Patent Application: US-82479907-A

Abstract:
the invention relates biosensor comprising a conductive or semi - conductive support and an intercalator polymer , wherein the intercalator polymer comprises monomer units of formula wherein p is an electropolymerizable moiety selected among the group consisting in pyrrole , acetylene , phenol , aniline , thiophene , carbazole and azulene ; sp is a spacer , and im is an intercalator . the invention also relates to a method for the detection of a dna sequence with the biosensor , comprising the steps of : hybridizing the dna sequence with a dna probe for forming a dsdna incubating the biosensor with the dsdna for anchoring the dsdna to the intercalator detecting the quantity of dsdna on the biosensor .

Description:
the original method of detection of a dna sequence according to the invention comprises the following steps . the first step is the preparation of a biosensor , comprising forming by electropolymerization an , intercalator polymer film onto a conductive or semi - conductive support . the covalent bonding between the polymer and an intercalator is made either prior or after polymerization , preferably after polymerization . methods for electropolymerization of polymers according to the invention are well known in the art and extensively disclosed in the scientific literature ( see cited publications ). the next step is the hybridization of the ssdna target with a ssdna probe in a mixture to form dsdna . methods for hybridization of ssdna with a probe are also well known in the art and extensively disclosed in scientific literature and other handbooks . the next step is the incubation of the biosensor in the mixture in order to allow the capture of the dsdna by the intercalator . then , a method of detection can be used in order to detect quantitatively or qualitatively the presence of dsdna on the biosensor . all direct or indirect detection methods known by one skilled in the art can be used . examples of use of cyclic voltammetry and amperometry will be described in detail in the experimental section below . it is also possible to use a label - free method relying on electrochemical impedance spectra ( eis ) for monitoring the interfacial property changes of modified electrodes upon hybridization event ( 20 ). a method based on the measurement of the frequency changes of a quartz crystal microbalance ( qcm ) can also be implemented ( 21 ). a device for the detection of a dna sequence in a sample can be elaborated . the device comprises a plurality of wells , each well containing a solution comprising the sample and a determined ssdna probe . hybridization occurs in the well ( s ) in which the ssdna probe is complementary to the dna sequence of the sample , thus forming a dsdna . the device also comprises a plurality of biosensors adapted for being immersed into the wells . according to a preferred embodiment , all the biosensors are fabricated by the method described above , on a single conductive support having as many spots as the number of wells . all the spots are formed on the conductive electrode by electropolymerization of an intercalator polymer . the spots are immersed into the corresponding wells and , if the wells contain dsdna , the dsdna is grafted to the intercalator of the corresponding biosensor ( s ). after detection , the support can then be washed into an acid or basic bath , which eliminates the dsdna , whereas the polymer , which is stable , remains . the support can therefore be re - used . the use of intercalators is widespread for the labelling after hybridization of dna probe and dna target and therefore their detection and quantification are well known ( 35 - 52 ). this represents a large family of compounds that have already been tested for the detection step . in fact , the present invention is applicable to all intercalators produced until now for insertion into the double dna helix . furthermore , this strategy of detection of a single stranded dna by hybridization in a solution with its complementary followed by the anchoring of the resulting double stranded dna by an intercalator , is applicable to every kind of sensor used for the detection of biomolecules ( electrodes , optical fibers , field effect transistors , etc .). this new concept of dna detection may also bring an increased sensitivity and more precisely an increased limit of detection for the direct detection methods . indeed , the classical direct detection of duplex formed on the surface on sensor that has been pre - functionalized by a ssdna — for example , impedance or gravimetric measurements — is based on a perturbation linked to the difference ( transfer resistance , weight , etc ) between a single stranded and a double stranded dna . in the grafted intercalator concept , the difference will be noticeably more important because it will concern a small molecule ( the intercalator ) and a dsdna ; a better detection sensitivity is therefore expected . another interesting advantage is the possibility to re - use the biosensor for several measurements of the same target , by a simple denaturation of the duplex . a specific advantage of this method compared to the other dna sensors is the possibility to re - use the sensor for other targets . indeed , the recognition specificity lies into the formation of the dsdna , the anchoring step being only specific of the duplex . this is an important point when expensive biosensors are used . at last , another advantage of the invention is that the functionalization of the surface of the sensor is very simple compared to grafting dna probes onto the surface . the approach was based on the covalent attachment of a synthetic planar intercalator , a redox acridone intercalator ( rad ) onto an electropolymerized polypyrrole film functionalized by activated esters groups . one example is a redox acridone derivate ( rad ) recently synthesized ( 11 ) that was used for the successfully determination of ssdna derived from west nile virus sequence ( ssdna - wnv ). for this purpose , the complementary dna strand ( target ) mixed into an aqueous solution with the dna probe were captured and thus , immobilized directly on the electrode surface via rad - duplex intercalation , rad being anchored onto a new electropolymerized polypyrrole n - hydroxysuccinimide film . because of the rising epidemy of wnv infections with no good antivirals or vaccines in sight , a greater reliance on early wnv diagnostics is needed ( 12 ). first isolated in uganda in 1937 , wnv is a single - stranded rna flavivirus transmitted to humans by an infected mosquito bite and is constituted of three structural proteins ( c , prm , e ). the envelope protein ( e protein ) is arranged in dimers on the surface of the mature virions and is one of the preferential targets for the diagnosis of wnv infection by rt - pcr or elisa due to its high immunogenity . human - to - human transmission via mosquito bites does not occur ; however , wnv can be transmitted from one person to another via blood transfusion , organ transplantation , breast - feeding , and transfer across the placenta ( 13 ). although most wnv infected persons are asymptomatic , 10 - 20 % develop symptoms such as fever , headache , rash , and malaise . a small number (& lt ; 1 %) of infected individuals develop severe neurological illness , including meningitis , encephalitis , and acute flaccid paralysis ; the infection proves to be fatal for 10 % of these patients with neurological illness ( 14 ). the elaboration of a new concept of dna sensor based on the revolutionary use of an intercalator molecule as a mechanical vector to extract a double stranded dna from an aqueous solution by its deposition on the transducer surface is reported here . in this context , a wnv genomic sensor was selected as a model . the target sequence is derived from a primer sequence used in rt - pcr and previously tested and published by huang et al . ( 15 ). the detailed steps in the elaboration of wnv - ssdna sensor are described below . a pyrrole - nhs monomer was synthesized as follows : 4 - pyrrol - 1 - ylbutanoic acid ( 298 mg , 1 . 9 mmol ), n - hydroxysuccinimide ( 228 mg , 2 . 0 mmol ), and 1 , 3 - dicyclohexylcarbodiimide ( 398 mg , 2 . 0 mmol ) were dissolved in dry tetrahydrofuran . the mixture was stirred under argon overnight at room temperature and then filtered . the organic phase was evaporated , and the residue was washed with ch 2 cl 2 . crystallization from ch 2 cl 2 gave 494 mg of monomer pyrrole - nhs ( 60 % yield ). 1 h nmr ( 250 mhz / cdcl 3 ): δ ( ppm ) 6 . 62 ( s , 2h ), 6 . 09 ( s , 2h ), 3 . 83 ( t , 2h ), 2 . 80 ( s , 4h ), 2 . 54 ( t , 2h ), 1 . 92 ( m , 2h ). pbs buffer was 0 . 1 m phosphaten buffer ( ph 7 ) containing 0 . 145 m nacl . to immobilize the intercalator on the pt disk electrode ( 5 mm - diameter ), a new pyrrole derivative functionalized with a n - hydroxysuccinimide group ( pyrrole - nhs ) ( represented on fig1 b ) was prepared by reaction of 4 - pyrrolylbutanoic acid with n - hydroxysuccinimide and characterized by nmr technique . electropolymerization of pyrrole - nhs ( 5 mm ) in solution of ch 3 cn + 0 . 1 m liclo 4 was accomplished by controlled potential oxidation ( 2 mc ) at 0 . 95v . upon transfer into ch 3 cn + 0 . 1 m liclo 4 solution free of monomer , the cyclic voltammogram of the resulting electrode exhibits a reversible peak system at 0 . 45 v ( shown on fig2 a ). the latter corresponds to the oxidation of the polypyrrolic film formed on the electrode surface . the apparent surface coverage ( γ ′= 1 . 55 × 10 − 8 mol · cm − 2 ) of electropolymerized pyrol - nhs was determined from the charge recorded under polypyrrole electroactivity , the electrochemical polymerization yield being 35 %. referring now to fig2 b , to increase the polymer permeability , an overoxidation of the polypyrrole film was performed by cycling the potential between 0 . 0 and 1 . 3 v . on this figure , ( a ) corresponds to the first scan whereas ( b ) corresponds to the fourth scan in ch 3 cn + 0 . 1 m liclo 4 ; the scan rate being 0 . 1 v · s − 1 . further , the covalent binding of the rad intercalator was carried out at room temperature , by spreading over the modified electrode an aqueous rad solution ( 20 μl , 140 μg / ml ) for an overnight incubation under a wet environment . the structure of the rad is shown on fig1 a . recognition ( hybridization ) of the ssdna target derived from a west nile virus sequence and ssdna probe hybridization between the denatured biotinylated oligonucleotide probe ( 140 μg / ml , 65 ° c . for 1 min ) and various wnv - dna target concentrations ( 1 pg to 100 μg / ml , 95 ° c . for 1 min ) were performed in “ a tube ” for 1 h , at room temperature . the volume of the hybridization mixture was 0 . 2 ml . dna hybridization buffer was based on 0 . 2 m phosphate buffer ( ph 7 ) containing 12 . 5 mm peg 8000 , 0 . 5 m nacl , 0 . 5 mm edta and 0 . 5 % ( w / v ) sds . denaturated salmon sperm solution 1 % ( v / v ) ( 95 ° c . for 1 min ) was added in the hybridization buffer . all buffers were sterilized by autoclaving for 30 min at 120 ° c . in house sterilized and deionized water was used in all solutions . the wnv oligonucleotides were provided by integrated dna technologies having the following sequences : target - dna : gctatttggctaccgtcagcatctctccaccaaag - 3 ′, (+) ( seq id no : 1 ); wnv - dna - biotin : 5 ′- cggtagccaaatagc / biotin /- 3 ′, (−) ( seq id no : 2 ). to investigate the anchoring properties of the rad into dsdna , a wnv - derived dna target was mixed with the biotinylated complementary oligonucleotide and the modified electrode was incubated with the hybridization solution for 2 h at 25 ° c . the modified electrodes were washed successively with 30 mm citrate buffer ( ph 7 ) containing 0 . 3 m nacl and 1 % ( w / v ) sds , then with 15 mm citrate buffer + 150 mm nacl + 1 % ( w / v ) sds , then with 15 mm citrate buffer + 150 mm nacl , and finally with 10 mm pbs containing 0 . 5 % sds . after incubation with the dsdna , the poly ( pyrrole - nhs )- rad electrodes were treated for 30 min with 20 μl of a blocking solution , a pbs buffer ( ph 7 ) containing 10 g · l − 1 bsa and 0 . 05 % ( v / v ) tween 20 . the resulting electrodes were then incubated for another 20 min with 20 μl of avidin ( 500 μg / ml ) and washed with pbs ( ph 7 ) containing 0 . 5 % ( w / v ) sds . finally , the modified electrodes were treated with 20 μl of biotinylated glucose oxidase ( gox ) solution ( 500 μg / ml ) for 20 min and rinsed with pbs ( ph7 ) containing 0 . 5 % ( w / v ) sds . the electrochemical behavior of modified dsdna - rad - poly ( pyrrole - nhs ) electrodes was examined by cyclic voltammetry in deaerated pbs ( ph 7 ) buffer . fig2 c shows a reversible peak system at − 0 . 35v whereas the poly ( pyrrole - nhs ) film is not electroactive in this potential range . on this figure , the curves ( a ), ( b ) and ( c ) correspond respectively to scan rates 20 , 50 and 100 mv · s − 1 . this redox signal is in good accordance with the potential value (− 0 . 25 v ) recorded for the one - electron reduction of rad in solution . in addition , the current intensity of this peak system varies linearly with the scan rate , corroborating thus the chemical grafting of rad . the apparent surface coverage of rad , 2 . 25 × 10 − 9 mol · cm − 2 , was estimated by integration of the charge under this peak system . taking into account that the theoretical maximum surface coverage with one close - packed rad layer corresponds to 2 . 37 × 10 − 10 mol · cm − 2 , it clearly appears that the intercalator molecules are grafted at the polymer - solution interface but also inside the polypyrrole structure . as shown on fig2 d , that shows cyclic voltammograms before ( a ) and after ( b ) incubation with wnv dsdna ( 10 ng / ml ) in pbs , the scan rate being 0 . 1 v · s − 1 , the dsdna - incubation step clearly induces a decrease in current intensity of the reversible peak system at − 0 . 35 v that was ascribed to the intercalation of the grafted rad into the formed wnv - dna duplex . indeed , the disappearance of the electrochemical signal of similar acridone derivatives in solution in the presence of dsdna was recently described and attributed to an intercalation process ( 17 ). in contrast , no peak evolution was observed on the cyclic voltammogram of a similar electrode before and after its incubation in pbs solution containing only the wnv - dna target ( 0 . 1 mg / ml ) and noncomplementary oligonucleotide . such decrease may be attributed to a reduced insertion rate of counterions into the film during the electroreduction of rad due to the steric hindrances , electrostatic repulsion , or both resulting from the duplex attachment at the polymer surface ( 18 ). as previously reported for oligonucleotides functionalized by redox groups ( 19 ), the intercalative binding of rad to dsdna and its strong interaction with the “ π - stack ” of the dna base pairs diminish its accessibility and hence may also prevent its electrochemical reduction by an electron hopping process . moreover , dsdna anchored onto rad - polypyrrolic film is stable since no evolution of the electrochemical signal was recorded after one - day storage in pbs . however , the electroactivity of covalently bound rad was not totally suppressed by the specific anchoring of hybridized wnv dna since the bulky duplex cannot penetrate inside the polymer film . as a consequence , the decrease in current intensity of the rad system cannot constitute an extremely sensitive method to quantify hybridization process . indirect dna detection by amperometry using labelled ssdna probe with biotin since the dna - probe was biotin labeled , the presence of the wnv duplex was electroenzymatically detected by the attachment of a biotinylated enzyme onto the duplex via avidin - biotin affinity interactions . for this purpose , the poly ( pyrrole - nhs )- rad electrodes were incubated with duplex solutions formed from dna target concentrations ranging from 1 pg up to 100 μg / ml . after the “ fishing step ”, a biotinylated gox was fixed by an avidin bridge onto the immobilized dsdna . since gox catalyzes the aerobic oxidation of glucose with the concomitant production of h 2 o 2 , its presence was detected via the electrochemical oxidation of h 2 o 2 at the underlying electrode surface potentiostated at 0 . 6 v . the current response of the poly ( pyrrole - nhs )- rad - dsdna electrodes toward glucose ( 20 mm ) was assayed in pbs ( ph 7 ). the results are shown on fig3 where the inset presents the current response of a dna electrode to glucose ( 20 mm ) for 1 ng / ml dna target and e = 0 . 6 v / ag / agcl . the current response reached a steady - state value in a very short time ( 30 - 40 s ). these current responses were proportional to the dna target concentration , the detection limit being 1 pg / ml ( 90 fm ). the repeatability and reproducibility of the current response of the poly ( pyrrole - nhs )- rad - dsdna electrodes were examined . for each dna target concentration , three measurements of 20 mm glucose were carried out leading to relative standard deviation ( rsd ) values ranging from 1 . 7 to 2 . 0 %. the elaboration of the dna sensor was also reproducible ; four poly ( pyrrole - nhs )- rad - dsdna electrodes were prepared from 1 ng / ml dna target and their amperometric responses led to a rsd of 3 . 7 %. to ensure that the amperometric detections are directly related to the dna hybridization phenomenon of various wnv - cdna target concentrations , the intercalator ( rad ) was replaced by glycine in the construction of the dna sensor . another control experiment consisted in the incubation of the poly ( pyrrole - nhs )- rad electrode with biotinylated ssdna probe instead of dsdna . both controls were treated for gox anchoring , and their amperometric responses to glucose were recorded . it clearly appears that these responses were very low (˜ 100 na ) compared to the current response characteristic to 1 pg / ml dna target concentration ( 3 μa ). these results demonstrate that an electrochemical wnv genomic sensor , using a dna intercalator , can be extremely sensitive and could be adapted to detect an early stage of a wnv infection . 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