THE ULTRATHIN POLYMER FILMS BASED ON POLYANILINE AS SPR SENSITIVE DIAGNOSTIC SYSTEMS

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1 THE ULTRATHIN POLYMER FILMS BASED ON POLYANILINE AS SPR SENSITIVE DIAGNOSTIC SYSTEMS ISAKOVA Alexndr 1, OMELCHENKO Olg 1, GARINA Ekterin 2, INDENBOM Andrey 1,3 1 A.N. Frumkin Institute of Physicl Chemistry nd Electrochemistry of RAS, Moscow, Russin Federtion iskov_leks@list.ru 2 N. F. Gmley FRCEM, Moscow, Russin Federtion 3 Moscow Institute of Physics nd Technology (Stte University), Moscow region, Russin Federtion Astrct In the present work the use of electrochemiclly synthesized films of polyniline (PANi/HCl) nd its complexes with polycids (PANi/PSSA nd PANi/PAMPSA) is proposed s sensing elements of SPR chips. Adsorption of influenz viruses nd ntiodies to them t the synthesized thin films of PANi ws studied. It is estlished tht these processes occur with higher rte on ultrthin films of PANi with polycids. It ws shown tht ntiodies to influenz viruses wekly ind to the surfce of stndrd PANi nd the rection "ntigen-ntiody" leds to desorption of the immunocomplex. The use of PANi/PAMPSA films provides more efficient inding with ntiodies due to their chemicl nture, moleculr structure nd rugged surfce. Keywords: SPR, influenz virus, conducting polymers, polyniline 1. INTRODUCTION Dignostics of viruses nd the development of new mterils for sensitive elements of sensors is one of the topicl prolems in modern iotechnology. One of the most promising mterils for sensors re conducting polymers, including polyniline. The chemicl structure of polyniline presents repeting N- phenyl-p-phenylenedimine nd quinondiimine locks, thus showing greter ffinity to proteins. PANi cn e esily synthesized on the surfce of the electrodes of the vrious nture, nd its physico-chemicl properties llow to crete devices with different type of registrtion response to protein inding (electrochemicl, opticl or impedimetric sensors). Erlier we estlished the fct tht polyniline is le to interct with influenz viruses [4]. In the present work we hve synthesized thin films of new complexes of polyniline with polysulfonic cids nd pplied them to detection of influenz viruses y surfce plsmon resonnce (SPR). 2. EXPERIMENTAL Polyniline (PANi) ws synthesized y electropolymeriztion of niline in queous solutions of polysulfonic cids on gilded SPR chips. Polycids of different structure: poly-2-crylmido-2-methyl-1- propnesulfonic cid (PAMPSA) (Aldrich, #CAS , Mw ~ 2, 15% queous solution); poly- 4-styrenesulfonic cid (PSSA) (Aldrich, CAS # , Mw ~ 75, 18% queous solution) ws used for the electrochemicl synthesis of PANi. The concentrtion of the polymeric cid in the solution ws 5 mm nd concentrtion of niline ws 25 mm. For comprison, we lso synthesized the film of "stndrd" PANi in 1 M HCl. The procedures of solution preprtion for the synthesis of PANi nd the film deposition on the sustrte were descried in [1]. The AFM investigtion ws crried out with AFM model Enviroscope nd controller Nnoscope V (Bruker). SPR studies were crried out using SPR refrctometer Biosuplr 6 (Mivitec, Germny) in flt two-chmer thermosttic flow cell ttched to gilded surfce of the chip. Influenz viruses nd ntiodies to them ws provided y NF Gmley FRCEM

2 3. RESULTS AND DISCUSSION 3.1. SYNTHESIS AND PROPERTIES OF THIN FILMS OF PANI Electrochemicl synthesis of PANi films ws crried out in the presence of polycids represented in Figure 1. ) ) Fig. 1 The structures of the monomer unit PAMPSA () nd PSSA (). The formtion of ultr-thin PANi films in the presence of PAMPSA nd PSSA, s well s the "stndrd" PANi films registered y growth of chrge coming to the working electrode. It hs een found tht the synthesis of PANi proceeds fster in the presence of polymeric cids, despite low concentrtion of regents. This is direct consequence of the ssocition of niline t polymer chin of these cids [1-3]. It hs een found tht the synthesis of PANi in the presence of PAMPSA runs little slower thn in the presence of PSSA. This my e due to high moleculr weight of PAMPSA nd the need to its proper sptil orienttion required for co-polymeriztion with polyniline. However, lmost the equl induction period ( sec.) ws oserved for ll types of cids. The set of these nd other dt otined erlier [1,3], indictes tht the film growth occur proly in the sme kinetic conditions. It cn e ssumed tht the properties of the films will vry nd depend on the type of used cids. Indeed, Figure 2 shows tht the morphology of the AFM films PANi-PAMPSA nd PANi-PSSA re different. Film PANi-PAMPSA hs 1.2 to 1.5 times rougher surfce compred to PANi-PSSA, with lrger individul grins (gloules). Fig. 2 AFM - investigtion of PANi films on gilded SPR chips: ) PANi-PAMPSA (Rq=1,84 nm); ) PANi- PSSA (Rq=1,59 nm) It ws found tht the SPR response of the films chnges with ph (Fig.3). This dependence hs chrcteristic S-shpe during step chnge of ph. This reltionship ws more smooth without the chrcteristic inflection point for the PANi-HCl film. This ehvior ppers, occurring due to structurl chnges in the films. Forwrd nd reverse ph dependency ws not the sme usully. Hysteresis ws

3 mplified with incresing of film thickness, nd decresing with the exposure time t ech ph vlue. However, the position of the inflection point in the curves of SPR responses during increse of the ph hrdly chnged if the exposure time t ech point exceeded 1 minutes. The most drmtic chnges in the properties of PANi-PAMPSA nd PANi-PSSA films were oserved t ph ner PANi_PSSA ph ph 1 Fig. 3 Chnges in SPR signl of PANi films depending on the solution ph. )PANi-PAMPSA, ) PANi-PSSA. The otined films were susequently used to study the dsorption of iologicl ojects, such s viruses nd ntiodies to them A study of dsorption of iologicl ojects in the PANi films y surfce plsmon resonnce We hve previously found tht the powders of PANi nd its complexes ctively interct with influenz viruses [4]. Therefore, we investigted the dsorption of influenz viruses nd ntiodies to them on thin films of PANi nd their complexes y SPR technique. Fig. 4 shows the kinetics of dsorption of the influenz virus A/Texs/5/212(H3N2) depending on the hemgglutinting titre in solution PANi_PAMPS A 8 HAE 4 HAE 2 HAE Rte of dsorption, RU/min 3 15 PANi_PSSA t, min HAE Fig. 4 The kinetics of SPR response growth () nd the chnge in the initil rte of dsorption () depending on the hemgglutinting titre (HAE) of influenz virus A/Texs/5/212(H3N2) For low concentrtions of influenz virus (2 HAE) dsorption rte did not depend on the type of PANi film. At higher concentrtions of virus dsorption proceeded fster t the films of PANi-PAMPSA. One cn offer some explntion for such high ffinity for these films to influenz viruses. As follows from the structure of monomer units (Fig.1) PAMPSA contins cronyl nd mino groups, which re close to proteins in nture, nd cple to mke hydrogen onds with them. In ddition PANi-PAMPSA hs more flexile nd moile structure tht contriutes to more close contct with iologicl ojects. Finlly, the

4 PANi-PAMPSA film hs greter surfce re, compred with PANi-PSSA (Fig. 2), which lso improves ility to dsor the virus prticles. In spite of significnt sorption signls of influenz virus, we could not chieve full coverge of the films surfce. As result, ntiodies where dsored not only on the viruses, ut lso on the free surfce of the film. Accordingly, it cn e registered dsorption of non-complementry ntiodies. Attempts to escpe surfce lyer tht ws not occupied y virus using BSA dsortion hs not yet yielded positive results. Therefore, we exmined nother method of registrtion of ntigen-ntiody interctions, offering to do sensitive proe on the sis of lyer of dsored ntiodies. They showed high ffinity for the surfce of the polymer films. The prospect of the iosensors sed on the films of PANi-PAMPSA with dsored ntiodies to influenz virus is determined y high ffinity of these polymers to proteins tht leds to full coverge of the sensor surfce y ntiodies. As result fter the complete surfce covering nother proteins nd other non-complementry ntiodies do not ound to the proe surfce nd do not distort the results of the determintion of ntigen-ntiody interctions with virl prticles. Figure 5 shows the kinetics of dsorption of ntiodies to the virus A/Texs/5/212(H3N2) on vrious polymer films. The speed of this process depended on the type of the film. For PANi-PAMPSA films the initil dsorption rte ws 3.5 times higher thn for PANi-HCl films. The resons for this is pprently the sme s for the dsorption of influenz virus (similrity of chemicl nture of the modifying cid, moility of its structure nd surfce roughness). For ll types of film, dsorption of ntiodies ws virtully irreversile. Wshing the cell with uffer solution did not led to pprecile desorption of the ntiodies PANi_HCl t, min 1 Fig. 5 Kinetics of dsorption of ntiodies to the influenz A/Texs/5/212(H3N2) virus on the films PANi- HCl (curve 1) nd PANi-PAMPSA (curve 2). C ntiody=,65 mg/ml The difference in the inding of different types of films with ntiodies to influenz virus ws demonstrted in study of susequent interction with the virus. It turned out tht fter the introduction of the virus solution short dsorption growth а considerle desorption of the pre-dsored ntiodies from PANi-HCl films ws oserved y SPR response (Fig. 6). Apprently, comprtively wek ond with the surfce nd the strong interction of virl prticles with the ntiodies resulted in desorption of immunocomplexes from the surfce. Wshing y uffer solution enhnces this effect.

5 9 desorption of "ntigen-ntiody" complex 85 norml sline 4HAE 8HAE 8 HAE influenz virus A/Texs/5/212(H3N2) norml sline t, min 1 Fig. 6 Sensogrm of interction of influenz virus A/Texs/5/212(H3N2) on SPR chips with PANi/ntiody sensor The formtion of immune complexes on the surfce of PANi-PAMPSA hs not een ccompnied y their desorption during the process of inding with virl prticles nd susequent wshing y the uffer solution due to higher ffinity of the polymer to the ntiodies. 4. CONCLUSION During the study, we hve shown tht films sed on PANi re promising for cretion of SPR sensors for influenz A viruses. The most convenient films re PANi-PAMPSA. Due to chemicl nture, moleculr structure nd rugged surfce they hve the highest ffinity to the ntiodies of influenz A. However, significnt effect of ph on the SPR response of these films in the region of ph 6.5 requires controlling the solution cidity nd the necessity to use uffer solutions. ACKNOWLEDGEMENTS This work ws prtilly supported y the RFBR grnt No _mol_ REFERENCES [1] Nekrsov A.A., Grikov O.L., Eremin T.V., Iskov A.A., Ivnov V.F., Tverskoj V.A., Vnnikov A.V. // Electrochim. Act. 28. V. 53. P [2] Grikov O.L., Nekrsov A.A., Iskov A.A., Ivnov V.F., Vnnikov A.V. // Russ. J. Electrochem., 26, Vol. 42, No. 1. P [3] Iskov A. A., Grikov O. L., Nekrsov A. A., Ivnov V. F., Tverskoi V. A., nd Vnnikov A. V. // Protection of Metls. 28.V. 44, No. 6, P [4] Ivnov V.F., Ivnov V.T., Kurochkin Y.E., Grikov O.L., Ilyin M.V., Mnykin A.A., Iskov A.A., Spurin I.Yu. // AIP Conference Proceedings. 21. V Р.46-48