Conducting polymers as a link between biomolecules and microelectronics

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Synthetic

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Conducting Polymers as a link between biomolecules and microelectronics G. Bidan, M. Billon, T. Livache’, G. Mathis’, A. Roget et L. M Torres-Rodriguez. CEA-GRENOBLE, .?J%IR5819 (CNRS-CEA-Universitk J. Fourier) 17, avenue des Martyrs, 38054 Grenoble Cedex 09, France ‘CIS Bio international. DIVr, BP 175, 30203 Bagnois sur Ckze Cedex, France.

Abstract Biosensors based on electronic conducting polymers (ECPs) appear particularly well suited to the requirements of modem biological analysis: multiparametric assays, high information density and miniaturization, We describe a new methodology for the preparation of addressed DNA matrices. The process includes an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing on their 5’ end a pyrrole moiety. The resulting polymer film deposited on the addressed electrode consists of pyrrole chains bearing covalently linked oligonucleotides. An oligonucleotide array was constructed on a silicon device bearing a matrix of 48 addressable 50 pm x 50 pm gold microelectrodes. This technology was successfully applied to the genotyping of Hepatitis C Virus in blood samples. Fluorescence detection results show good sensivity and a high degree of dimensional resolution. The need for versatile processes for the immobilization of biological species on surface led us to extend our methodology. A biotinylated surface was obtained by co-electropolymerization of pyrrole and biotin-pyrrole monomers. The efficiency for recognition (and consequently immobilization) of R-phycoerythrin-avidin was demonstrated by fluorescence detection. Copolymerization of decreasing ratios of ovrrole-biotin over uvrrole allowed us to obtain a decreasing scale of fluorescence. 1. Introduction There is a need of microanalysis devices increasing the number of analysed parameters in a sample with a low cost while decreasing the sample volume. In order to reach the high information density on a small surface necessary for this, it is essential to develop a versatile addressing system. Biosensors based on electronic conducting polymers [l] (ECPs) appear particularly well suited to these requirements of modem biological analysis. Indeed the ECPs were attractive materials in order to elaborate a sensitive layer at the surface of an electrode. This is due to their key properties, i. e. (i) an easy electrodeposition on the electrode surface allowing miniaturization and (ii) a versatile fimctionalization either by grafting or doping enabling one to perform selective recognition. Many biosubstances such as enzymes [2] or peptides [3,4], have been immobilized on an ECPs matrix which is widely a polypyrrole film. Indeed the conditions of the electrodeposition of pyrrole monomers (low potential, buffered aqueous solution)

Monomers

in solution

Scheme I: Synthetic stratew for the immobilization of biological enrities (BE) by copolymen’zation

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were compatible with the stability of the biological entities. Thanks to the biocompatibility of the polypyrrole, it has even been possible to include single-stranded DNA in the bulk or at the surface of polypyrrole films. The immobilisation of singlestranded DNA can be realized either by postfunctionnalization [5] or by copolymerization [6] (scheme 1). This last technic was based on the electrooxidation of a mixture of pyrrole and ODNpyr (pyrrole covalently linked to an oligonucleotide via a spacer arm). The process leads in one step to the irreversible immobilization of ODN units (in a solid copolymer film formed by an oligonucleotide grafted on a polypyrrole chain) on an electrode. This copolymerization functionalization was extended to the grafting of peptides and was illustrated by an immunodetection on a polypyrrole peptide-chip [7]. More recently, we took up this concept for the biotin avidin system. From the copolymerization of pyrrole monomers and pyrrole linking covalently biotin entity through a spacer arm, we have synthesised [g] a polypyrrole film bearing biotin units; anchoring points of avidin conjugates via the strong affinity between avidin and biotin. In addition to the versatility of functionalization, the copolymerization process allows the elaboration of microbiosensors [9]: an oligonucleotide array constructed on a silicon device bearing a matrix of addressable 50 pm x 50 pm microelectrodes. The DNA chip was prepared by successive electrochemical copolymerizations. Each gold microelectrode being addressed by selective switching, the resulting polymer growth is strictly limited to the selected electrode surface and takes place on the activated electrode and not on its neighbour. Therefore one can cover each dot by a polypyrrole grafted by a different ODN. In order to illustrate the features presented by the coelectropolymerisation process, we reported herein two examples. The first concerned the genotyping of hepatitis C virus in blood samples using a silicon chip bearing gold microelectrodes. The reserved.

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genotyping of hepatitis c virus is carried out through the detection of generic DNA sequences and specific sequences namely Tl and T2. These three sequences were specifically recognized via the hybridization by ODN-G, ODN-Tl and ODNT2 probes respectively immobilized in the polypyrrole film. Afterwards the hybridization detection is analysed by fluorescence. The second example concerned the preparation by copolymerization of polypyrrole layers timctionalized with biotin units and carried out on microelectrodes arrayed on a silicon chip. From different ratios of biotmylated pyrrole to pyrrole monomers, we have obtained different polymers where the amount of biotin inserted in the polypyrrole matrix should be modulated from the amount of biotinylated monomers involved in the copolymerization reaction. 2. Experimental 2.1. Silicon chips The matrix of 48 (4x12) gold microelectrodes (50 pm x 50

unr) was made by microelectronic technologies on a silicon support (CEA/Leti, France) as previously described [9]. Each electrode is individually connected. 2.2. Preparation

of grafted

biomolecules

ODNpyr were synthesized according to the previously described procedure by using a pyrrole-phosphoramidite building block in the course of the ODNs synthesis [lo]. Biotinylations of ODNs were carried out in the same way. The biotinylated pyrrole is synthesized by coupling an amino-alkyl pyrrole and an activated ester of biotin [8]. 2.3. Synthesis of the polypyrrole

support

Electrochemical reactions were carried out in a lml teflon cell including a platinum wire as counter electrode and a saturated calomel electrode as reference electrode. For oligonucleotides the copolymerization reactions were carried out in 500 pl of a solution containing 20 mM pyrrole (Tokyo Kasei), 0.1 M LiC104 (Flucka) and 1 uM of ODNpyr. The films were synthesized on the working microelectrode by cyclic voltamperometry (potential sweeping between -0.35 and +0.85 V vs SCE, scanrate 100 mv/s). The reaction was stopped when the charge reached 250 nC. After each synthesisthe support and the cell were rinsed, another electrode was selected and swiched on and the following copolymerization was carried out. For biotin immobilization, the electro-copolymerization conditions are the same as for oliginucleotides. Biotinylated pyrrole concentrations was from 100 pM to 0.01 pM. 2.4. Molecular material

recognition

and detection

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The biotin detection was carried out in a similar way. Namely, the biotinylated chip was incubated for 10 mn in a buffered solution of 5% streptavidin-R-phycoerythrin. However, to avoid any side immobilization of the streptavidin-Rphycoerythrin by non-specific adsorption on polypyrrole, the biotinylated support was previously blocked by washing with Denhardt’s reagent. 3. Results and Discussion 3.1. HCV Genotyping on DNA Chips

Before to undertake the HCV genotyping procedure, optimizations of the support preparation have been carried out using the ODN-G probe: - Film thickness (evaluated through the charge passed for the synthesis) was varied from 10 to 30 mn for co-poly(pytrole ODN-Gpyr) film. After a fast increase the fluorescencereached a plateau for a thickness of 15 nm. The optimization of the film thickness (250 nC, 20 mn) led to a rapid synthesis of an homogeneouspolymer layer and to a perfect covering of the gold microelectrode. - The ratio between pyrrole and ODN bearing a pyrrole group (ODN-pyr) is of major importance. The concentration of pyrrole for electropolymerization in aqueous media camrot be decreasedbelow 20 mM. Conversely only the concentration in ODN-Gpyr had been modulated from 0.12 to 3 pM. Results of fluorescence analysis shown that a saturation of the polypyrrole surface occured when the concentration in ODN-Gpyr reached 0.6 pM. - The specificity of the addressing was demonstrated by performing hybridization using a specifically synthesized biotinylated complementary ODN-G under saturating conditions (250 pM in the hybridization buffer). No cross-contamination with ODN-Tlpyr or ODN-T2pyr was evidenced. - The sensitivity level was evaluated by end point dilutions of the same complementary ODN-G. A high fluorescencesignal was observed for concentrations greater than 10.” M; however, it decreased for lo“* M (12 x lo6 copies in 20 pl) and was negative for lo-l3 M. Reported to the electrode area this correspondsto 4800 molecules/w*. - To increase the hybridization signal for long DNA fragments, the efficiency of a 5’-end polythymidine arm was evaluated. HCV probes bearing 3, 5 or 10 thymidines at their 5’ end were essayed. Results showed that the fluorescence was higher with 5 thymidines or more. Pentathymidine arms (5’ end) were further used for all pyrrole HCV probes.

of the bound

The DNA chips were prepared with three HCV typing oligoprobes and a negative control (polypyrrole alone). These probes were individually copolymerized with pyrrole on the chip and were hybridized with clinical sample as following. The HCV RNAs were extracted from known patient sera. They were reversedtranscribed and amplified by a PCR reaction in the presence of a sense biotinylated primer. The obtained biotinylated Gagments were hybridized to the probes immobilized on the microelectrodes. The genotyping was realized by successivehybridizations on the DNA chip. After each hybridization the chip was incubated for 10 mn in a buffered solution of 5% streptavidin-R-phycoerythrin, then rinsed and finally the fluorescence was recorded for 1 s with an epifluorescence microscope (BX 60, Olympus) equipped with a Pelt&r cooled CCD camera (Hamamatsu) and image analysis software. Each detection was followed by a 1 mn denaturation step in 0.1 M NaOH in order to regenerate the chip.

Figure I: (A) Pattern of probe repartition on the microelectrode array: PP. polypywole hornopolymer; G, Tl, T2, polypyrrole copolymers bearing probes ODN-Gmr, ODNTlpyr and ODN-TZpyr, respectively. (@) fluorescence result of genotyping of a clinical sample positive for HCV T2 on the array described above.

For HCV genotyping of clinical samples, the DNA chips were prepared according to the procedure described above with

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the three HCV typing oligoprobes (i.e.: ODN-Gpyr, ODN-Tlpyr and ODN-T2pyr) and a negative control (polypyrrole alone). These probe were copolymerized on the areas of the chip according to the pattern shown in Figure 1A. Figure 1B shows the fluorescence results obtained with type 2 RNA: hybridization of G and T2 probes. Therefore, an easy determination of HCV genotype 2 was obtained without any ambiguity and was in accordancewith results obtained by other methodologies. The DNA chips can be stored for at least 3 months without loss of activity under moist conditions at 4°C. 3.2. The biotinylated

polypyrrole

In accordance with the copolymerization hmctionalization, biotinylated poypyrrole films have been electrosynthesizedfrom pyrrole and biotinylated pyrrole on microelectrodes. The biotinylated pyrrole used was a biotin entity linked to the nitrogen atom of a pyrrole unit by a hydrophilic arm (alkoxyl chain composedby three oxygen and ten methylene groups). The hydrophobic/hydrophilic balance as well as the length of the spacer arm have been selected to allow good solubility and accessibilityof the hanging biotin [81.

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4. Conclusion

This technology appearsto be versatile and very precise with routine synthesis on SO-pm electrodes. There is no need for specific instrumemation for the manufacturing of the biochip (copolymerization) in contrast with other methodologies involving photochemistry or robotic deposition. The electrochemical addressing concept needs the synthesis of tailor-made monomers and implies a number of electrochemical deposition experiments identical to the number of modified microelectrodes. However, it results in a perfect control of the purity of the immobilized DNA sequence(contrary to (( in situ )) synthesis of ODN) and is perfectly adapted for targetted genotyping. The immobilization of biotin opens the route for the elaboration of a large number of biosensors due to the commercial availability of a wide variety of avidin conjugates of biomolecules. In addition the fluorescence scale demonstrates the feasability of the quantification of the titration.

Figure 2 : A) Pattern of the biotin repartition on the microelectrode array. Electrosvntheses were car&d out at 100 mV s-l by repetitive potential scans between -0.35 and 0.85V vs SCE f rom 20mMpyrrole and a decreasing range of biotin pyrrole with a synthesis charge of 18mC.cmv2 (about 60 nm thickness) in 0.1 M LiClOd /‘Hz0 containing 3% of CHsCN on microelectrodes (50x50 pm2) arrayed on a silicon chip. pp. polypyrrole homopolymer ; a. b, c. d. e, biottnylated polypvrrole synthesized in presence of lOO$J IO&I, IN, 0.1 pM and 0.01 pM of biotin pvrrole respectively. Each electropolymerization was followed by a thorough washing step and blocking step (see text).

The accessibility of immobilized biotin entities toward avidin conjugated has been demonstrated by quartz microbalance experiments [ 111. Using various concentrations of biotinylated pyrrole for the same concentration of pyrrole monomers, we obtained different copolymerscontaining variable amount of biotin. Figure 2 shows the pattern of the deposition of these areas. The presence of biotin was analysed from a fluorescence measurement thanks to streptavidine entities bearing each of them a fluorescent phycobiliproteine. The anchoring of the streptavidius to the immobilized biotins was easy via the strong biotiu/avidin affinity. Figure 3 clearly shows that the intensity of the fluorescence is related to the amount of biotin involved in the copolymerization reaction and yields the amount of accessible biotin at the surface of the fihn.

5. References.

[l] G. Bidan in Gabor Harsanyi (Ed.), Polymer films in sensor applications, Ph. D. Gabor Harsanyi, Hungary, (1995) 206. [2] W. Schumann,Mikrochim. Acta, 121(1995) 1. [3] F. Gamier, H. K. Youssouti, P. Srivastava,A. Yasser, J. Am. Chem. Sot., 116 (1994) 8813. [4] P. Bauerle, M. Hiller, S. Scheib, M. Sokolowski, E. Umbach, Adv. Mater., 8 (1996) 2 [5] H. K. Youssoufi, F. Gamier, , P. Srivastava,A. Yasser, J. Am. Chem. Sot., 119 (1997) 7388. [6] T. Livache, A. Roget, E. Dejean, C. Barthet, G. Bidan, R. Teoule, Nucleic Acids Res., 22 (1994) 2915. [7l T. Livache, H. Bazin,P. Caillat, A. Roget, Biosensors & Bioelectronics, in press. [8] L. M. Torres-Rodriguez,A. Roget, M. Billon, T. Livache, G. Bidan, J. Chem. Sot., Chem. Commun., in press. [9] T. Livache, B. Fouque, A. Roget, J. Marchand, G. Bidan, R. Teoule and G. Mathis, Anal. B&hem., 255 (1998) 188. [lo] A., Roget, H. Bazin, and R. Teoule, Nucleic Acids Res., 1, (1989) 7643-7652.

[l l] L. M. Torres-Rodriguez, M. Billon, A. Roget and G. Bidan, this Journal, Proceedings of ICSM’98, Montpellier 12-18 juillet 1998.