Conducting polymers on microelectronic devices as tools for biological analyses

Conducting polymers on microelectronic devices as tools for biological analyses

Clinica Chimica Acta 278 (1998) 171–176 Conducting polymers on microelectronic devices as tools for biological analyses ´ Thierry Livache a , *, Herv...

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Clinica Chimica Acta 278 (1998) 171–176

Conducting polymers on microelectronic devices as tools for biological analyses ´ Thierry Livache a , *, Herve´ Bazin b , Gerard Mathis b a

CIS bio international, DRFMC, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France b ` Cedex, France CIS bio international, DIVT, BP 175, 30203 Bagnols /Ceze

Abstract In the field of biological analysis, the need for multiparametric analysis has prompted the development of supports bearing a series of biomolecules linked to a support in a precise location (addressed). To reach a high information density, miniaturization of this kind of support has to be carried out. We describe in this paper an approach involving the use of electro-conducting polymers such as polypyrrole. This technology is based on an electro-directed copolymerization of pyrrole and oligodeoxynucleotides (ODN) linked to a pyrrole residue. The process allows the grafting of the selected ODN at the surface of the successively addressed microelectrodes. In this way, the syntheses are carried out on 50 mm electrodes on passive chips or on active (multiplexed) chips bearing 48 or 128 gold microelectrodes, respectively. The detection of biological targets recognized by the biochip is carried out by using fluorescent tracers. This technology, involving prepurified materials precisely addressed, allows better reproducibility of the biochip preparation and, then, an easy interpretation of the fluorescence results. The versatility of this technology is illustrated by ODN or peptide copolymerizations leading to DNA chips or peptide chips, respectively. This would open the field for other biological interaction studies.  1998 Elsevier Science B.V. All rights reserved. Keywords: DNA chip; Peptide chip; Polypyrrole; Silicon device

1. Introduction In the field of biological analysis, the need for multiparametric analysis has prompted the development of supports bearing a series of biomolecules linked to *Corresponding author. E-mail: [email protected] 0009-8981 / 98 / $ – see front matter  1998 Elsevier Science B.V. All rights reserved. PII: S0009-8981( 98 )00143-0

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a support in a precise location (addressed). To reach a high information density, miniaturization of this kind of support has been carried out and different strategies have been described, i.e. mechanical spotting of oligodeoxynucleotides (ODNs) or in situ synthesis via a photochemical process [1–3]. In this paper, we describe an approach involving the use of electro-conducting polymers such as polypyrrole [4,5]. This technology is based on an electrodirected copolymerization of pyrrole and a biomolecule linked to a pyrrole residue. In brief, electrochemical oxidization of pyrrole gives, in one step, a solid polypyrrole film laid on the surface of the electrode. The copolymerization of pyrrole and an ODN tethered to a pyrrole group allows the formation of a solid copolymer constituted by an ODN grafted onto a polypyrrole chain. The synthesis of polypyrrole is limited to the surface of the electrode, so that the size of the organic support is the same as that of the electrode. The same process was successfully applied to pyrrole-peptides. Successive copolymerizations with the selected biomolecules allow the construction of a biological array from an electrode array. Miniaturization of this process was achieved by performing electropolymerization on devices bearing an array of 50 mm microelectrodes. Biological applications of these chips have been carried out either in the field of DNA hybridization or in the field of peptide immunoassays. In this way, the detection of biological recognition is performed by fluorescence microscopy.

2. Materials and methods The synthesis of pyrrole–ODNs involved the preparation of a nucleoside phosphoramidite bearing a pyrrole with a spacer arm [4]. This monomer was used for the synthesis of 59 end-tethered ODNs. After deprotection, the ODNs modified with pyrrole were purified by reversed-phase high-performance liquid chromatography (HPLC). The pyrrole peptides were constructed by coupling a pyrrolyl residue onto a synthetic peptide via a linker and were purified by the same process. The polypyrrole syntheses were performed on two kinds of silicon chips constructed by the classical microelectronic technologies on silicon wafers. The first was a 4 cm 2 passive chip bearing an array of 48 gold microelectrodes (50 mm) including 48 gold contact pads, ensuring external connection. The second was a 10 mm 2 active multiplexed chip bearing 128 electrodes for only 9 gold outlets. All of the silicon chips were constructed by the CEA-Leti (Grenoble, France). Electrosyntheses of polypyrrole film were carried out by potential sweeping between 2 0.35 and 0.85 V/ SCE (Saturated Calomel Electrode) in a 0.1 mol / L LiClO 4 solution containing 20 mmol / L pyrrole and 1 mmol / L pyrrole–ODN.

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The addressing of the successive electropolymerizations was ensured by the selective switching of a given electrode and by the selection of the modified ODN used for the copolymerization. Following syntheses, the chip was rinsed with water and stored at 48C. Hybridization of the chip with biotinylated polymerised chain reaction (PCR)amplified samples and detection with a streptavidin–phycoerythrin conjugate (Molecular Probe) were performed in phosphate buffered saline (PBS), according the procedure described elsewhere [5]. In order to regenerate the chip, the detection step was followed by a 1-min denaturation treatment in 0.1 M NaOH. Biotinylated standard (ODN tethered both to pyrrole and biotin groups) and pyrrole-peptides were copolymerized on the chip by the same electrochemical process. Their detection was carried out with biotinylated antibodies in PBS containing 1% bovine serum albumin (BSA]. 3. Results and discussion

3.1. Polypyrrole copolymerization The copolymerization of polypyrrole on the surface of the gold microelectrodes leads to thin homogeneous films (50 to 100 nm thickness). The spacial resolution is also very good (less than 2 pm). The amount of hybridizable ODN linked to the support is estimated to be 200 fmol / mm 2 [5]. The supports used for the electrochemistry are 50 mm microelectrodes arrayed on silicon devices (Fig. 1).

3.2. Construction and use of a DNA chip Biotinylated PCR-amplified products or ODN can be hybridized on the chip. The following example, described elsewhere in detail [5], shows an application of the DNA chip dealing with hepatitis C virus (HCV) genotyping. In this way, probes for HCV genus (G), or specific for type 1 (T1) and type 2 (T2), were immobilized according to the pattern shown in Fig. 2A. RNA from serum samples was reverse-transcribed and amplified in the presence of a biotinylated primer and hybridized on the chip. Fluorescence analysis was carried out after incubation with a streptavidin-R–phycoerythrin conjugate. A typical result is shown in Fig. 2B. This demonstrates the ability of these DNA chips to be used with real biological samples.

3.3. Construction and use of a peptide chip Peptide fragments from adrenocorticotropic hormone (ACTH), namely pyrrole-peptide(18 – 39) and pyrrole-peptide(11 – 24), were copolymerized on

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Fig. 1. (A) View of the passive silicon chip bearing 48 microelectrodes and 48 outlets. (B) View of the multiplexed silicon chip bearing 128 microelectrodes for only nine outlets.

the chip according to the pattern shown in Fig. 3A. A first detection step was carried out with a biotinylated monoclonal antibody recognizing the region 34–39 of the ACTH (Mab 34 – 39 ) and followed by a streptavidin–phycoerythrin detection process (Fig. 3B). Addition of a Mab18 – 24 recognizing the region (18–24) followed by a revelation process leads to the results shown in Fig. 3C. Moreover, the use of a pyrrole–biotin ‘probe’ allows semi-quantification of the fluorescence level. These preliminary results show that the copolymerization process is compatible with peptide immobilization and with their immunodetection. This demonstrates the possibility of the construction of peptide-microarrays.

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Fig. 2. HCV genotyping: (A) pattern of distribution of the probe on the microelectrode array. pp, polypyrrole homopolymer; G, T1, T2, polypyrrole-bearing ODN probes for HCV genus, Type 1 and Type 2, respectively. (B) Fluorescence results of hybridization of an amplified clinical sample containing HCV type 1.

4. Conclusion The process of pyrrole copolymerization with biomolecules leads to the synthesis of polymer films bearing biological molecules that can be addressed on the surface of a given electrode belonging to an array of microelectrodes. The easy electrosynthesis of polypyrrole films and their high chemical stability

Fig. 3. Peptide detection: (A) pattern of peptide distribution. a, polypyrrole homopolymer; b, c, d, polypyrrole-bearing peptide 18 – 39 ; g, h, polypyrrole-bearing peptide 11 – 24. (B) and (C) Fluorescence results of immunodetection with biotinylated Mab(34 – 39) followed by detection with biotinylated Mab(18 – 24).

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makes these materials very attractive candidates for biological arrays. The robustness of the copolymerization procedure allows the synthesis of welldefined copolymers (composition, thickness, etc.) including a pre-purified biological molecule. Moreover, the very mild coupling conditions (water at pH | 7) allow the copolymerization of many biological molecules, resulting in a highly versatile arraying process. With this methodology, the need to synthesize all of the products to be arrayed leads to a limitation of the number of parameters to be analyzed. However, as pointed out elsewhere [6], this kind of chip involving prepurified materials allows for better reproducibility of the biochip preparation and then, an easy interpretation of the fluorescence results. Integration of such a bio-array in a total analysis system would be possible through the use of micro-devices such as those described recently [7]. Routine syntheses are carried out on 50 pm microelectrodes. Due to its high dimensional resolution, further miniaturization would be reached via reduction of the electrode size, leading to the exciting field of biorecognition on very small surfaces [8].

Acknowledgements We thank the CEA / DRFMC for helpful support. This work was supported in part by the Ministere de l’Industrie, France (grant 95.493.0183).

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