Synthetic Metals 161 (2011) 812816

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Synthetic Metals
journal homepage: www.elsevier.com/locate/synmet

Microwave assisted click chemistry on a conductive polymer lm

Anders E. Daugaard a , Thomas S. Hansen b , Niels B. Larsen b , Sren Hvilsted a,
a b

Danish Polymer Centre, Department of Chemical and Biochemical Engineering, Technical University of Denmark, Building 227 Sltofts Plads, DK-2800 Kgs. Lyngby, Denmark Department of Micro- and Nanotechnology, Technical University of Denmark, DTU Nanotech, Frederiksborgvej 399, 4000 Roskilde, Denmark

a r t i c l e

i n f o

a b s t r a c t
Microwave (MW) irradiation has been used to accelerate the functionalization of an azide functional poly(3,4-ethylenedioxythiophene) lm by click chemistry. The absorption of MW energy by the conductive polymer has been exploited for localized activation of the reaction on the polymer surface. By use of an alkyne modied uorescein derivative the reaction conditions have been optimized in a conventional MW oven, enabling the use of different sizes of substrates. The optimization resulted in a reduction of reaction times of approximately 20 h to only 2 min for bulk lm functionalization. The method has been applied for anchoring of the chelating agent nitrilotriacetic acid (NTA) on the conductive polymer. The chelating linkage ability of NTA on the surface was investigated through a sandwich ELISA study conrming the selective bonding of a histidine tagged protein. 2011 Elsevier B.V. All rights reserved.

Article history: Received 3 November 2010 Received in revised form 24 January 2011 Accepted 3 February 2011 Available online 5 March 2011 Keywords: Conductive polymers Microwave reaction Click chemistry Poly(3,4-ethylenedioxythiophene) (PEDOT) Nitrilotriacetic acid (NTA)

1. Introduction During the last decades research on conductive polymers has been rapidly expanding, giving rise to many new applications in areas such as photovoltaic devices [1], light emitting diodes [2], sensors [3,4] and strain gauges [5]. In order to expand this area with new interesting materials, preparation of new monomers that can be functionalized prior to or after polymerization are required. One example of such a new monomer is our recently published click approach to functionalized poly(3,4-ethylenedioxythiophene) (PEDOT) by use of an azide functional monomer, 3,4-(1-azidomethylethylene)dioxythiophene (EDOT-N3 ) that can be polymerized under chemically oxidative conditions [6]. The polymer was post functionalizated through copper catalyzed Huisgen 1,3-dipolar cycloadditions of the azide polymer with alkynes (CuAAC) giving bulk functionalized lms in approximately 20 h. The modularity of the method was exploited to prepare conductive polymers with different surface properties, illustrating the versatility of the method as the many publications within the area of click chemistry also shows [710]. Simultaneously a similar approach to functionalization of PEDOT was published by Buerle et al. who prepared the same monomer and showed that it could be polymerized under electrochemically oxidative conditions [11].

Corresponding author. Tel.: +45 4525 2965; fax: +45 4588 2161. E-mail addresses: adt@kt.dtu.dk (A.E. Daugaard), thomas.steen.hansen@nanotech.dtu.dk (T.S. Hansen), niels.b.larsen@nanotech.dtu.dk (N.B. Larsen), sh@kt.dtu.dk (S. Hvilsted). 0379-6779/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.synthmet.2011.02.005

Long reaction times seem to be a general trend in lm functionalization by click chemistry, where reaction times of about 20 h or longer are quite common [1113]. The shortest reaction times are found for monolayers, where a few cases present reaction times of about 2 h [1416]. Films are most often functionalized through placement of a reaction mixture on top of the sample, which is left without agitation until the reaction has been completed. It is difcult to reduce these reaction times without immersion of the samples into a reaction bath, which results in the use of large amounts of reagents and could cause layered structures to delaminate. We have previously shown that the PEDOT-N3 can be functionalized using electrochemistry with high spatial resolution and in short time (15 min) using electrochemistry [17,18]. Due to the relative complex setup with counter electrodes and the need for a highly well-dened geometry, this approach is not suited for uniform functionalization of large lm areas in the cm2 range. In solution, the reaction time of CuAAC have been reduced by use of MW irradiation to heat reaction vessels at high temperature and pressure, giving reaction times on the order of minutes. There are many examples of applications of this methodology in solution [19,20], though only one example of the procedure on a surface [21]. In this case a monolayer on a small silicon substrate was reacted while immersed into a reaction mixture in a MW reactor. This setup limits the size of the substrate and requires a large amount of reactant. To our knowledge there are no reports of the use of MW irradiation for CuAACs on polymer substrates or surfaces. Here we present a setup, where an ordinary MW oven can be used to functionalize a conductive polymer through CuAAC within minutes. This allows the reaction of a conductive polymer lm on differently shaped and sized sub-

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strates of polymer or glass, with the use of only small amounts of reactants. 2. Experimental 2.1. General methods The conductivity was measured with a four-point probe (Jandel Engineering Ltd., Linslade, UK) connected to a four-point source meter (Keithley 2400, Cleveland, US). UV/vis spectra were acquired on a Perkin Elmer Lambda 5 spectrometer. XPS analysis was performed on a Thermo Fisher Scientic K Alpha (East Grinstead, UK) using monochromatized aluminium K radiation in a 400 m spot on the sample. Survey and high resolution spectra were acquired and analyzed using the manufacturers Avantage software package. Spectra were generally acquired with electron charge compensation in operation to avoid sample charging, except for a series of measurements to determine the detrimental effects of electron ooding on azide functional groups. A standard kitchen MW oven was used for the reactions. 2.2. Chemicals Chemicals except for Baytron C were acquired from Aldrich and were used as received unless otherwise specied. Baytron C was purchased from H.C. Starck. Methyl 2-(3-oxo-6-(prop-2-ynyloxy)xanthen-9-yl)benzoate (1) was prepared as described elsewhere [6]. 2.3. PEDOT-N3 functionalization through CuAAC, general procedure The reaction mixture was prepared from a solution of 1 (2.2 mg, 5.7 mol) in DMF (0.2 mL) mixed with a solution of CuSO4 (10 L, 0.1 M, 1 mol) and sodium ascorbate (20 L, 0.1 M, 2 mol) in DMF (0.1 mL). A PEDOT-N3 sample (2.5 cm2 exposed area) was reduced with an aqueous solution of sodium ascorbate (0.1 M) and the reaction mixture was placed on the reduced PEDOT-N3 (dark blue), and the sample was reacted in the MW oven at 800 W for 2 min. Subsequently the surface was rinsed with H2 O and DMF and nally dried with pressurized air. The lms were reoxidized by immersion in 10 mL of 10% Baytron C in water (approximately 4 w/w% Fe(III)tosylate) for 5 min at room temperature (light blue/orange), followed by rinsing with H2 O and drying with pressurized air. 2.4. Detection of his-tagged protein by Sandwich ELISA Holes with a diameter of 6 mm were cut in transfer adhesive using a CO2 laser and placed on the sample. These holes dene the active area. The NTA functionalized PEDOT (PEDOTNTA) [18] samples prepared by the standard method were treated with 100 mM ethylenediaminetetraacetic acid (EDTA) for 30 min followed by treatment with 200 mM NiSO4 for 30 min. The samples were then blocked with Bovine Serum Albumine (BSA) in phosphate buffered saline (PBS) for 30 min and then washed with PBS + 0.05% Tween. An aqueous solution containing 1 g/mL histagged Protein A/G in PBS with 0.1% BSA were then placed on the sample over night at 4 C. Protein A/G has the ability to bind most types of mammal immunoglobulin G (IgG). The samples were again thoroughly washed with PBS/Tween and then treated with a solution of 10 g/mL Horse Radish Peroxidase (IgGHRP) in PBS/0.1% BSA. 30 L of commercially available premixed H2 O2 /tetramethylbenzidine (TMB) solution was added to each hole forming a drop. After 10 min the TMB solution was transferred to a 96 well plate and mixed with 50 L 1 M H2 SO4 to stop the reaction. The adsorption was then measured in an ELISA reader.

3. Results and discussion MW chemistry is based on dielectric heating and therefore relies on the ability of the substrate or the reaction mixture to absorb energy and convert it to heat. In the presented approach the idea is that the conductive polymer can absorb the MW energy and convert it to heat at the place of reaction. The ability of conductive polymers to absorb MW energy is well documented and has been applied for electromagnetic interference shielding [22,23]. In addition to this, it has been shown by Tran et al. [24] that the MW absorption of a conductive polymer can be used to weld polymers together through localized heating in the welding zone. In the present case, an initial experiment with MW heating showed that the conductive PEDOT-N3 became too warm when exposed to MW irradiation for a few seconds, which resulted in cracking and destruction of the polymer lms. This is in accordance with Mathew et al. [25] who showed that conductive polymers generally absorb high amounts of energy from MW irradiation. The degree of absorption, and thus heating of the polymer, is related to the conductivity, dielectric constant, dielectric loss and absorption coefcients of the polymers. Polymers with lower conductivity generally absorb less energy than more conductive polymers. The prepared PEDOT-N3 lm is in its conductive state after polymerization and would therefore absorb a high amount of energy in the MW oven. By chemical reduction with a solution of sodium ascorbate the DC conductivity was reduced from approximately 60 S/cm to 0.2 S/cm. It is well known that conductivity increases with frequency, and therefore this reduction in the conductivity does not directly correspond to a reduction in conductivity at the MW frequency. However, conductive polymers with lower DC conductivity seem to have a lower conductivity at MW frequencies [25,26]. The reduction in DC conductivity was sufcient for the lm to remain intact under MW irradiation for several minutes. Test reactions were performed using an alkyne derivative of uorescein as shown in Scheme 1. Through chemically oxidative polymerization EDOT-N3 yields a lm of PEDOT-N3 on the polymer substrate (A in Scheme 1). This polymerization method facilitates the use of different types of substrates and in the experiments conducted here a copolymer of ethylene and norbornene (Cyclic Olen Copolymer COC) was used. In principle other polymer substrates would be applicable to the same procedures. Before reaction the PEDOT-N3 lm is reduced with sodium ascorbate, which caused a decrease in the lm conductivity to 0.2 S/cm (B in Scheme 1). The reagents are placed on the polymer lm and the reaction is performed under MW irradiation (C in Scheme 1). When the reaction has taken place the lm was reoxidized with Fe(III)tosylate to regain a conductivity of approximately 15 S/cm (D in Scheme 1). The procedure was tested with an alkyne functional uorescein as shown in Scheme 1(C). A sample investigated by XPS gave the results reported in Table 1. As reported earlier the azide degrades in the XPS during analysis, which gives rise to a certain reduction in the nitrogen signal. Other than this, the XPS result for the pristine PEDOT-N3 and the uorescein reacted sample correspond to the calculated response.
Table 1 XPS data from PEDOT-N3 and uorescein derivatized PEDOT (atom%). PEDOT-N3 calculated C O N S 56 18 18 8 PEDOT-N3 a 56.3 20.9 14.9 8.0 PEDOT-uorescein calc 73 17 7 3 PEDOTuorescein 69.7 21.0 6.1 3.2

a The analysis has been performed without charge compensation, in order to minimize the degradation of the azide. However, as earlier reported [6] some degradation is unavoidable, which gives rise to the reduced nitrogen content.

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Fig. 1. UV/vis data showing the extent of reaction through absorption of the reacted uorescein derivative at 450 nm on a 0.8 cm2 sample (100 L of reaction mixture). (A) UV/vis spectra of a sample prepared at 400 W after 2 and 6 min of irradiation. (B) Increased absorbance at 450 nm as an effect of reaction time and input power. Due to overheating the sample reacted for 6 min at 800 W has been omitted.

Scheme 1. Preparation of PEDOT-N3 and subsequent click reactions with the uorescein derivative. (A) The EDOT-N3 monomer is chemically oxidized to yield a uniform PEDOT-N3 lm with a conductivity of 60 S/cm. (B) The lm is reduced using sodium ascorbate in order to decrease the conductivity. (C) A reaction solution containing the alkyne modied uorescein is added and the sample is treated under MW irradiation facilitating the CuAAC reaction. (D) The sample is reoxidized to regain conductivity.

The uorescein reagent has earlier been used to monitor the extent of bulk reaction on PEDOT-N3 , where it has been conrmed that only reacted reagent remains in the lm [6]. Therefore UV/vis spectroscopy can be used for determination of the extent of reaction in the bulk lm with varied input powers and irradiation times as shown in Fig. 1(A) and (B). The UV/vis spectra clearly show that the uorescein derivative is present in the lm, which conrms that the reaction has taken place. It is clear from Fig. 1(A) that an increase in the irradiation time

from 2 to 6 min gives an increase in absorbance, which corresponds to an increase of the loading of uorescein in the product. To get an overview of a variation in irradiation time and input power the increased absorbance of a sample under varied conditions is plotted in Fig. 1(B). This overview illustrates how an increased irradiation time, as well as an increase in input power directly corresponds to an increase in the loading of uorescein. Unfortunately samples cannot stand irradiation for 6 min at the highest input power, therefore only the result of a 2 min irradiation has been included at 800 W. The amount of absorbed energy is dependent on the ratio of the reaction volume to the area of the sample, thus a sample with a smaller reaction volume would react faster. Since the degree of reaction is dependent on volume to area ratio it is necessary to optimize the reaction for the specic size of sample if complete bulk reaction is required. To illustrate this, the reaction was optimized at 800 W on a 2.5 cm2 sample with 200 L reaction volume to identify the shortest possible time needed in order to achieve full bulk reaction. The UVvis spectra from the optimization are shown in Fig. 2. After 2 min at 800 W the absorption has reached 1.3, which correlates directly to a bulk functionalized lm of the same size prepared by the conventional method after 20 h. Further variation of the reaction time shows some interesting effects. By repetition of the reaction for two times 1.5 min, only a slight increase compared to irradiation for 1 min was observed as shown above. It is interesting that the combined reaction time of 3 min only affords a slightly

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Fig. 2. UV/vis spectra, showing the absorption from uorescein in 2.5 cm2 samples (200 L reaction mixture) reacted at 800 W with varied reaction time.

Fig. 3. Absorption of oxidized TMB on a pristine PEDOT compared to a PEDOT-NTA prepared under equivalent conditions.

higher loading compared to irradiation for 1 min. This shows that there is a substantial initiation delay before the reaction is accelerated. An explanation could be that the conductive polymer warms up the solvent on the lm surface and at a certain temperature the reaction is conducted at a much higher rate. A reference reaction was carried out on a standard hot-plate, and this conrmed that an increase in rate could be obtained through conventional heat transfer. It was unfortunately not possible to measure the exact reaction temperature as the reaction mixture cools down rapidly after reaction. If the temperature at the reaction site becomes sufciently high, the reaction that takes place could be a purely thermal cycloaddition. This was investigated with a blank sample prepared as the others but without copper. As can be seen in Fig. 2 MW irradiation for 3 min at 800 W resulted in no increase in the UV/vis absorbance of the blank sample. This conrms that the reaction occurring is a CuAAC and not just a thermal cycloaddition. However, it is not possible to investigate the regioselectivity as the conductive polymer is insoluble. In addition to the presented uorescein derivative a number of commercial alkynes are now available, which enables a large number of different functional entities on the surface of the conductive polymer. Other alkyne derivatives have also been tested, e.g. a uorocarbon, an end-functional poly(ethylene glycol), a rhodamine derivative and different receptors. The combination of the conductive polymer and the variations possible in post functionalization has a large potential in development of a new sensor system. In pursuit of this application a number of different alkyne

functional receptors including crown ethers and biotin have been tested with only moderate success. Lately a NTA functionalized PEDOT (PEDOT-NTA) was prepared and this has been tested for the ability to selectively bond histidine tagged proteins as sketched in Scheme 2. Prepared PEDOT-NTA was exposed to Nickel ions, which enables the complex to selectively bond histidine tagged proteins on the surface. In order to prevent unspecic bonding a number of washing steps are included in the process. After preparation of the surface it was exposed to a solution containing a histidine tagged Protein A/G (antigen). In order to detect the amount of immobilized Protein A/G the sample was exposed to Horse Radish Peroxidase (IgGHRP), which was used to catalyze the oxidation of tetramethylbenzidine with hydrogen peroxide. After termination of the oxidation the amount of oxidized TMB can be detected by UV/vis spectroscopy and this corresponds to the presence of IgGHRP and thereby histidine tagged protein. The result is shown in Fig. 3. There is a clear difference between the two samples, though the sample without NTA still shows some absorption from oxidized TMB. This is due to unspecic adsorption of the histidine tagged protein. Histidine tagged proteins are known to adsorb to hydrophilic surfaces, and in this case the protein probably adsorbs to the edge of the transfer adhesive, which was used to control the exposed area. The NTA reacted surface does however have a significantly higher amount of Protein A/G as a result of the NTANickel complex on the polymer surface. A detection scheme based on the conductive polymer is currently under investigation.

Scheme 2. Schematic of sandwich ELISA on PEDOT-NTA, for detection of selective immobilization of histidine tagged Protein A/G.

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4. Conclusion In conclusion we have shown that it is possible to use an ordinary MW setup to accelerate click reactions on a conductive polymer surface. The method was investigated, and a correlation between loading and input power was found. Optimization of the setup gave a reduction of the reaction time from >20 h to only 2 min. There was some indication of a short delay before the reaction could be accelerated, which is believed to be due to heating of the reaction media on the sample. The decreased reaction time observed here substantially reduces the preparation time of functional conductive polymer surfaces. As a proof of concept, the selective immobilization of a histidine tagged Protein A/G on a PEDOT-NTA surface has been shown through a sandwich ELISA study. Acknowledgement The Danish Research Council for Technology and Production Sciences (through the framework program Design and Processing of Polymers for Microuidic Applications, grant 26-04-0074) is thanked for nancial support. References
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