EP1138048A1 - Polymeres conjugues fonctionnalises capables d'electroconductivite et d'electroactivite, et utilisations de ces polymeres - Google Patents

Polymeres conjugues fonctionnalises capables d'electroconductivite et d'electroactivite, et utilisations de ces polymeres

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Publication number
EP1138048A1
EP1138048A1 EP99972771A EP99972771A EP1138048A1 EP 1138048 A1 EP1138048 A1 EP 1138048A1 EP 99972771 A EP99972771 A EP 99972771A EP 99972771 A EP99972771 A EP 99972771A EP 1138048 A1 EP1138048 A1 EP 1138048A1
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Prior art keywords
polymer
group
antiligand
electrochemical
different
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German (de)
English (en)
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Francis Garnier
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Biomerieux SA
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Biomerieux SA
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Priority claimed from US09/195,544 external-priority patent/US6201086B1/en
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Publication of EP1138048A1 publication Critical patent/EP1138048A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G61/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G61/12—Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule
    • C08G61/122—Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule derived from five- or six-membered heterocyclic compounds, other than imides
    • C08G61/123—Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule derived from five- or six-membered heterocyclic compounds, other than imides derived from five-membered heterocyclic compounds
    • C08G61/124—Macromolecular compounds containing atoms other than carbon in the main chain of the macromolecule derived from five- or six-membered heterocyclic compounds, other than imides derived from five-membered heterocyclic compounds with a five-membered ring containing one nitrogen atom in the ring

Definitions

  • Conjugated polymers such as polypyrroles , polythiophenes, polyanilines , polyphenylenes and derivatives thereof are known for their electroactive nature, which is widely described in review works such as the "Handbook of Organic Conducting Polymers” (T.J. Skotheim Editor, Marcel Dekker, New York, 1986). These polymers are obtained in the form of a film on an electrode, in the form of self-supporting films or alternatively in the form of a composite when combined with a polycationic or polyanionic polymer and behave like organic electrodes, which charge up according to an anodic oxidation process, by insertion of ions from the electrolytic medium.
  • the subject of the invention is thus an electrically conductive electroactive functionalized polymer which corresponds to the formula (I) :
  • n is a non-zero integer and i is an integer ranging from 2 to n-1, and
  • Rl, Ri and Rn which may be identical or different, each represent H or a functional group capable of covalently bonding with a first biological molecule or antiligand, and in that said polymer has a conductivity and an electroactivity which are substantially of the same order as that of the corresponding non-functionalized conjugated polymer, that is to say of the corresponding polymer of formula I, in which Rl, Ri and Rn each represent H.
  • the functional group ( ⁇ ) is (are) independently chosen from the following set of functional groups:
  • Yp-C-X where X represents H, OH, a substituted or unsubstituted lower O-alkyl radical, or a halogen, in particular Cl; Yp-NHZ, Z representing H or an alkyl radical; Yp-NH-CO-CF3; Yp-X where X corresponds to the above definition, p being an integer preferably equal to 0, 1 or 2; -Si(alkyl)3, -Si (alkoxyl) 3 or an activated ester group such as COON-hydroxysuccinimide .
  • Y preferably represents a group chosen from alkyls having from 1 to 5 carbon atoms, alkoxyls having from 1 to 5 carbon atoms and polyethers corresponding to the general formula (CH2-CH2-0) m- (CH2) m ' - , m representing an integer ranging from 1 to 3 and m 1 an integer equal to 1 or 2.
  • the invention also concerns an electrically conductive, electroactive functionalized conjugated polymer of formula (I 1 )
  • each R which may be identical or different from one monomer unit to one another, is selected from the group consisting of H and functional groups capable of covalently bonding with a first biological molecule or antiligand with the proviso that (a) at least one said R of formula (I 1 ) represents said functional group or (b) if each YpR in formula (I 1 ) is identical, they are different from CH2-COOH, each Yp, which may be identical or different from one monomer unit to one another, is a coupling arm wherein p is zero or an integer, wherein said polymer has a conductivity and an electroactivity which are substantially of the same order as a conductivity and an electroactivity of a corresponding polymer of formula (III), in which each said R represents H.
  • Preferred polymers of formula (I 1 ) are the following :
  • R is selected from the group consisting of COX, where X represents H, OH, a substituted or unsubstituted lower O-alkyl radical or an halogen ; activated esters ; NHZ where Z represents H, an alkyl radical or CO-CF3 " ; Si(alkyl)3 ; Si(alkoxyl)3 ; electrochemical probes ; electrochemical probes bound to an activated ester ; electrochemical probes are preferably selected from the group consisting of ferrocene and quinone and/or activated esters are selected from the group consisting of COON-hydroxysuccinimide, COON- hydroxyphtalimide, and COOpentafluorophenol ; the halogen is preferably chlorine ;
  • p is at least one and R is selected from the group consisting of H, OH, a substituted lower O-alkyl radical, and a halogen ;
  • Y is selected from the group consisting of alkylene groups having from 1 to 5 carbon atoms ; oxy-alkylene groups having from 1 to 5 carbon atoms ; polyethers having the formula [ (CH2-CH2-0)m (CH2)m'] where m is an integer ranging from 1 to 3 and m* is an integer equal to 1 or 2 ; (CH2)m CONH (CH2)m" where each of m and m" identical or different is an integer ranging from 1 to 3 ; (CH2) CON (CH2)m"' where m is an integer ranging from 1 to 3 and m 1 " is 2 or 3 ;
  • Said antiligand is able to form an antiligand/target molecule complex.
  • said complex is selected from the group consisting of peptide/antibody, antibody/haptene, hormone/receptor, polynucleotide hybrids/polynucleotide and polynucleotide/nucleic acid couple.
  • the target molecule may comprise a histidine tag.
  • More preferred polymers are the following : - polymers (A) wherein p is 1, Y is selected from the group consisting of CH2, CH2-CH2 and CH2-CH2-CH2, and at least one said R is COOH ; this substituent Y P R is able to show a reactivity toward amino functions of a biological probe (for example, oligonucleotide, oligonucleotide derivatized with an amino function, amino acid, peptide), allowing the further grafting of this biological probe on the homo- or co-polymer ; such substituent is also very useful for increasing the hydrophilicity of the homo- or co-polymer, while it is essential for the electrochemical behavior in aqueous solution ;
  • each of m and " identical or dif erent is an integer ranging from 1 to 3 , and at least one 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 its sharp electrochemical signal, which improves the electrochemical detection sensivity of the biological sensor ;
  • Those preferred mono- or co-polymers may be obtained by electropolymerization of the corresponding monomer units, in various solvents such as acetonitrile or propylene carbonate, in presence of an electrolyte such as LiC104, with a concentration of about 0.1 M/l, at a constant potential of about 0.9 V/SCE, or a constant curreint density of some mA/cm , or by cyclic voltammetry.
  • Typical concentration of monomer units is ranging from 10 ⁇ 2 to 10 ⁇ 1 M/l. They can be polymerized alone leading to homopolymers .
  • copolymers in which one can associate various properties ; for instance, a copolymer of poly (A, B,C) allows to associate the hydrophilic property of A, required for a sensing in aqueous solution, with the sharp signal of the electrochemical probe C together with the recognition property exerted by the biological probe of B with the biological target in solution.
  • A, B,C a copolymer of poly (A, B,C) allows to associate the hydrophilic property of A, required for a sensing in aqueous solution, with the sharp signal of the electrochemical probe C together with the recognition property exerted by the biological probe of B with the biological target in solution.
  • Another subject of the invention is an electrically conductive electroactive conjugated polymer comprising at least one functional group which is covalently bonded to a first biological molecule or antiligand corresponding to the formula (II):
  • n is a non-zero integer and i is an integer ranging from 2 to n-1, and
  • R'l, R*i and R'n which may be identical or different, each represent H or a functional group capable of covalently bonding with, or covalently bonded to, a first biological molecule or antiligand.
  • the functional groups bonded to a biological molecule or ligand are advantageously chosen, before reaction with the latter, from the following set of functional groups:
  • Yp-C-X where X represents H, OH, a substituted or unsubstituted lower O-alkyl radical, or a halogen, in particular Cl; Yp-NHZ, Z representing H or an alkyl radical; Yp-NH-CO-CF3; Yp-X where X corresponds to the above definition, p being an integer preferably equal to 0, 1 or 2; -Si(alkyl)3, -Si (alkoxyl) 3 or an activated ester group such as COON-hydroxysuccinimide .
  • the functional groups bonded to a biological molecule are identical and consist, before reaction with the first biological molecule (s) , of -(CH2)- COOH, said first biological molecules or antiligands being chosen from peptides or peptide derivatives, in particular Gly-Phe, Phe-Pro and Phe-HEA-Pro, and from polynucleotides such as the oligonucleotide of sequence: CCTAAGAGGGAGTG .
  • the invention further concerns an electrically conductive, electroactive conjugated polymer of formula (II*)
  • each R 1 which may be identical or different from one monomer unit to one another, is selected from the group consisting of H and functional groups capable of covalently bonding with, or covalently bonded to, a first biological molecule or antiligand with the proviso that at least one said R' of formula (II') represents one said functional group covalently bounded to one said first biological molecule or antiligand
  • each Yp which may be identical or different from one monomer unit to one another, is a coupling arm wherein p is zero or an integer, wherein said first biological molecule comprises a polynucleotide or peptide sequence.
  • Preferred polymers of formula (II 1 ) are the following :
  • Y is selected from the group consisting of CH2, CH2-CH2 and CH2-CH2-CH2, and at least one said functional group, before being bonded to a first biological molecule or antiligand, is COOH ;
  • Y is selected from the group consisting of CH2, CH2-CH2 and CH2-CH2-CH2, and at least one said functional group, before being bonded to a first biological molecule or antiligand, is an activated ester ;
  • a preferred activated ester is selected from the group consisting of COON-hydroxysuccinimide, COON- hydroxyphtalimide, and COOpentafluorophenol ;
  • Y is (CH2)m CONH (CH2)m" where each of m and m" identical or different is an integer ranging from 1 to 3 , and at least one said functional group, before being bonded to a first biological molecule or antiligand, is an electrochemical probe ;
  • a preferred electrochemical probe is selected from the group consisting of ferrocene and quinone ;
  • the first biological molecule or antiligand is preferably selected from the group consisting of amino acids, peptides, oligonucleotides, antigens.
  • Another subject of the invention is the use of a conjugated polymer as defined above for detecting or assaying, in vitro or in vivo, a second biological molecule or ligand, which is different from the antiligand and which interacts specifically with the latter, said ligand being detected and/or assayed by observation and/or measurement of a potential difference or of a variation in current between the conjugated polymer not bonded to the ligand and the conjugated polymer bonded to the ligand.
  • the polymers of the invention are used to detect and/or assay an enzyme, such as a proteolytic enzyme and in particular carboxypeptidase A, or a polynucleotide, or to extract, in vitro or in vivo, a second biological molecule or ligand, which is different from the antiligand and which interacts specifically with the latter.
  • an enzyme such as a proteolytic enzyme and in particular carboxypeptidase A, or a polynucleotide
  • a second biological molecule or ligand which is different from the antiligand and which interacts specifically with the latter.
  • the conjugated polymer is deposited on a conductive substrate, such as metal or a carbon derivative, or in the form of a self- supporting film.
  • the invention relates to an electrode and to a self-supporting film which consists of a conductive substrate such as a metal or a carbon derivative and of a polymer as defined above.
  • the antiligand is specific for the ligand or target molecule.
  • the antiligand is chosen in particular in order to form an antiligand/target molecule complex.
  • the complex may be represented in particular by any peptide/antibody, antibody/haptene, hormone/receptor , polynucleotidehybrids/polynucleotide , polynucleotide/ nucleic acid couple or the like.
  • polynucleotide denotes a seguence of at least five deoxyribonucleotides or ribonucleotides optionally comprising at least one modified nucleotide, for example a nucleotide containing a modified base such as inosine, 5- methyldeoxycitidine, 5-dimethylamino-deoxyuridine, deoxyuridine, 2 , 6-diaminopurine, 5-bromodeoxyuridine or any other modified base which allows hybridation.
  • a modified base such as inosine, 5- methyldeoxycitidine, 5-dimethylamino-deoxyuridine, deoxyuridine, 2 , 6-diaminopurine, 5-bromodeoxyuridine or any other modified base which allows hybridation.
  • This polynucleotide may also be modified at the internucleotide bond (for example such as phosphorothioate, H-phosphonate and alkylphosphonate bonds) , or on the skeleton, for example alpha-oligonucleotides (FR 2,607,507) or PNAs (M. Egholm et al., J. Am. Chem. Soc, (1992), 114, 1895-1897). Each of these modifications may be taken in combination.
  • peptide refers in particular to any peptide of at least two amino acids, in particular a protein, protein fragment or oligopeptide, which is extracted, separated or substantially isolated or synthesized, in particular those obtained by chemical synthesis or by expression in a recombinant organism; any peptide in whose sequence one or more amino acids from the L-series are replaced by an amino acid from the D-series, and vice versa; any peptide in which at least one of the CO-NH bonds, and advantageously all of the CO-NH bonds, of the peptide chain is (are) replaced by one (or more) NH-CO bonds; any peptide in which at least one of the CO-NH bonds, and advantageously all of the CO-NH bonds, is or are replaced by one or more NH-CO bond(s) , the chirality of each aminoacyl residue, whether or not this is involved in one or more abovementioned CO-NH bonds, being either conserved or inverted with respect to the aminoacyl residue
  • peptides may be grafted, as shown by the non-exhaustive list below: adrenocorticotropic hormones or fragments thereof; angiotensin analogs or inhibitors thereof (components of the renin-angiotensin system which regulate renal hypertension) ; natriuretic peptides; bradykinin and peptide derivatives thereof; chemotactic peptides; dynorphin and derivatives thereof; endorphins or the like; encephalins or derivatives thereof; inhibitors of enzymes (such as proteases); fragments of fibronectin and derivatives; gastrointestinal peptides; peptides associated with the release of growth hormones; neurotensins and the like; opioid peptides; oxytocin, vasopressin, vasotocin and derivatives; kinase proteins.
  • angiotensin analogs or inhibitors thereof components of the renin-angiotensin system which regulate renal hypertension
  • Peptides and polynucleotides have high biological activity, and are known to control many biological functions (A.S. Dutta, Advances in Drug Research, B. Testa Editor, Academic Press, New York, 1991, 21, 145) .
  • peptides show very considerable therapeutic potential as agonist or antagonist receptors, and as very powerful inhibitors which bind strongly to enzymes, this being the principle upon which affinity chromatography is based.
  • polynucleotides may give rise to advantageous recognition phenomena, allowing in particular the development of novel gene scavengers.
  • a functionalized polymer which is at least partially bonded to an antiligand polynucleotide is placed in contact with a sample liable to contain the target, and the hybridization reaction is then detected if it takes place, either directly by measuring a potential difference or a variation in current between the non-bonded polymer and the bonded polymer which has reacted with the target, or indirectly by the same measurement as above, but using an additional detection polynucleotide which is capable of reacting with the target, said additional polynucleotide preferably adjoining the antiligand polynucleotide and being labeled with an electroactive molecule.
  • antibody refers to any monoclonal or polyclonal antibody, any fragment of a said antibody such as an Fab, Fab ' 2 or Fc fragment, as well as any antibody obtained by genetic modification or recombination. Functionalization of the polypyrrole in the 3- or
  • 4-position of the pyrrole ring may be carried out either on the monomer units with a subsequent polymerization step, or on the monomer units of a presynthesized polymer.
  • Any suitable functionalizing agent may be used, provided that it comprises at least one reactive function capable of reacting with atoms 3 and/or 4 of the pyrrole ring.
  • the functionalizing agent may thus be a monofunctional agent, provided that after the step of grafting on to the pyrrole ring the novel reactive function is introduced for subsequent reaction with the antiliqand, and that this reactive function is multifunctional, such as bifunctional agents and in particular homo- or heterobifunctional agents.
  • the functionalizing agent is chosen from substituted or unsubstituted alkyl or alkoxyl or polyether chains ending with a group bearing a reactive function.
  • the reactive function is represented in particular by a functional group such as a carboxylic, hydrazide, amine, nitrile, aldehyde, thiol, disulfide, iodoacetyl, ester, anhydride, tosyl, mesyl, trityl or silyl group or the like.
  • a conjugate resulting from the covalent coupling of an antiligand for example a polynucleotide
  • a functionalized polypyrrole for example a polynucleotide
  • a polynucleotide is synthesized having a reactive function on any site of the nucleotide chain such as, for example, the 5' end or the 3' end, or on a base or on an internucleotide phosphate, or on the 2' position of a sugar.
  • the polynucleotide is then coupled with the polymer, which is prepared beforehand and contains a reactive function complementary to the above, that is to say one which allows the formation of a covalent bond by reaction between the two complementary reactive functions, one borne by the polynucleotide and the other by the functionalized polymer.
  • a reactive function complementary to the above that is to say one which allows the formation of a covalent bond by reaction between the two complementary reactive functions, one borne by the polynucleotide and the other by the functionalized polymer.
  • primary amines may be coupled with an activated carboxylic acid or an aldehyde or alternatively a thiol function may be coupled with a haloalkyl.
  • the reactive function of the polynucleotide for the coupling to the polymer is at the 5 ' or 3 ' end.
  • the polynucleotide and the polymer each bear a reactive function, it being possible for these reactive functions to be identical to or different from each other, these two functions not being complementary but being capable of reacting with an intermediate coupling agent which is a bifunctional reagent (homobifunctional if the two functions are identical or heterobifunctional if the two functions are different) .
  • an intermediate coupling agent which is a bifunctional reagent (homobifunctional if the two functions are identical or heterobifunctional if the two functions are different) .
  • homobifunctional coupling agents which may be mentioned are DITC (1,4- phenylene diisothiocyanate) , DSS (disuccinimidyl suberate) or analogs thereof.
  • heterobifunctional coupling agents which may be mentioned are SMCC (succinimidyl-4-(N- maleimidomethyl) cyclohexane-1-carboxylate) or SMPB (succinimidyl-4-(p-maleimidophenyl) butyrate) , which are capable of reacting with a primary amine, on the one hand, and with a thiol, on the other hand.
  • SMCC succinimidyl-4-(N- maleimidomethyl) cyclohexane-1-carboxylate
  • SMPB succinimidyl-4-(p-maleimidophenyl) butyrate
  • Figure 1 represents examples of conjugated polymers such as 1) polyacetylene; 2) polypyrrole; 3) polythiophene; 4) polyphenylene; 5) polyaniline;
  • Figure 2A represents a polypyrrole substituted in the 3-position with acetic acid (1) and Figures 2B, 2C, 2D, 2E and 2F represent polypyrroles substituted in the 3- position with various peptides (2 to 6 respectively) ;
  • Figure 3 represents the voltammograms of four functionalized polymers, identified below, in 0.5 M H20- NaCl medium poly (pyrrole-acetic acid) in (1), poly (pyrrole (Gly-DPhe) ) in (2), poly (pyrrole (Val) ) in (3) and poly (pyrrole (Phe) ) in (4);
  • Figure 4 represents the voltammogram of poly(2), in 0.5M H2 ⁇ -NaCl medium, in the presence of carboxypeptidase A at concentrations respectively of 0. Omg in 5 cm 3 of electrolyte (a), 1.2 mg in 5 cm 3 of electrolyte (b) , 2.4 mg in 5 cm 3 of electrolyte (c) and 5.0 mg in 5 cm 3 of electrolyte (d) ;
  • Figure 5 corresponds to the amperometric response of an electrode, poly (2) , as a function of the amount of enzyme, carboxypeptidase A, in nanomoles, present in the medium.
  • the linear relationship between the current observed and the amount of enzyme, at a potential of 0.3 V, is given with reference to a saturated calomel electrode
  • Figure 6 is a theoretical diagram of a field effect microelectrochemical transistor for the (amplified) detection of the presence of a biological species recognized by a functionalized conductive polymer.
  • P polymer.
  • Sub substrate.
  • S and D source and drain electrodes respectively.
  • CE counterelectrode acting as a grille G. R, reference electrode. Potent., potentiostat ,
  • Figure 7 is a theoretical diagram of a 2- compartment electrochemical cell containing a functionalized polypyrrole membrane, for the extraction of biological species recognized by a substituent grafted on to an electroactive conjugated polymer chain,
  • Figure 8 represents the voltammogram of poly[N-3- hydroxysuccinimidepyrrole] , in 0.1 M LiC10 4 ⁇ acetonitrile medium with a saturated calomel reference electrode, showing high electroactivity and high electrochemical reversibility
  • Figure 9 represents the voltammogram of a poly (pyrrole-ODN] [pyrrole-COOH] ) copolymer electrode, with ODN of sequence CCTAAGAGGGAGTG as polynucleotide or oligonucleotide. No modification is observed after incubation of this polymer with a non-target seguence, GGTGATAGAAG ATC , and
  • Figure 10 represents the voltammogram of a poly ( [pyrrole-ODN] [pyrrole-COOH] ) copolymer electrode, with the sequence: CCTAAGAGGGAGTG as oligonucleotide ODN. This electrode was incubated in the presence of a target, 335 nmol CACTCCCTCTTAGG, at 37 °C for 2 h. This electrode was then rinsed and analysed in electrochemical medium. A potential shift is observed with respect to the previous voltammogram.
  • Figure 11 represents the cyclic voltammogram of a film P[Py-NHR] , poy(B) , (0.9 V/SCE with grown charge 40 mCcm- 2 , in 0.1 M LiClOA acetonitrile solution, scan rate 20 roVs" 1 .
  • Figure 13 represents the voltammogram of copoly [pyNHFe(Cp) 2-pyNHP] , copolyf (B) (C) ] in an aqueous solution of 0.5M NaCl.
  • Fiqure 14 represents an electrochemical characterisation of films of PEG and of poly [pyrrole-ODN, pyrrole-COOH] in aqueous medium, before (1) or after hybridization with ODN for different target ODN proportions [(2) 66 nmoles, (3) 165 nmoles, (4) 500 nmoles) ] .
  • Figure 15 represents voltammograms of copoly [pyNHFe(Cp) 2, PyODN] , in aqueous solution of 0.5M NaCl. Initial voltammogram and after incubation with non- target ODN (-) and after incubation with complementary target ODN (0.02 nmole/5 ml) ( —).
  • the polymers according to the invention may be used in particular for the detection of biologically active species which may be present in a sample and which may react with the antiligand or grafted antiligands. Indeed, as shown above, it is observed that the conjugated polymers functionalized in the 3-position of their heterocycle and on to which are grafted one or more antiligands, after reaction with one or more ligands, exhibit a modification of the electrochemical response with respect to a reference polymer which has not reacted with the ligand or ligands of a biological medium, this being visualized by a change in the oxidation potential.
  • This variation in the oxidoreduction of the polymer in the electrochemical voltammogram inparts a scavenger-type function and may thus be used for a quantitative measurement of the biologically active species, either by variation of the potential, at fixed current, or by variation of the current at fixed potential, or alternatively by the production of field effect microelectrochemical transistors .
  • the polymers of the invention may also be used for the extraction of biologically active species in solution.
  • the biologically active species in solution combines strongly with the antiligand grafted on to the polymer chain, such as a bioactive peptide or a polynucleotide, thereby making it possible to extract the biologically active species selectively from a medium.
  • This type of extraction may be performed in vitro or even in vivo when the support polymer is biocompatible, such as polypyrrole for example.
  • the polymers of the invention may be a source of release, from one medium into another medium, of biologically active species (enzymes or the like) .
  • the functionalization of the electroactive conductive polymers of the invention, such as polypyrroles, by groups showing recognition with respect to compounds of biological interest may be extended to recognition of nucleic acids (NAs) .
  • NAs nucleic acids
  • the grafting of polynucleotides or oligonucleotides, ODN, along the conjugated chain of polymers should allow the discrimination of corresponding NAs or NA fragments within a biological medium.
  • This recognition will be performed by selective hybridization between the ODN, grafted on to the polymer, and the corresponding NA present in the external medium, in which the film of functionalized polymer is immersed, just like the "peptide/enzyme” recognition described later.
  • the "ODN/NA” complexation results in a modification of the physicochemical properties of the conjugated polymer, characterization of which will make it possible to confirm the presence of the desired NA.
  • the essential point relates to the nature of the physicochemical properties of the polymer destined for modification during the "ODN/NA" recognition. Indeed, in order to develop a rapid, sensitive and quantitative method for measuring the presence of NA, the aim of the present invention relates to the development of electroactive materials whose electrochemical response will be modified after "ODN/NA” hybridization.
  • the modification will relate to a potentiometric-type variation, such as variation of the oxidation potential of the polymer, or an amperometric-type variation, by variation of the oxidation (or reduction) current observed at a given potential.
  • electrochemical response may be measured quantitatively, the functionalized polymer films being used either as electrochemical scavengers of amperometric or potentiometric type, or alternatively in a field effect microelectrochemical transistor structure, as has been described above in the case of enzymatic recognition starting with peptides grafted on to polypyrrole.
  • the advantages of measurements of this type are the speed, the sensitivity and the possibility of readily producing matrix cards of 2n measuring elements, containing n target and nontarget ODNs, which are thus capable of rapidly discriminating between the presence and absence of genes in a medium.
  • a second essential point relates to the fact that in order to obtain an electrochemical response to a recognition phenomenon, the functionalization in the 3-position of a heterocyclic (pyrrole) ring is essential.
  • the polymers of the invention are electroactive polymers in which either all of the monomer units are functionalized with an antiligand such as an oligonucleotide, or only some of the monomer units are thus functionalized.
  • the monomer units may be functionalized with identical or different antiligands, and in the latter case the polymers of the invention may be used for the detection of several target ligands within the same sample.
  • the polymers of the invention may be prepared by the following different routes: a) Totally functionalized polymers.
  • the first step relates to the functionalization of the monomer, such as pyrrole, by an antiligand such as a given oligonucleotide.
  • the second step then relates to the polymerization of this monomer, resulting in a film of polymer in which all the monomer units are functionalized. b) Partially functionalized copolymers.
  • the partial functionalization of a polymer film may also be performed starting with a conjugated polymer film, into which chemical groups compatible with the grafting of an antiligand such as an oligonucleotide are introduced beforehand.
  • a monomer containing a grafting synthon is first produced, such as [N-3- hydroxysuccinimidepyrrole] .
  • the synthon [N- hydroxysuccinimide] is known to allow the subsequent grafting of an oligonucleotide.
  • This monomer is subsequently polymerized or copolymerized with another pyrrole derivative.
  • the polymer film obtained is then immersed into the reaction medium containing an oligonucleotide, and the reaction to graft this oligonucleotide to the pyrrole monomers is then carried out.
  • This grafting in fact only involves some of the pyrrole monomer units constituting the polymer.
  • the polypyrrole (1) was chosen as conjugated polymer support on account of its biocompatibility (H. Naarmann, personnel communication) .
  • An acetyl spacer arm A is grafted between carbon atom 3 of the pyrrole ring and the peptide substituent in order to preserve the conductivity and the electroactivity of the corresponding functionalized polypyrrole.
  • Various peptides, with their carboxylic end function unprotected or protected in methyl ester form, were chosen for their biological pertinence and were grafted on to a pyrrole-acetic acid monomer, PyA (1).
  • pyrrole-acetic acid PyA (1)
  • pyrrole (Glycine- dPhenylalanine) Py(Gly-DPhe) (2)
  • proteolytic enzymes such as carboxypeptidase A (Sigma) and trypsin (Sigma)
  • Example 2 Polymerization These monomers were polymerized electrochemically on a 0.7 cm 2 platinum electrode, as well as on a platinum grille of surface area 10 cm 2 in propylene carbonate medium with 0.5 M NaCl, at a constant potential of 0.8 V/SCE. Thick polymer films are obtained, in thicknesses of up to 10 ⁇ m. As shown in Figure 3, the electroactivity of these polymers was confirmed by cyclic voltammetry in 0.5 M H20-NaCl medium, at a neutral pH of 7. The oxidation potential values, of the order of 0.30 V/SCE, close to that of unsubstituted polypyrrole, confirm the electroactivity of these polypyrroles functionalized with dipeptides .
  • Example 3 Recognition of carboxypeptidase A
  • carboxypeptidase A with which (Gly-DPhe) is known to form stable complexes at neutral pH. Solutions of increasing concentration of carboxypeptidase A, ranging from 1 mg to 5 mg in 5 cm3 of 0.5 M H20-NaCl, were analysed.
  • nonspecific electrodes such as unsubstituted polypyrrole, or poly (3, 4, 5 or 6) are immersed in this solution, a voltammogram identical to that obtained in Example 2 is observed, without any modification.
  • poly (pyrrole (Gly-DPhe) ) poly
  • the released enzyme was characterized conventionally by the Bradford test, involving measurement of the enzymatic activity with Coomassie brilliant blue, and by the use of a standard bovine serum albumin.
  • a poly (Gly-DPhe) film is used containing 5 x 10-6 monomer units, corresponding to a polymerization charge of 1 coulomb, a significant amount, 400 icrograms, of carboxypeptidase A was obtained after release into an acidic medium.
  • this amount of released enzyme shows that about 1 molecule of enzyme is complexed per 200 monomer units of (pyrrole-dipeptide) , which appears to be reasonable given the difference in size (of about a factor of 100) .
  • This result also shows that the enzyme must be distributed inside the polymer film, thereby demonstrating the permeability of this film with respect to the enzyme. Comparable results were obtained when trypsin was used as the enzyme.
  • this grille electrode When this grille electrode is placed at a potential at which the variation of the voltammogram as a function of the enzyme concentration is at a maximum, at about 0.2 V in the case represented in Figure 4, the conductivity of the polymer varies considerably with the enzyme concentration. A potential then applied between source and drain electrodes makes it possible to obtain an amplified signal, this transistor operating according to the principle of a field effect transistor.
  • This process consists in using a membrane or electrode based on the abovementioned materials, and in placing it in contact with the medium in which the desired biologically active species coexists with other species.
  • the selective affinity provided by the grafted peptide with respect to this desired species will cause complexation of the latter with the peptide grafted on to the polymer of the electrode or of the membrane.
  • This membrane or electrode is then removed from the analysis medium and introduced into another medium, referred to as the recovery medium.
  • the recovery medium containing a support salt of NaCl type, the electrode or membrane is subjected to an electrochemical oxidation, which causes the expulsion of protons, by means of the carboxylic acid groups, up to a pH which is sufficient for the dissociation and release of the desired species.
  • the poly(l, 2) copolymer, or the poly (2) polymer with its carboxylic function free is prepared in membrane or electrode form, optionally using a polycationic or polyanionic support polymer of the polystyrene sulfonate type or alternatively perfluoro membrane type such as NAFION.
  • This possibly composite electrode or membrane constitutes the junction between two compartments A and B, as represented schematically in Figure 7. An example of the continuous extraction of carboxypeptidase A by this process is described below.
  • the bioselective element consists of a poly(l, 2) copolymer described above, polymerized in a NAFION (Aldrich) membrane, obtained by evaporation of 10 ael of a 5 % solution of NAFION in a mixture of linear higher alcohols (Aldrich) on a very fine platinum grille.
  • NAFION Aldrich
  • a first step 1 10 grams of carboxypeptidase A are introduced into the compartment A, in 0.5 M H20-NaCl medium.
  • a complexation on the [poly ( 1 , 2 )] -NAFION composite membrane then occurs immediately on the peptide units of (2), as described above in Example 3.
  • An oxidation is subsequently performed, using a counter-electrode and a reference electrode which are introduced into the compartment B.
  • the electrochemical oxidation leads to the release of protons in this compartment B, up to a pH of about 4, and to the immediate release of carboxypeptidase A.
  • 1.2 grams of carboxypeptidase A were then recovered in the compartment A over one cycle of this continuous process, as was determined by assaying the enzyme activity.
  • Example 5 Synthesis of a polypyrrole functionalized with a polynucleotide or oligonucleotide
  • the electropolymerization solution contains 0.5 M LiC104 and 0.1 M monomer (III) in acetonitrile.
  • the electropolymerization is performed on a platinum electrode of surface area 0.7 cm 2 , at a potential of 0.9 V relative to a saturated calomel electrode, SCE, using an electropolymerization charge of 30 mC.
  • a black film appears on the electrode, corresponding to poly (III) , the thickness of which is about 200 nm.
  • the grafting is also accompanied by hydrolysis of the succinimide groups, which are not substituted with the oligonucleotide, in acetic acid.
  • the polymer obtained on the electrode thus corresponds to a pol ( [pyrrole-ODN] [pyrrole-COOH] ) copolymer.
  • the recognition phenomenon was confirmed by electrochemical characterization of the poly ( [pyrrole- ODN] [pyrrole-COOH] ) polymer film obtained in Example 5c.
  • the electrochemical analysis was first carried out directly on the electrode obtained after synthesis, and then after this electrode had been placed in the presence of the target ODN and the non-target ODN.
  • the hybridization reaction of this polymer was thus performed in the presence of complementary ODN (target) and non- complementary (ODN) (non-target) , in an agueous solution buffered with PEG.
  • the voltammogram obtained shows no modification.
  • the voltammogram obtained, Figure 10 is different, with an increase of the oxidation potential to 0.40 V/SCE, equivalent to an increase of 60 mV. This increase is entirely indicative of a hybridization which has taken place between the ODN and the corresponding target.
  • This increase in the oxidation potential is attributed to a phenomenon of complexation of the arm hanging along the polypyrrole chains, thus accompanied by an increase in the energy required to oxidize this polymer.
  • the mixture is stirred for 15 minutes and the tosyl chloride solution is added in excess (32 g in 100 ml of THF) and drop by drop so as to have an excess of radicals and to encourage the reaction. Vigorous stirring is maintained during the addition and then for 1 hour. The product changes from an orange colour to a brown colour.
  • 2 phases are seen to form. After adding 300 ml of water, they are allowed to separate. The aqueous phase is extracted with ethyl acetate. The first organic phase is evaporated to remove the THF, the second organic phase is added to it and the ethyl acetate is evaporated.
  • the crystals are dissolved in dichloromethane and this organic phase is washed with water until the washing water is neutral.
  • the organic phase is dried over anhydrous MgS0 4 , - filtered and the dichloromethane evaporated.
  • Recrystallisation is carried out to purify the crystals.
  • the 1-tosyl pyrrole is dissolved in 300 ml of heptane by heating under reflux for 30 minutes. On filtering at a Buchner, the impurities (coloured brown) remain on the filter and a pale yellow product is obtained which gives white crystals once the solution has cooled.
  • the reaction is allowed to proceed for an hour under agitation and at ambient temperature.
  • the entire mixture is then poured into 500 ml of iced water.
  • the organic phase is extracted and the aqueous phase is washed with dichloromethane. This operation is repeated several times.
  • the organic phases are washed with 1M NaOH to make the Al soluble and remove it with the water washings. At the end of the washing process, the neutral pH of the aqueous phase is checked.
  • the organic phases are dried over anhydrous magnesium sulphate and the dichloromethane is evaporated.
  • a recrystallisation is then carried out to purify the crystals obtained: the 3-acetyl 1-tosyl pyrrole is dissolved in heptane (500 ml) . It is heated under reflux, filtered at a Buchner and the filtrate placed in a freezer. The material in the bottom of the flask is crystallised several times. The heptane is evaporated and violet crystals are obtained.
  • the esterification catalyst is prepared. This consists of depositing Tl (NO 3 ) 3 .3H 2 0 on clay. In a single necked flask, 55 ml of trimethyl orthoformate, is mixed with 45 ml of methanol and 22 g of Tl (NO 3 ) 3 .3H 2 0. This is stirred for 5 minutes and then 45 g of clay is added in such a way that a greyish beige suspension is obtained. This is stirred for a further 15 minutes and the solvents evaporated using a rotary vaporiser. Once it is well dried, the catalyst on clay is recovered in the form of a beige powder.
  • the stirring is stopped and two phases can then be seen (an orange phase above and a thick phase with a greyish beige colour below)
  • the organic phase is recovered after filtration.
  • the methanol is evaporated using a rotary vaporiser and then CH 2 CI 2 and water are added.
  • the aqueous phase is washed with CH 2 CI 2 and the organic phase (a blood red colour) with water in order to remove the thallium salts.
  • This organic phase is dried over MgS ⁇ 4 and evaporated using a rotary vaporiser.
  • ester methoxy-3-acetyl 1-tosyle pyrrole dimethoxy 3-acetyl 1-tosyle pyrrole the desired ester will be isolated on a silica gel column. Having made a chromatographic plate it may be noted that this ester is already isolated but nevertheless a column is used to remove excess clay (which remains at the head of the column) . For the column, it is necessary to use a mixture of two solvents of different polarity as eluent since using a suitable composition of the two solvents, better separation is obtained. Therefore a mixture made up of 30% ethyl acetate (polar solvent) and 70% heptane (non-polar solvent) is used. The solvent is evaporated and a yellow oil is collected.
  • polar solvent ethyl acetate
  • heptane non-polar solvent
  • a rotary vaporiser is then used and a silica gel column is used to separate the- acid and the clay.
  • the solvent is evaporated and a yellow liquid is obtained that crystallises rapidly. It is advisable to store it in ether in the freezer in order to restrict the risk of polymerisation. 2.7 g of product is obtained representing a yield of 82.6%.
  • a product of mass 1.04 g is obtained representing a yield of 35%.
  • reaction medium is acidified with 15 ml of 6N sulphuric acid.
  • the two phases are separated and the aqueous phase is washed with anhydrous Et 2 0.
  • the product is then in the NH 3 form and is therefore to be found in the aqueous phase.
  • the product is then in the NH 2 form and therefore in the organic phase and is recovered, dried over potassium carbonate and evaporated.
  • Homo- and co-polymers are obtained by electropolymerisation of corresponding monomers or monomer mixtures, in the following conditions : solvent : acetonitrile or propylene carbonate electrolyte : LiC104 0.1M potential : 0.9 V/SCE or current : 2-5 mA.cm- 2 or potential scanning between 0-0.9 V/SCE electrode : Pt, Ag, Pd, Sn ⁇ 2, Fe, Al concentration of monomer (s) : 10 "1 -2.10" 1 M.l "1

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Abstract

Cette invention concerne un polymère conjugué fonctionnalisé capable d'électroconductivité et d'électroactivité, représenté par la formule générale (I'). Dans cette formule, nst un entier ou zéro. Chaque R, qui peuvent être identiques ou différents d'une unité monomère à l'autre, est H ou groupe fonctionnel capable de se lier par covalence à une première molécule biologique ou à un antiligand du groupe des COON-hydroxyphtalimides, COO-pentafluorophénols, sondes électrochimiques et sondes électrochimiques liées à un ester activé. Toutefois, (a) l'un au moins desdits R de la formule (I') doit être ledit groupe fonctionnel et, (b) si chacun des YpR de la formule (I') est identique, ils ne doivent pas être CH2-COOH. Chacun des Yp, qui peuvent être identiques ou différents d'une unité monomère à l'autre, est une liaisone couplage où p est zéro ou un entier. Ledit polymère a une conductivité et une électroactivité qui sont presque du même ordre que celles d'un polymère correspondant de la formule (I'), dans laquelle chaque R représente H.
EP99972771A 1998-11-19 1999-11-18 Polymeres conjugues fonctionnalises capables d'electroconductivite et d'electroactivite, et utilisations de ces polymeres Withdrawn EP1138048A1 (fr)

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US09/195,544 US6201086B1 (en) 1994-04-22 1998-11-19 Electrically conductive electroactive functionalized conjugated polymers, and uses thereof
PCT/IB1999/001947 WO2000031750A1 (fr) 1998-11-19 1999-11-18 Polymeres conjugues fonctionnalises capables d'electroconductivite et d'electroactivite, et utilisations de ces polymeres

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FR2808024B1 (fr) * 2000-04-21 2006-09-15 Bio Merieux Complexe electroactif, sonde electroactive et procede de preparation
FR2833014B1 (fr) * 2001-11-30 2005-07-08 Bio Merieux Procede selectif de detection, d'identification et de dosage d'un cation metallique divalent dans un echantillon
FR2833013B1 (fr) * 2001-11-30 2005-06-24 Bio Merieux Sonde electroactive comportant un agent chelatant et un cation metallique
FR2835836B1 (fr) 2002-02-14 2006-03-17 Bio Merieux Metallocenes bifonctionnalises, procede d'obtention, utilisation pour le marquage de molecules biologiques
US6987164B2 (en) 2002-03-09 2006-01-17 Samsung Electronics Co., Ltd. Electrically conductive polymer, sensor using the same, and method for detecting target molecule using the sensor
SE0201468D0 (sv) * 2002-05-13 2002-05-13 Peter Aasberg Metod att använda luminescenta polymerer för detektion av biospecifik växelverkan
FR2849038B1 (fr) * 2002-12-19 2005-03-11 Apibio Nouveaux pyrroles substitues avec des oligonucleotides, polymeres electroactifs et leurs utilisations
KR100580621B1 (ko) * 2003-03-07 2006-05-16 삼성전자주식회사 전도성 화합물, 이를 포함하는 전극 및 센서, 상기 센서를이용한 표적 물질 검출방법
US7863455B2 (en) 2004-02-24 2011-01-04 Japan Science And Technology Agency Electrochemically active ligand for sequence-specific detection of double-stranded nucleic acid molecule
FR2892723B1 (fr) * 2005-11-03 2009-04-24 Biomerieux Sa Nouveaux monomeres electropolymerisables, solubles en solution aqueuse, comportant une metalloporphyrine.
FR2921517B1 (fr) * 2007-09-26 2010-12-03 Commissariat Energie Atomique Membranes conductrices de protons pour pile a combustible presentant un gradient de protons et procedes de preparation desdites membranes
KR101580318B1 (ko) * 2008-05-14 2015-12-28 삼성전자주식회사 나노 금속 결합용 단량체, 전도성 고분자 복합체 및 그의제조방법
JP2016045064A (ja) * 2014-08-22 2016-04-04 国立大学法人山形大学 トランジスタ型アミンセンサ
JP6714278B2 (ja) * 2014-09-19 2020-06-24 国立大学法人 新潟大学 基質抗原同時検出バイオセンサ、基質抗原同時検出方法、および、プログラム

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JPH03205422A (ja) * 1990-01-08 1991-09-06 Nippon Oil Co Ltd ポリ［（３―ピロリル）酢酸］
FR2720832A1 (fr) * 1994-04-22 1995-12-08 Francis Garnier Electrodes et membranes électroactives à base de peptides bioactifs, pour la reconnaissance, l'extraction ou le relargage d'espèces biologiquement actives.

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