py (A), (1) and Py (G
ly-DPhe), the copolymer between (2) was prepared electrochemically under the same conditions as described above. When subjected to electrical oxidation at 0.3 V / SCE, this poly (1,2)
) The copolymer leads to a large change in pH between pH = 4 in the region of the electrode.

Starting with this poly (1,2) copolymer or with a poly (2) polymer whose carboxylic acid functionality is free, two methods can be carried out for the extraction; batchwise and continuous Is the way.

A) Batch-wise method This method involves the use of a membrane or electrode based on the materials described above and the contact of it with a medium in which the desired biologically active species coexists with other species. Including. The selective affinity provided by the grafted peptide to the desired species will cause complex formation of the desired species with the peptide grafted to the electrode or membrane polymer. The membrane or electrode is then removed from the analysis medium and introduced into another medium, called the recovery medium, in which the electrode or membrane is electrochemically oxidized in a recovery medium containing a support salt of the NaCl type. Which causes the liberation of protons by the carboxylic acid groups to a pH sufficient to dissociate and release the desired species.

B) Continuous Method Poly (1,2) copolymer or poly (carboxylic acid group free thereof)
2) The polymer is prepared in membrane or electrode form, optionally using a polycationic or polyanionic support polymer of the polystyrene sulfonate type, or alternatively a perfluoromembrane type such as NAFION. This considered synthetic electrode or membrane constitutes a coupling between the two compartments A and B, as schematically represented in FIG. An example of a continuous extraction of carboxypeptidase A by this method is described below.

In the case represented in FIG. 7, the bioselective element is a 10% solution of NAFION in a mixture of linear higher alcohols (Aldrich) on a very homogeneous platinum lattice.
Consists of a poly (1,2) copolymer polymerized on a NAFION (aldrich) membrane, obtained by evaporation of μl. N resulting therefrom having a thickness of about 5 μm
The AFION film then functions as a support for the electropolymerization of the poly (1,2) copolymer, resulting in a poly (1,2) -NAFION electroactive synthetic membrane.

In a first step 1, 10 grams of carboxypeptidase A was added to 0.5M
Introduce compartment A in H2O-NaCl medium. Next, [poly (1,2)]-NAF
Complex formation on the ION synthetic membrane occurs rapidly at the peptide unit of (2) as described above in Example 3. Thereafter, oxidation is performed using the counter electrode and the reference electrode introduced into the section B. In a second step 2, protons are released into this compartment B to a pH of about 4 by electrochemical oxidation, and carboxypeptidase A is rapidly released. Then 1.2 grams of carboxypeptidase A was recovered in compartment A in one cycle of this continuous method as measured by assaying for enzyme activity. Thus, these results confirm the phenomenon of recognition of these electrodes for enzymes, as well as the ability to extract these enzymes. This behavior is coupled in a one-to-one manner with the chemistry of the dipeptide grafted onto the polypropylene chain.

Example 5 Synthesis of Polypyrrole Functionalized with Polynucleotide or Oligonucleotide This synthesis is performed according to the following three steps. a) Synthesis of [N-3-hydroxysuccinimide pyrrole] monomer 0.4 mol of pyrrole-acetic acid (I), 30 ml of chloroform, and 0.4 mol of N-hydroxysuccinimide, NHS (II) were added to a three-necked flask. Add to
The mixture is stirred and a solution of 0.4 mol of dicyclohexylcarbodiimide, DCC in 20 ml of chloroform is added by titration. After stirring for 2 hours, a white solid is formed, which is filtered and washed with chloroform. After evaporation and washing with acetonitrile to remove unwanted reaction products, the product is recrystallized from chloroform. The desired compound [N-3-hydroxysuccinimidopyrrole] (III) is thus obtained in the form of a white powder.

Embedded image Melting point, m.p. p. = 135 ° C. 1H NMR (ppm): (NH, 1H, 9, s); pyrrole (2H, 6.7)
, m): pyrrole (1H, 6.1, s); CH2 (2H, 3.8, s); hydroxysuccinimide
(4H, 2.8, s).

B) Synthesis of polymer by electropolymerization The electropolymerization solution contains 0.5 M LiClO 4 and 0.1 M monomer (III) in acetonitrile. The electropolymerization is carried out on a platinum electrode with a surface area of 0.7 cm2 at a voltage of 0.9 V against the saturated calomel electrode, SCE, using an electropolymerization charge of 30 mC. A black film corresponding to the poly (III) appears on the electrode and its thickness is about 200 nm.

Embedded image

As shown in FIG. 8, the electrical activity of this polymer was 0.5 M LiClO 4, showing an oxidation peak at 0.28 V / SCE and a reduction peak at 0.24 V / SCE.
-Analyzed in acetonitrile medium. 40 m between these oxidation and reduction peaks
The small separation of V confirms the very large electrical activity and reversibility of this polymer.

C) Grafting of oligonucleotide ODN The above electrode with a polymer film was prepared by mixing a mixture of dimethylformamide with 10% 0.1 M borate buffer at a pH of 9.3 and a 26.2 nanomolar array.
CCTAAGAGGGAGTG as well as a 14-base oligonucleotide containing a 5 'amine function that allows the coupling reaction to be performed at the N-hydroxysuccinimide group generated by the pyrrole unit of the polymer, immersed in the reaction medium formed with ODN. Liquid. Grafting is also achieved by hydrolysis of the succinimide group, which is not displaced by the oligonucleotide in acetic acid. The polymer thus obtained on the electrode corresponds to a poly ([pyrrole-ODN] [pyrrole-COOH]) copolymer.

Embedded image

Example 6 Characterization of Recognition Phenomenon The cognition phenomena were determined using the poly ([pyrrole-ODN] [pyrrole-CO
[OH]) was confirmed by electrochemical characterization of the polymer. Electrochemical analysis was first performed directly on the electrode obtained after the synthesis, and this electrode was then later applied to the target ODN.
And in the presence of a non-target ODN. The hybridization reaction of the polymer was thus performed in an aqueous solution buffered with PEG in the presence of a complementary ODN (target) and a non-complementary ODN (non-target). 335 nanomolar concentration of target O
DNCACTCCCTCTTAGG or non-target ODNGGTGATAGAAGTAT at 272 nanomolar concentration
Incubation is performed at 37 ° C. for 2 hours in the presence of each of C. After the reaction, the films were rinsed with PEG buffer and analyzed by circular volt current. The obtained voltage and current values are shown in FIGS. The results are the first in FIG. 9 where after synthesis and before placement in the presence of target or non-target sequences, the polymer had 0.34 V / SC
E indicates a reversible oxidation peak, confirming the electrical activity of this functionalized polymer. After the incubation reaction in the presence of the non-target sequence and after the rinsing, the resulting voltage-current values show no change. On the other hand, when the polymer is incubated in the presence of the target, the volt-ampere values obtained in FIG. 10 are different, with an increase in the oxidation voltage at 0.40 V / SCE equal to a 60 mV increase. This increase is
It fully illustrates the hybridization that has occurred between N and the corresponding target. This increase in oxidation voltage is attributed to the phenomenon of complexation of the arms that snag along the polypyrrole chain, and is thus achieved by the increase in energy required to oxidize the polymer.

This result indicates that this new class of electroactive materials of conjugated polyheterocycles substituted at position 3 with oligonucleotides effectively results in the phenomenon of recognition of complementary DNA, and furthermore these materials Confirm that this allows electrochemical reading of this selective hybridization. This electrochemical readout is a method for electrochemical, amperometric or voltometric type gene search, as well as a method for electric field effect microelectrochemical transistor type gene search based on amperometric response. It opens the way.

Example 7: Synthesis of preferred polymer (A) 1) Synthesis of 1-tosylpyrrole

Embedded image Method 8 ml of pyrrole, previously distilled in 100 ml of THF, were placed in a 500 ml two-necked flask equipped with a magnetic stirrer and condenser. After 15 minutes, when the mixture became homogeneous, 6 ml of tetrabutylammonium hydroxide (reaction catalyst) was added. The colorless solution turned orange. While homogenizing the mixture, a 50% solution of sodium hydroxide was prepared (62.5 g in 125 ml of water), which was then added dropwise to the mixture. This procedure moved the proton away from the nitrogen on the pyrrole, optimizing the substitution by the tosyl group.

The mixture is stirred for 15 minutes and the tosyl chloride solution is added in excess (3 ml in 100 ml of THF).
2g) was added drop by drop to have excess radicals and to accelerate the reaction. Vigorous stirring was maintained during the addition and for an additional hour. The product turned from orange to brown.

When the stirring was stopped, two phases were found to form. After adding 300 ml of water,
These were separated. The aqueous phase was extracted with ethyl acetate. Evaporating the first organic phase,
The THF was removed, a second organic phase was added to this and the ethyl acetate was evaporated. The crystals were dissolved in dichloromethane and the organic phase was washed with water until the wash water was neutral. The organic phase was dried over anhydrous MgSO4, filtered and the dichloromethane was evaporated. The crystals were purified by recrystallization. 1-Tosylpyrrole was dissolved in 300 ml heptane by heating at reflux for 30 minutes. After filtration through Buchner, impurities (brown
) Remained on the filter and a pale yellow product was obtained, which upon cooling the solution gave white crystals. 22.3 g of crystals were obtained, and the yield was 87.5%.

2) Synthesis of 3-acetyl 1-tosylpyrrole

Embedded image Method 94.6 g of aluminum chloride (reaction catalyst) and 600 ml of dichloromethane were mixed in a three-necked flask equipped with a magnetic stirrer and a condenser with a moisture guard of CaCl2. The mixture was stirred until homogeneous and then 37.5 ml of acetic anhydride (excess reactant) was added dropwise. After 15 minutes, a clear brown solution was obtained. A solution of 1-tosylpyrrole (80 ml
31.4 g) in dichloromethane was added dropwise.

The reaction was allowed to proceed for 1 hour at room temperature with stirring. Then 500ml of the whole mixture
Of ice-cold water. After separation, the organic phase was extracted and the aqueous phase was washed with dichloromethane. This operation was repeated several times. The organic phase is 1M NaO
Washed with H to dissolve Al3 +, which was removed with a water wash. At the end of the washing process, it was confirmed that the pH of the aqueous phase was neutral.

The organic phase was dried over anhydrous magnesium sulfate and the dichloromethane was evaporated. Next, crystallization was performed, and the crystals already obtained were purified. 3-Acetyl 1-tosylpyrrole was dissolved in heptane (500 ml). This was heated at reflux, filtered on a Buchner and the filtrate was placed in a freezer. This material on the bottom of the flask crystallized several times. Heptane was evaporated to give purple crystals. 19.61 g of crystals were obtained with a yield of 52.4%.

3) Synthesis of methyl 1-tosylpyrrole-3 acetate

Embedded image Method In the first step, an esterification catalyst was prepared. This is on the clay
It consists of depositing T1 (NO3) 3.3H2O. In a one-neck flask, 55 ml of trimethyl orthoformate are mixed with 45 ml of methanol and 22 g of T1 (NO3) 3.3H2O. This was stirred for 5 minutes and then 45 g of clay was added to give a grayish beige suspension. This was stirred for a further 15 minutes and the solvent was evaporated using a rotary evaporator. Once thoroughly dried, the catalyst on the clay, in the form of a beige powder, was recovered. You can then proceed with the synthesis of the ether. 11 g of acetyltosylpyrrole (400 m
(dissolved in 1 l of methanol) was mixed with the thallium catalyst on clay in a one-neck flask. Note that a condenser was attached, which was left stirring overnight.

When the stirring was stopped, two phases were found: an upper orange phase and a lower beige grayish phase. The organic phase was collected after filtration. The methanol was evaporated using a rotary evaporator, then CH2Cl2 and water were added. The aqueous phase is CH2Cl2
And the organic phase (reddish brown) was washed with water to remove thallium salts. The organic phase was dried over MgSO4 and evaporated using a rotary evaporator.

The reaction produces a number of by-products, eg,

Embedded image The desired ester is isolated on a silica gel column since If the chromatographic plate was prepared, it would be apparent that the ester would no longer be isolated, but the column was used to remove excess clay, which remained at the head of the column. In the case of the column, it is necessary to use a mixture of two solvents of different polarities, which, with the appropriate composition of the two solvents,
This is because better separation can be obtained. Therefore, 30% ethyl acetate (polar solvent) and 7%
A mixture consisting of 0% heptane (a non-polar solvent) was used. Upon evaporation of the solvent, a yellow oil was recovered. 9.23 g of ester was obtained with a yield of 75.6%.

4) Synthesis of pyrrole-3-acetic acid

Embedded image Method 7.65 g of 1-tosylpyrrole-3-acetate, 100 ml of methanol, and 100 ml of 5M
The sodium hydroxide was mixed in a one-neck flask equipped with a condenser. The medium was stirred and heated at reflux for 2 hours 30 minutes. It was left to cool to room temperature. Sodium carboxylate was obtained. The methanol was evaporated and 100 ml of ether and water were added. The two phases separated and the aqueous phase contained the carboxylate. This phase was washed several times with ether. Then, concentrated hydrochloric acid was added very slowly, taking care to control the temperature of the reaction medium, using an ice bath to prevent polymerization. The product was then found in the organic phase in the form of the acid. The aqueous phase was extracted with ether, and the organic phase was dried over sodium sulfate.

The acid and clay were then separated using a rotary evaporator and using a silica gel column. Evaporation of the solvent gave a rapidly crystallizing yellow liquid. It is desirable to store in ether and in a freezer so as not to polymerize. 2.7 g of product was obtained, with a yield of 82.6%.

Example 7: Synthesis of preferred polymer (B) 1) Synthesis of 3-acetate N-hydroxyphthalimide

Embedded image Method 1.38 g of pyrrole-3-acetic acid, 1.98 g of N-hydroxyphthalimide, and 50 ml of chloroform were mixed in a three-neck flask. Using an additional ampoule, the DCC solution (
2.67 g DCC) in 30 ml CHCl3 was added slowly. During the addition of DCC, the mixture formed a solution.

This was left overnight with stirring. DCU formed. This is filtered and CHC
13 was evaporated. New white crystals of DCU formed. Put this in the refrigerator for one hour,
Filtered and evaporated. This operation was repeated until the DCU was completely removed. Plate chromatography was performed using a 2/98 solvent of dichloromethane / methanol. It was found that the mark corresponding to NHP appeared. To remove this, a silica gel column was used, with the same eluate used for the plate. The product was recrystallized in chloroform. 1.04 g of product was obtained with a yield of 35%.

Example 8: Synthesis of preferred polymer (C) 1) Synthesis of β-ferroceneethylamine

Embedded image Method 1.36 g of AlCl3 in 12 ml of anhydrous Et2O was placed in a three-necked flask. 0.388 g of LiAlH4 was added slowly. When the addition was yielded, 1.36 g of ferrocene-1-cyanomethyl (1
(Diluted in 2 ml of anhydrous Et2O) was added. The solution was stirred at reflux for 2.5 hours. The reaction medium was then acidified with 15 ml of 6N sulfuric acid. The two phases separated and the aqueous phase was washed with anhydrous Et2O. The product then took the NH3 + form and was therefore found in the aqueous phase. KOH was added until pH = 9. Then the product is NH2
And thus recovered in the organic phase, dried over potassium carbonate and evaporated. 0.45 g of a viscous orange liquid was obtained with a yield of 15%.

2) NHP-ferrocene ethylamine coupling

Embedded image Method 160 g of NHP-pyrrole, 150 mg of ferroceneethylamine, and 10 ml of CH3CN were mixed in a three-neck flask. The solution was stirred for 48 hours. CH3CN was evaporated and the residue was dissolved in CHCl3. The solution was washed twice with 10% NaHCO3 solution and then with water to pH7. The organic phase was dried over MgSO4 and NaHCO3 was evaporated. 120 mg of product was obtained with a yield of 60%.

Example 9: Synthesis of preferred polymer (D) 1) The following synthesis

Embedded image 2) Coupling

Embedded image

Example 10: Synthesis of preferred polymer (E)

Embedded image

Example 11: Synthesis of preferred polymer (F)

Embedded image

Embedded image

Example 12: Electropolymerisation Homo- and co-polymers were obtained by electropolymerisation of the corresponding monomers or monomer mixtures under the following conditions: Solvent: acetonitrile or propylene carbonate Electrolyte: LiClO 40.1 M Potential : 0.9V / SCE or current: 2-5mA.cm-2 or potential scanning range 0-0.9V / SCE Electrode: Pt, Ag, Pd, SnO2, Fe, Al Monomer concentration: 10-1-2.10-lM.l -1.

[Brief description of the drawings]

FIG. 1 shows examples of composite polymers such as 1) polyacetylene; 2) polypyrrole; 3) polythiophene; 4) polyphenylene; 5) polyaniline.

FIG. 2A shows polypyrrole (1) substituted at position 3 with acetic acid, and FIGS. 2B, 2C, 2D, 2E and 2F show polypyrrole substituted at position 3 with various peptides (2 to 2 respectively). 6).

FIG. 3 shows the following four functionalized polymers of 0.5 M H 2 O—Na
1 represents a voltammogram in a Cl medium. (1) Poly (pyrrole-acetic acid) (2) Poly (pyrrole (Gly-DPhe)) (3) Poly (pyrrole (Val)) (4) Poly (pyrrole (Phe))

Figure 4, respectively, electrolyte 5 cm ³ in 0.0 mg (a), the electrolyte 5 cm ³ in 1.2 mg (b), the electrolyte 5 cm ³ in 2.4 mg (c) and electrolyte 5 cm ³ in 5.0mg (
0.5% of poly (2) in the presence of carboxypeptidase A at a concentration of d)
1 shows a voltammogram in H2O-NaCl medium.

FIG. 5 corresponds to the current response of the electrode, poly (2), as a function of the amount (nanomoles) of the enzyme carboxypeptidase A present in the medium. The linear relationship between the current observed at a potential of 0.3 V and the amount of enzyme is shown for a saturated calomel electrode.

FIG. 6 is a theoretical diagram of a field effect microelectrochemical transistor for (amplified) detection of the presence of an organism species recognized by a functionalized conductive polymer. The following abbreviations mean P-polymer, Sub-substrate, S and D, respectively, source and drain electrodes, counter electrode acting as CE-grill G, R-reference electrode, Potent-potentiostat. Have.

FIG. 7 is a theoretical diagram of a two-compartment electrochemical cell containing a functionalized polypyrrole membrane for the extraction of species recognized by substituents grafted on electroactive composite polymer chains.

Figure 8, 0.1 M LiClO ₄ ^- acetonitrile medium, with a saturated calomel reference electrode, represents the voltammogram of poly [N-3- hydroxysuccinimide pyrrole, high electrical activity and high electrochemical reversibility Is shown.

FIG. 9 represents a voltammogram of a poly (pyrrole-ODN] [pyrrole-COOH]) copolymer electrode using an ODN of sequence CCTAAGAGGGAGTG as a polynucleotide or oligonucleotide. After incubation of this polymer, no modification with the non-target sequence GGTGATAGAAGTATC was observed.

FIG. 10 shows the sequence: CCTA as oligonucleotide ODN.
Poly (pyrrole-ODN) [pyrrole-COOH using AGAGGGAGTG
]) Represents the voltammogram of the copolymer electrode. This electrode is coupled to the target, 335
Incubated for 2 hours at 37 ° C. in the presence of nanomolar CACTCCCTCTTAGG. The electrodes were then rinsed and analyzed in an electrochemical medium. A potential shift was observed for the voltammogram.

FIG. 11 shows a film P [Py-NHR], poly (B), (0.9
V / SCE, cyclic charge voltammogram at 40 mCcm ⁻² , 0.1 M LiClO _{4 in} acetonitrile, scan rate 20 mVs ⁻¹ ).

FIG. 12 is a graph showing a scanning speed of 5 in CH ₃ CN / 0.1 M LiClO _4.
P [Py-FeCp2], poly (C) (thin, e = 1 nm) in the range of 130 mVs ^-1
3) represents a cyclic voltammogram of the thin film of FIG.

FIG. 13 shows copoly [pyN] in an aqueous solution of 0.5 M NaCl.
₃ shows voltammograms of HFe (Cp) ₂ -pyNHP] and copoly [(B) (C)].

FIG. 14 shows PEG and poly [pyrrole- in an aqueous medium.
[(2) 66 nmol before (1) or after hybridization of ODN, pyrrole-COOH] film with ODN for various target ODN ratios;
(3) 165 nmol, (4) 500 nmol].

FIG. 15 shows copoly [pyN] in an aqueous solution of 0.5 M NaCl.
HFe (Cp) ₂ , pyODN]. After incubation with the first voltammogram and the non-target ODN (-) and the complementary target ODN (0.
(-Nm) after incubating with (2 nmol / 5 ml).

[Procedure amendment]

[Submission date] May 29, 2001 (2001.5.29)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 1

[Correction method] Change

[Correction contents]

Embedded image Wherein n is an integer or zero, each R may be the same or different from each other , is H, or -CO
A functional group capable of covalently binding to a first biological molecule or an anti-ligand selected from the group consisting of ON-hydroxyphthalimide, -COO pentafluorophenol , an electrochemical probe and an electrochemical probe coupled to an activated ester, Provided that (a) at least one of the Rs in the formula (I ′) represents the functional group, or
(B) when YpRs of the formula (I ′) are the same, they are different from CH ₂ —COOH , and (c) R is —COON-hydroxyphthalimide, —COOpentafluorophene.
Nord or if it is ferrocene and electrochemical consisting probe group or al selection as quinone,, Y is -CH ₂ - unlike, each Yp may be the same or different from each other, p Is a linking arm wherein is zero or an integer, wherein the polymer has conductivity and electroactivity of substantially the same order of magnitude as the conductivity and electroactivity of the corresponding polymer of formula (I ') when R represents H .

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Claim 15

[Correction method] Change

[Correction contents]

Embedded image Wherein n is an integer or zero, each R ′ can be the same or different from each other , is H , or can be covalently linked to a first biological molecule or anti-ligand, or A covalently bonded functional group, wherein the functional group is selected from the group consisting of COON-hydroxyphthalimide, COO pentafluorophenol, an electrochemical probe and an electrochemical probe bonded to an activated ester, provided that the formula (II ′) wherein R 'represents a functional group at least one covalently attached to the first biological molecule or antiligand, and, R' is -COON- hydroxyphthalimide, -COO pentafluorophenol
, Or if it is selected from ferrocene and the group consisting of electrochemical probes such as quinone, Y is -CH ₂ - unlike, each Yp may be the same or different from each other, p is zero Or a binding arm that is an integer, wherein the first biological molecule comprises a polynucleotide or peptide sequence.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0053

[Correction method] Change

[Correction contents]

Example 5 Synthesis of Polypyrrole Functionalized with Polynucleotide or Oligonucleotide This synthesis is performed according to the following three steps. a) Synthesis of [N-3-hydroxysuccinimide pyrrole] monomer 0.4 mol of pyrrole-acetic acid (I), 30 ml of chloroform, and 0.4 mol of N-hydroxysuccinimide, NHS (II) were added to a three-necked flask. Add to
The mixture is stirred and a solution of 0.4 mol of dicyclohexylcarbodiimide, DCC in 20 ml of chloroform is added by titration. After stirring for 2 hours, a white solid is formed, which is filtered and washed with chloroform. After evaporation and washing with acetonitrile to remove unwanted reaction products, the product is recrystallized from chloroform. Thus the desired compound, [3- (acetato -N- hydroxysuccinimide) pyrrole] (III) are obtained in the form of a white powder.

Embedded image Melting point, m.p. p. = 135 ° C. 1H NMR (ppm): (NH, 1H, 9, s); pyrrole (2H, 6.7)
, m): pyrrole (1H, 6.1, s); CH ₂ (2H, 3.8, s); hydroxysuccinimide (
4H, 2.8, s).

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0067

[Correction method] Change

[Correction contents]

The reaction produces a number of by-products, eg,

Embedded image The desired ester is isolated on a silica gel column because it can yield If a chromatographic plate was prepared, this ester would no longer be isolated, but the column was used to remove excess clay, which remained at the top of the column. In the case of the column, it is necessary to use a mixture of two solvents of different polarities, which, with the appropriate composition of the two solvents,
This is because better separation can be obtained. Therefore, 30% ethyl acetate (polar solvent) and 7%
A mixture consisting of 0% heptane (a non-polar solvent) was used. Upon evaporation of the solvent, a yellow oil was recovered. 9.23 g of ester was obtained with a yield of 75.6%.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0075

[Correction method] Change

[Correction contents]

Example 10: Synthesis of preferred polymer (E)

Embedded image

[Procedure amendment 6]

[Document name to be amended] Drawing

[Correction target item name] Figure 2

[Correction method] Change

[Correction contents]

FIG. 2

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 27/414 G01N 27/30 301K

Claims (22)

[Claims] 1. A conductive, electroactive functionalized binding polymer of the formula (I ′), comprising: Wherein n is an integer or zero, and each R can be the same or different from each other and can be H, or an electrical bond to COON-hydroxyphthalimide, COO pentafluorophenol, an electrochemical probe and an activated ester. A functional group capable of covalently binding to a first biological molecule or an anti-ligand selected from the group consisting of chemical probes, provided that (a) at least one of said Rs in formula (I ') represents said functional group Or (b) when each YpR in formula (I ') is the same, they are different from CH2-COOH; each Yp may be the same or different from each other, and p is zero or an integer. Wherein said R and H have substantially the same order of magnitude of conductivity and electroactivity as the conductivity and electroactivity of the corresponding polymer of formula (I '). Polymer. 2. The polymer according to claim 1, wherein the electrochemical probe is selected from the group consisting of ferrocene and quinone. 3. Y is an alkylene group having 1 to 5 carbon atoms; an oxyalkylene group having 1 to 5 carbon atoms; m is an integer of 1 to 3, and m ′ is 1 or 2; The formula [(CH2-CH2-O) m (CH2
) m ']; m and m''are the same or different and are an integer in the range of 1 to 3; (CH2) mCONH (CH2) m''; An integer in the range of 3 and m ′ ″ is 2 or 3 (CH
2) The polymer of claim 1, wherein the polymer is selected from the group consisting of mCON (CH2) m '". 4. The polymer according to claim 1, wherein p is 0, 1 or 2. 5. The polymer of claim 1, wherein said anti-ligand is capable of forming an anti-ligand / target molecule complex. 6. The polymer of claim 5, wherein said conjugate is selected from the group consisting of peptides / antibodies, antibodies / haptens, hormones / receptors, polynucleotide hybrids / polynucleotides and polynucleotide / nucleic acid pairs. 7. The polymer according to claim 5, wherein the target molecule comprises a histidine tag. 8. p is 1 and Y is CH2, CH2-CH2 and CH2-C
H2-CH2 and at least one of said R is COO
The polymer of claim 1, wherein the polymer is H. 9. p is 1 and Y is CH2, CH2-CH2 and CH2-C
H2-CH2 and at least one of said R is COO
The polymer according to claim 1, which is N-hydroxyphthalimide or COO pentafluorophenol. 10. p is 1, Y is (CH2) mCONH (CH2) m ″, wherein m and m ″ are the same or different and are integers in the range of 1 to 3, and Y is at least one. The polymer of claim 1, wherein one of said R is an electrochemical probe. 11. The polymer according to claim 10, wherein the electrochemical probe is selected from the group consisting of ferrocene and quinone. 12. The method according to claim 12, wherein the electrochemical probe is conjugated to an activated ester.
The polymer according to claim 10. 13. p is 1 and Y is (CH2) mCONH (CH2) m ″ where m and m ″ are the same or different and are an integer in the range of 1 to 3, and at least one The polymer of claim 1 wherein one R is NH2 or any substituent having a terminal amino group NH2. 14. (CH2) mCON (CH2) m ′ ″ wherein p is 1, Y is m is an integer in the range of 1 to 3, and m ′ ″ is 2 or 3. Yes,
The polymer of claim 1, wherein at least one R is COOH. 15. A conductive, electroactive binding polymer of the formula (II ′), comprising: Wherein n is an integer or zero, and each R ′ can be the same or different from each other and can be, or are, covalently bound to H, or a first biological molecule or anti-ligand, Functional group,
The functional group is selected from the group consisting of COON-hydroxyphthalimide, COO pentafluorophenol, an electrochemical probe and an electrochemical probe attached to an activated ester, provided that at least one of the R ′ of formula (II ′) is A functional group covalently linked to one biological molecule or an anti-ligand, each Yp may be the same or different from each other, and p is zero or an integer; A polymer whose biological molecule comprises a polynucleotide or peptide sequence. 16. p is 1 and Y is CH2, CH2-CH2 and CH2-
Wherein at least one functional group selected from the group consisting of CH2-CH2 and before being bound to the first biological molecule or antiligand is COON-hydroxyphthalimide or COO pentafluorophenol. Item 16. The polymer according to Item 15, 17. p is 1, Y is (CH2) mCONH (CH2) m ″, wherein m and m ″ are the same or different and are integers in the range of 1 to 3, and Y is 16. The polymer of claim 15, wherein the at least one functional group before being attached to a biological molecule or anti-ligand of is an electrochemical probe. 18. The polymer according to claim 17, wherein the electrochemical probe is selected from the group consisting of ferrocene and quinone. 19. The method according to claim 19, wherein the electrochemical probe is conjugated to an activated ester.
19. The polymer according to claim 18. 20. p is 1, Y is (CH2) mCONH (CH2) m ″, wherein m and m ″ are the same or different integers in the range of 1 to 3, and Y is At least one functional group prior to attachment to the biological molecule or anti-ligand of is NH2 or any substituent having a terminal amino functional group NH2,
The polymer according to claim 15. 21. (CH2) mCON (CH2) m '''wherein p is 1; Y is m is an integer ranging from 1 to 3; and m''' is 2 or 3. Yes,
16. The polymer of claim 15, wherein the at least one functional group before being attached to the first biological molecule or anti-ligand is COOH. 22. The polymer of claim 15, wherein said first biological molecule or anti-ligand is selected from the group consisting of amino acids, peptides, oligonucleotides, antigens.