pubs.acs.org/Langmuir © 2010 American Chemical Society
Biomolecular Hybrid of Poly(3-thiophene acetic acid) and Double Stranded DNA: Optical and Conductivity Properties Pratap Mukherjee, Arnab Dawn, and Arun K. Nandi* Polymer Science Unit, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700 032, India Received January 22, 2010. Revised Manuscript Received May 1, 2010 A new biomolecular hybrid of poly(3-thiophene acetic acid) (PTAA) and double stranded deoxyribonucleic acid (ds-DNA) is prepared. The transmission electron microscopy (TEM) images exhibit fibrillar network morphology making a nanostructured self-assembly of PTAA-DNA hybrid. The confocal fluorescence image of PTAA shows green fluorescence exhibiting agglomeration in the pure state but the spreading of green fluorescence over the network superstructure in the hybrids indicating the immobilization of PTAA on DNA surfaces. Fourier transform infrared (FTIR) spectra indicate hydrogen bonding between -COOH groups of PTAA and PdO groups of Na-DNA. Circular dichroism (CD) spectra denote that DNA conformation remains unaltered during hybrid preparation. A blue shift of the π-π* absorption peak of PTAA in the hybrid solutions occurs with aging time. The photoluminescence intensity in the hybrid solution increases with a concomitant blue shift of the emission peak with aging time, and it is faster with increased DNA concentration. Possible reasons of different optical behavior are discussed in the light of duplex and triplex hybrid formation. Dynamic light scattering study indicates an increased particle size of PTAA with addition of DNA favoring the hybrid particles to remain in solution. The dc-conductivity of the hybrids decreases from that of PTAA with an increase of Na-DNA concentration, and the current (I )-voltage (V ) curves indicate a semiconducting nature of the hybrids.
1. Introduction Recently, considerable research interest in optical and electrochemical deoxyribonucleic acid (DNA) detection/hybridization sensors, bioelectronics, and bioactive communication systems has grown to understand and to monitor the biological processes.1-20 The discovery of conducting polymers plays an important role, as the electroactive nature of these polymers helps to design bioactive communication systems and biosensors. In our previous works, we reported poly(o-methoxy aniline)-DNA hybrid systems which are good semiconductors and may be useful in *To whom correspondence should be addressed. E-mail psuakn@ mahendra.iacs.res.in. (1) Wallace, G. G.; Kane-Maguire, L. A. P. Adv. Mater. 2002, 14, 953. (2) Nilsson, K. P. R.; Herland, A.; Hammarstrom, P.; Inganas, O. Biochemistry 2005, 44, 3718. (3) Cougnon, C.; Gautier, C.; Pilard, J.-F.; Casse, N.; Chenais, B. Biosens. Bioelectron. 2008, 23, 1171. (4) Prevette, L. E.; Kodger, T. E.; Reineke, T. M.; Lynch, M. L. Langmuir 2007, 23, 9773. (5) Asberg, P.; Nilsson, K. P. R.; Inganas, O. Langmuir 2006, 22, 2205. (6) Dawn, A.; Nandi, A. K. Macromol. Biosci. 2005, 5, 441. (7) Dawn, A.; Nandi, A. K. Macromolecules 2005, 38, 10067. (8) Dawn, A.; Nandi, A. K. Langmuir 2006, 22, 3273. (9) Dawn, A.; Nandi, A. K. J. Phys. Chem. C 2007, 111, 6268. (10) Lokshin, N. A.; Sergeyev, V. G.; Zezin, A. B.; Golubev, V. B.; Levon, K.; Kabanov, V. A. Langmuir 2003, 19, 7564. (11) Nagarajan, R.; Liu, W.; Kumar, J.; Tripathy, S. K.; Bruno, F. F.; Samuelson, L. A. Macromolecules 2001, 34, 3921. (12) Ho, H.-A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore, K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002, 41, 1548. (13) Ho, H.-A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168. (14) Satz, A. L.; Bruice, T. C. J. Am. Chem. Soc. 2001, 123, 2469. (15) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 1942. (16) Nilsson, K. P. R.; Inganas, O. Nat. Mater. 2003, 2, 419. (17) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 896. (18) Gibbs, J. M.; Park, S.-J.; Anderson, D. R.; Watson, K. J.; Mirkin, C. A.; Nguyen, S. T. J. Am. Chem. Soc. 2005, 127, 1170. (19) Thompson, L. A.; Kowalik, J.; Josowicz, M.; Janata, J. J. Am. Chem. Soc. 2003, 125, 324. (20) Korri-Youssoufi, H.; Garnier, F.; Srivastava, P.; Godillot, P.; Yassar, A. J. Am. Chem. Soc. 1997, 119, 7388.
Langmuir 2010, 26(13), 11025–11034
developing bioelectronics.6-9 Here, we report the preparation and properties of a DNA-poly(thiophene acetic acid) (PTAA) hybrid, whose optical and conducting properties might be useful for DNA hybridization, gene therapy, and DNA diagnostic study. Poly(3-alkyl thiophenes) (P3ATs) are important conducting polymers because of their solubility and fusibility.21,22 Replacing the alkyl substituent by a carboxylic acid group, the utility of the polymer may increase significantly to produce a biomolecular hybrid.23,24 The most important member in this family is PTAA (Scheme 1), and its anion is reported to bind with many biomolecules such as insulin, lysozyme, and so on.2 The conformational change in biomolecules induces conformational change of PTAA, altering its optical properties (e.g., UV-vis, PL, etc.).5,24 Thus, PTAA and other ionomers of polythiophenes may be useful to prepare good biosensors and to produce various biochips.5,12,13,24 Here, we report the change in physical, optical, and conductivity properties of PTAA-DNA hybrids. PTAA is a weak acid (the pKa value of this acid is 5.53 and 5.33 in 0.2 and 1 M NaCl solutions, respectively)23 where a major part of the -COOH group remains undissociated in water and would be available for H-bonding with the PdO group of the DNA anion. This interaction may therefore make it immobilized on the DNA periphery. Previously, ionic interaction between a DNA anion and a cation of a polythiophene derivative was used by Leclerc and co-workers12,13 to make the hybrid. They observed a red shift in π-π* absorption peak of the UV-vis absorption spectra for the duplex formation of single stranded (ss)-DNA and polythiophene (21) Roncali, J. Chem. Rev. 1992, 92, 711. (22) McCullough, R. D.; Ewbank, P. C. In Handbook of Conducting Polymers, 2nd ed.; Skotheim, T. A., Elsenbaumer, R. L., Reynolds, J. R., Eds.; Marcel Dekker: New York, 1998; p 225. (23) Kim, B. S.; Chen, L.; Gong, J. P.; Osada, Y. Macromolecules 1999, 32, 3964. (24) Nilsson, K. P. R.; Rydberg, J.; Baltzer, L.; Inganas, O. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10170.
Published on Web 05/25/2010
DOI: 10.1021/la101215v
11025
Article
Mukherjee et al. Scheme 1. Poly(3-thiophene acetic acid)
conducting polymer may uncoil/coil slowly as observed in the poly(o-methoxy aniline)-DNA (POMA-DNA) system.6-9 Here, we present the morphology from transmission electron microscopy (TEM) and confocal fluorescence microscopy studies to understand the hybrid formation. The UV-vis and PL properties are investigated in detail to observe any conformational change of PTAA occurring on the DNA surface. Any change in optical property might be useful for developing biosensors. The conductivity and current-voltage (I-V) characteristics of the complexes are reported to make an idea for its probable application in bioelectronics and bioactive communication system.
2. Experimental Section
cation; however, in the triplex formation during the hybridization with another DNA molecule, they observed a blue shift. Photoluminescence (PL) quenching took place in the duplex formation, but PL enhancement occurred in the triplex formation. For the different mismatches in the series of oligonucleotides complexed with the cationic polythiophene derivatives, they also detected changes in UV-vis and fluorescence spectra. Thus, the above results indicate the importance and applicability of the polyplexes in the advancement of genomics and proteomics research. Further, the polycation-DNA complexes are reported to protect DNA from enzymatic degradation,25-28 and the polyplexes are shown to enter into cells via endocytosis.26,29,30 Thus, the polyplexes would be useful for the understanding of biological processes inside the cells through their optical properties.26,29 In this Article, we report the preparation and properties of complexes of double stranded (ds)-DNA and PTAA at three different compositions. In the polyplexes formed by strong ionic interaction as in polycation-DNA systems, the release of DNA is difficult during gene delivery.31,32 Here, we are interested to use nonionic interactions such as H-bonding/π-π interaction in the polyplex formation, facilitating easy detachment of DNA in the process of gene delivery.33 PTAA is a weak electrolyte, so a major portion of carboxylic acid groups would remain un-ionized in the aqueous medium, making it available for H-bonding with phosphate groups of DNA. Also use of ds-DNA in the polyplex formation would be more realistic than using ss-DNA polyplexes to understand the biological processes accurately. In ss-DNA, more H-bonding sites are available than those in ds-DNA, so bonding with a conducting polymer would be higher. However, in living cells, ds-DNA is of longer existence than ss-DNA, which is produced from the ds-DNA only during replication, genetic recombination, and transcription processes. So we chose to work on ds-DNA to appropriately monitor the cell processes from the change of optoelectronic properties. Further, in the polyplex, the (25) Liu, Y.; Reineke, T. M. Bioconjugate Chem. 2006, 17, 101. (26) Nisha, C. K.; Manorama, S. V.; Ganguli, M.; Maiti, S.; Kizhakkedathu, J. N. Langmuir 2004, 20, 2386. (27) Gao, X.; Huang, L. Biochemistry 1996, 35, 1027. (28) Abdelhady, H. G.; Allen, S.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M. Nucleic Acids Res. 2003, 31, 4001. (29) (a) Vuorimaa, E.; Urtti, A.; Sepp€anen, R.; Lemmetyinen, H.; Yliperttula, M. J. Am. Chem. Soc. 2008, 130, 11695. (b) Ruponen, M.; Yl€a-Herttuala, S.; Urtti, A. Biochim. Biophys. Acta 1999, 1415, 331. (30) Mislick, K. A.; Baldeschwieler, J. D. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 12349. (31) Hardy, J. G.; Kostiainen, M. A.; Smith, D. K.; Gabrielson, N. P.; Pack, D. W. Bioconjugate Chem. 2006, 17, 172–178. (32) Kostiainen, M. A.; Smith, D. K.; Ikkala, O. Angew. Chem. 2007, 46, 7600– 7604. (33) Ma, H.; Chen, G. Nature and Science 2005, 3, 25–31.
11026 DOI: 10.1021/la101215v
2.1. Samples. Calf thymus DNA (type-1 sodium salt, Sigma Chemicals), 3-thiophene acetic acid (Aldrich), and anhydrous ferric chloride (E. Merck, Mumbai) were used as received. Chloroform (E. Merck, Mumbai) on keeping overnight over calcium chloride is refluxed with solid P2O5 for 6 h and is distilled. Methanol is distilled after drying over CaH2. 3-Thiophene acetic acid is esterified with dry methanol by refluxing for 24 h in the presence of concentrated H2SO4 (0.05 mL) to protect the oxidative decomposition of carboxylic acid groups during polymerization. The protected monomer is polymerized by oxidative polymerization using anhydrous FeCl3 in dry chloroform. The molar ratio of FeCl3 and monomer is 4:1, and the mixture is stirred for 24 h at 0 °C under N2 atmosphere.23 The polymer is poured into a large excess of methanol to precipitate poly(3-thiophene methyl acetate) (PTMA). The precipitate is washed with fresh methanol and deionized water. The PTMA is hydrolyzed by refluxing with 2 M NaOH solution for 24 h at 100 °C. The water-soluble part is neutralized with dilute HCl solution, and the precipitate is carefully washed with water and dried in vacuum at 60 °C for 3 days. Each step is characterized by 1H NMR spectroscopy. The molecular weight (M w) of the polymer (PTAA) is found to be 6900, and polydispersity index = 3.57, measured from gel permeation chromatography (Waters) in tetrahydrofuran (THF) at 30 °C. Polystyrene has been used as standard in the GPC measurements. The regioregularity of PTAA is measured from the 1H NMR spectra presented in Figure 1 in the Supporting Information. The peak at 7.26 ppm is considered for R protons of H-H linkages, and the peak at 7.31 has been considered for R protons of H-T linkages of thiophene rings.23 From the ratio of the peak area, the % H-T linkage is calculated and has the value 43.9 mol % for the present PTAA. 2.2. Hybrid Preparation. A 0.01% (w/v) Na-DNA solution is made in double distilled water by keeping at 30 °C for 24 h. The PTAA solution (0.01% w/v) is made by sonication for 30 min in a sonication bath (model AVIOC, Eyela, 60 W). They are mixed by taking appropriate volumes of polymer solution and Na-DNA solution to make the hybrids of components 3:1, 1:1, and 1:3 (by weight). The three mixtures are designated as PD31, PD11, and PD13, respectively. The mixtures are kept under spectroscopic and light scattering investigations for 15 days and are then freeze-dried. 2.3. Microscopy. TEM micrographs of the samples are made by dropping a drop of the above 15 days aged solution on a carbon coated copper grid and drying at 30 °C in air and finally in vacuum. Samples are then observed through a high-resolution transmission electron microscope (JEOL, 2010EX) operated at an accelerated voltage of 200 kV fitted with a CCD camera. The fibrillar thickness (t) values are measured using Image J software at 50 different positions. From the fibrillar thickness, the fibrillar √ diameter (d) is calculated using the relation d = 2t. The average values and standard deviations are measured using a statistical program. For confocal fluorescence microscopy studies, the PTAA/ hybrid solutions (15 days aged) are drop-cast on microscopic glass slides and dried at 30 °C. These are then finally dried in a Langmuir 2010, 26(13), 11025–11034
Mukherjee et al.
Article
Figure 1. TEM micrographs of (a) calf thymus DNA [Na-salt form], (b) PTAA, and (c) DNA-PTAA hybrid (PD11). vacuum-desiccator at 30 °C. The samples are then covered by glass coverslips and are observed through a Leica Confocal microscope (model-TCS-SP2) using a 63 lens. 2.4. Spectroscopy. The circular dichroism (CD) spectra of the aqueous solutions of Na-DNA, PTAA, and the hybrids are studied in a spectropolarimeter (JASCO, model J-815) in a 1 cm quartz cuvette at 30 °C. The UV-vis spectra are taken in aqueous solutions of the hybrids for different aging times from 190 to 1100 nm using a UV-vis spectrophotometer (Hewlett-Packard, model 8453) at 30 °C. The Fourier transform infrared (FTIR) spectra of the freeze-dried samples are obtained using a Nicolet FT-IR instrument [Magna IR-750 spectrometer (series II)]. KBr pellets of the solid samples are made to run the scan. Subtraction of PTAA spectra from the DNA-PTAA hybrid spectra is done using the Omnic software attached to the instrument to understand the interaction between PTAA and DNA. Photoluminescence (PL) study of the hybrid solutions is performed using a quartz cell of 1 cm path length with a Horiba Jobin Yvon Fluoromax-3 instrument. The solutions are excited at 370 nm, and the emission scans are taken from 385 to 725 nm using a slit width of 3 nm with an increment of 1 nm wavelength having an integration time of 0.1 s. Fluorescence lifetimes are determined from time-resolved intensity decays by the method of time correlated single photon counting (TCSPC) using a picosecond diode laser of 403 nm (IBH, UK, nanoLED-07) as the light source and TBX-04 as the detector. The decay curves are analyzed using IBH DAS-6 decay analysis software. The goodness of fit is evaluated by χ2 criterion and the randomness of the residuals. Mean fluorescence lifetimes (τf) for biexponential iterative fitting are calculated from the decay times (τi) and the relative amplitudes (ai) using the following relation
conducting strips of 1 mm width placed perpendicularly.34 The sandwiching of the samples between the two ITO strips is made under pressurized conditions by holding glass slides with four springed clips to make the contact best. The thickness of the samples is measured by using a screw gauge. The area of the samples is 0.01 cm2. The conductivities of the sandwiched samples are measured with an electrometer (Keithley, model 617).34 The dc conductivity of PTAA obtained by the above technique is verified by casting a film from dimethyl sulfoxide (DMSO) solution on thoroughly cleaned glass substrate. Then gold was deposited on the top of the film by masking the middle of the top to produce the two lateral electrodes. The conductivity value in the undoped state is then measured by the two-probe method. The conductivity value at 30 °C is found to be 2.22 10-8 S/cm which is close to the value reported by the sandwich method (1.02 10-8 S/cm). The conductivity measured by this technique was also verified earlier with poly(3-hexyl thiophene) film using two-probe method.34 So the sandwich technique of conductivity measurement between two ITO electrodes yields satisfactory results. The PTAA and PTAA/DNA hybrid do not produce good films from aqueous solutions due to the low solubility of PTAA, and the use of organic solvents would denature DNA. Under this restriction, the conductivity measurements of hybrids are made using the above sandwich technique between two ITO electrodes which is proven to yield satisfactory results. For current-voltage (I-V) measurements, the same sandwiched samples are used with the same electrometer by applying voltages between þ5 and -5 V. And the I-V plot of PTAA obtained by the lateral method is found to be similar to that of the above procedure.
τf ¼ a1 τ1 þ a2 τ2
3. Results
ð1Þ
2.5. Dynamic Light Scattering Studies. The effective particle size of pure PTAA and its hybrids with DNA in aqueous medium (concentration 0.01% w/v) are measured at different aging times using a dynamic light scattering (DLS) instrument (Brookhaven Instruments Corporation, Model BI-APD). The temperature of DLS measurement is 30 °C. The scattering intensity is plotted with particle diameter to get the size distribution curve which shows the particle size distribution of scattering objects. The maximum intensity in the Gaussian type of scattering plot corresponds to the effective particle diameter. 2.6. Conductivity Measurements. The dc conductivities of the freeze-dried samples are measured by a two-probe method by sandwiching the samples between two indium-tin oxide (ITO) Langmuir 2010, 26(13), 11025–11034
3.1. Morphology. The hybrid morphology can be clearly understood from the TEM micrographs presented in Figure 1 and Supporting Information Figure 2. The fibrils of Na-DNA (Figure 1a) have average diameter of 14.5 ( 0.8 nm, forming a network structure, and the PTAA sample has irregular spheroidal morphology (Figure 1b). The fibrillar network structure is present in all the hybrids, but the network density is somewhat lower than that of pristine Na-DNA. The average fibrillar diameter values are 23.6 ( 6.5, 26.8 ( 6.4, and 36.0 ( 8.3 nm for PD13, PD11, and PD31, respectively. Though the uncertainty in the diameter values is somewhat large, certainly there is an increase in average fibrillar (34) Dawn, A.; Nandi, A. K. J. Phys. Chem. B 2006, 110, 18291.
DOI: 10.1021/la101215v
11027
Article
Figure 2. Schematic model of PTAA/Na-DNA hybrid (PD11) fibril. Direction of arrows indicates direction of charge transfer.
diameter of the hybrid with increasing PTAA concentration. The reason for the increase of fibrillar diameter with increasing PTAA concentration may be that the DNA interacts with PTAA chains and larger numbers of PTAA chains can self-assemble in the PTAA rich hybrids on the DNA periphery, producing thicker fibrils. A model for the nanostructured self-assembly can be given based on the quantitative estimation of the experimental results obtained from TEM micrographs.35 The fibrillar nature of the PTAA-DNA hybrids drives us to choose a nanostructured selfassembly for a circular cross section of the hybrid fibrils. The appearance of the fibers is same except they are fatter and their density is lower than that of DNA because of dilution with PTAA. In the solution, PTAA gets adsorbed on the outer periphery of the DNA double helix and then this conjugate pair produces fibrils of larger diameter than that of pure DNA. The mode of fibril formation in the two cases may differ to some extent, but it may be approximated that the mode of hybrid fibril formation is very much like that of DNA because of similar morphology. So one scenario that is consistent with the electron microscopic data is presented in an approximate model (Figure 2), and it may be argued that each ds-DNA self-assembles with 13 PTAA chains and 37 such self-assemblies aggregate to produce the hybrid fibril in the PD11 system.35 However, the other hybrids cannot be represented by a single structured model. In PD13 and PD31 hybrids, 9 and 28 PTAA chains self-assemble with single ds-DNA, respectively, and 37 such self-assemblies aggregate to produce the hybrid fibril. There might be some other models which may also (35) The number of PTAA chains surrounding a DNA double helix is calculated from the cross-sectional area of the PTAA chain (0.785 nm2 determined from molecular mechanics model (Supporting Information Figure 3) and cross-sectional area of B-DNA (4.41 nm2) (9). In each Na-DNA fibril (diameter 14.5 nm), there are 37 ds-DNA. The PTAA contribution of cross-sectional area in the hybrid is calculated by subtracting the cross-sectional area of the DNA fibril from that of each hybrid. Dividing this contribution by the cross-sectional area of a PTAA chain, we obtain the number of PTAA chains in the hybrid fibril. Assuming that 37 ds-DNA are also present in the hybrid fibril, the number of PTAA chains selfassembled with each ds-DNA has been calculated by dividing number of the PTAA chains with 37.
11028 DOI: 10.1021/la101215v
Mukherjee et al.
fit, but we think that the present model gives the best fit of our experimental outcome. In the model (Figure 2), it is apparent that DNA is surrounded by many PTAA chains at a cross section point, but these PTAA cross sections may originate from the PTAA chains converging from different directions toward the DNA. At a point, only one PTAA chain is interacting through H-bonding but not the others which are interacting at different points on the outer periphery of ds-DNA. Since polyplex formation is a self-assembly phenomenon of the DNA double helix and PTAA chains (both having nanometer size cross sections), so it may be called nanostructured self-assembly. In Figure 3, the confocal fluorescence images of PTAA and PD31 hybrid are presented. The PTAA exhibits fluorescent green spots (Figure 3a), but the hybrid exhibits a green fluorescent network structure (Figure 3b). This green network structure is also observed in the PD11 and PD13 hybrids (Supporting Information Figure 4). PTAA has a good fluorescence property, and the green spots in Figure 3a indicate that the PTAA remains in an agglomerated state. However, in the hybrids, the green fluorescence spreads throughout the whole space, showing a fibrillar network structure of DNA. The green fibrillar network superstructure is very clearly observed in the PD31 hybrid, and due to the increasing network density with increasing DNA concentration the green fluorescence spreads isotropically and uniformly throughout the space for PD11 and PD13 hybrids. This spreading of the fluorescent PTAA on the surface of DNA fibrils suggests that there is certainly some interaction present between DNA and PTAA. Thus, PTAA achieves the morphology of a DNA network due to its immobilization on DNA making the hybrid more fluorescent (Figure 3b) than that of pure PTAA (Figure 3a). 3.2. FTIR Study. To ascertain the nature of interaction, the FTIR spectra of Na-DNA and the PTAA subtracted PD31 and PD11 hybrids are presented in Figure 4. The PdO stretching vibration peak of pure Na-DNA (1238 cm-1)4 shifts to lower wavenumber (1220 cm-1) in the hybrids. The shift to lower energy indicates lowering of the bond order of the PdO bond, and it may occur from H-bonding interaction of the PdO bond through the H atom of the undissociated -COOH group of PTAA. However, we have not observed any change in the peak position of the O-H stretching frequency (3433 cm-1, Supporting Information Figure 5) because PTAA itself remains H-bonded (inter/intramolecularly) through the -OH group of carboxylic acid.23 So this result clearly indicates that a new intermolecular H-bonding interaction between PTAA and Na-DNA molecules is acting in the blends. The H-bonding interaction, though weaker than ionic interaction with cationic polythiophene derivatives,13 is certainly effective to make the PTAA-DNA hybrid, as is evident from the confocal fluorescence microscopy images (Figure 3). 3.3. CD Spectroscopy. The ellipticity versus wavelength plot of the CD spectra of the 15 days aged sample solutions are presented in Figure 5, and it is apparent that Na-DNA and its hybrids with PTAA have almost similar patterns; that is, the nature of the CD spectrum of Na-DNA does not change with mixing. The present CD spectrum of Na-DNA shows that DNA corresponds to a B polymorphic structure.36 In the hybrids, the polymorphic nature remains the same, indicating no conformational change of DNA due to the hybrid formation. In Figure 5, the CD spectrum of PTAA is also shown exhibiting the absence of a chiral center in it. 3.4. UV-vis Spectra. In Figure 6a and b, the UV-vis spectra of PTAA and PD11 hybrid are shown for different aging (36) Sprecher, C. A.; Baase, W. A.; Johnson, W. C. Biopolymers. 1979, 18, 1009.
Langmuir 2010, 26(13), 11025–11034
Mukherjee et al.
Article
Figure 3. Confocal fluorescence microscopy images of (a) pure PTAA and (b) PD31 hybrid.
Figure 4. FTIR spectra of Na-DNA and PTAA subtracted PTAA-DNA hybrids (PD31 and PD11).
Figure 5. CD spectra of calf thymus DNA [Na-salt form], PTAA, and DNA-PTAA hybrid (PD31, PD11, and PD13) solutions.
times. The 262 nm peak of the DNA base pairs remains unchanged in position and in intensity (Figure 6b), indicating DNA is not denatured during the hybrid formation. However, the UV-vis peak of the π-π* transition band of the polymer changes with time, showing a blue shift of the band from 429 to ∼365 nm (inset of Figure 6b), and this is also true for the other two hybrids (Supporting Information Figure 6). This is an interesting observation and a slower and lower blue shift of π-π* transition band (429 to 403 nm) is also observed in pure PTAA solution (Figure 6a) having the same PTAA concentration as that in PD11 solution. The blue shift of the π-π* transition band of Langmuir 2010, 26(13), 11025–11034
PTAA with aging time is compared in Figure 6c for all the hybrids and for pure PTAA solution having the same concentration as that in PD11 hybrid solution. It is apparent that the PD13 system shows a faster blue shift than those of PD11, PD31, and pure PTAA systems; the blue shift is highest in the DNA rich hybrid. This effect is quite opposite in nature to that observed in POMADNA hybrids where a sigmoidal increase of λ versus log(time) was observed.6-9 A possible explanation of the blue shift is that the effective conjugation length of the polythiophene chain decreases with increasing aging time and it may occur due to the twisting of PTAA chains arising from conformational transition along the backbone. The different rates of the blue shift may arise from the different rates of conformational change of PTAA chains anchored from the DNA surface. In pure PTAA solution, intra/ intermolecular H-bonding might be possible, causing twisting of PTAA chains and resulting in a blue shift.23 However, such a possibility is lesser in the case of the PTAA-DNA hybrid, as the hydrogen atom of the carboxylic acid group of PTAA would preferably attach to the more electronegative oxygen atom of the phosphate group of DNA due to the higher polarizability of phosphorus than that of carbon. Once a -COOH group of PTAA is hydrogen bonded with the phosphate group of DNA, the PTAA chain goes through a strained rotation along the single bond between the thiophene rings. It takes a longer time for completion of the process, causing a slow twisting of the polymer backbone and resulting in slow blue shift with aging time. Thus, the complete binding of PTAA and DNA is a very slow process. The slowness is dependent on the DNA concentration, and a higher DNA concentration makes the conformational change of PTAA faster because of an increased choice of interacting sites. In pure PTAA solution, the blue shift is lesser and slower because thermal fluctuations hamper the stable conformational transition, resulting in lower twisting of the backbone chain than that in the hybrids. In the case of the POMA-DNA system, the repulsion between radical cations of attached POMA chains with DNA was the cause for uncoiling of POMA chains to show red shifting with time.6-9 Thus, it may be argued that the attachment of polymer chains on the DNA periphery may be an easy way to study the segmental rotation of polymer chains resulting in uncoiling/ coiling. DNA chains have a high molecular weight and very large surface area causing absorption of polymer chains. Due to the DOI: 10.1021/la101215v
11029
Article
Mukherjee et al.
Figure 6. UV-vis spectra of (a) pure PTAA solution of the same concentration as that in PD11 hybrid solution for different aging times and (b) PTAA-DNA hybrid solution (PD11) at different aging times. In the inset of (b), the shift of the 429 nm peak of PTAA in the PD11 hybrid to lower wavelengths with aging time is shown. (c) Plot of wavelength of absorption maximum of π-π* transition of polythiophene chain versus aging time in PD31, PD11, and PD13 hybrids and pure PTAA solution of the same concentration as that in PD11 hybrid.
attachment with higher weight species (DNA), the effect of thermal fluctuations on the segmental conformational changes of the polymer is reduced, facilitating detection of such fine conformational properties of polymer chains. In the PTAAinsulin and PTAA-lysozime systems, a similar blue shift was observed.2,5 However, there the reason is different because the addition of proteins to PTAA causes polyplex formation and the conformational change of protein forces PTAA chains to adopt a nonplanar coiled conformation showing the blue shift. An additional support of the blue shift may be given from conductivity data (presented later) which is also dependent on conjugation length. 3.5. Photoluminescence Spectra. In Figure 7, the PL spectra of pure PTAA solution (having the same polymer concentration as that in the PD11 hybrid) and PD11 hybrid solution are presented. In the pure PTAA solution (Figure 7a), the λmax value decreases with aging time; however, the PL intensity shows a different behavior. Initially, it decreases with time showing PL quenching (up to 218 h), and then it increases showing PL enhancement. This is an interesting new observation in the PTAA solution. A probable reason is the aggregation (both intramolecular and intermolecular) of PTAA chains through H-bonding producing initially a duplex. Due to the aggregation process, conformational change occurs, twisting the PTAA chains and hence shortening the conjugation length. So the energy level of excitons increases, and during decay of excitons higher energy emission occurs, producing a blue shift with time. The PL quenching in this pure system is interesting, and it occurs due to 11030 DOI: 10.1021/la101215v
the creation of more nonradiative decaying paths due to interchain aggregation. However, after 218 h, the PL intensity slowly increases. No definite reason for this intensity increase is known, and a possible reason is the onset of aggregation of higher order of PTAA chains, that is, from duplex to triplex that leads to an efficient energy transfer to the neighboring acceptor causing enhancement of PL intensity.13 In the case of hybrids, the situation is really different as presented in Figure 7b (see also Supporting Information Figure 7). Here, the PL intensity shows a continuous enhancement with time, and the blue shift of λmax occurs. The blue shift of λmax may be explained from the conformational changes of PTAA chains during aggregation with ds-DNA, and the PL enhancement may also be explained from the attachment of PTAA chains on the outer periphery of ds-DNA causing triplex formation.13 Due to this binding, such PTAA chains cannot aggregate with the other PTAA chains and they become far apart from each other. Consequently, the nonradiative decaying paths of the excitons of PTAA chains decrease, resulting in PL intensity enhancement.13,37 The increase of PL intensity of the PD11 system with time is sigmoidal in nature (Figure 7c); that is, initially the rate of increase is very slow, and at intermediate the increase is very sharp showing autocatalytic nature and finally there is a leveling of intensity. This is also true for PD31 and PD13 hybrids (Supporting Information Figure 8). This cooperative nature of the process is similar to that of shifting (37) Dore, K.; Dubus, S.; Ho, H.-A.; Levesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M. G.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 4240.
Langmuir 2010, 26(13), 11025–11034
Mukherjee et al.
Article
Figure 7. Emission spectra of (a) pure PTAA solution of the same concentration as that in PD11 and (b) PD11 hybrid solution. (c) Plot of emission intensity versus time of PD11 hybrid solution. (d) Plot of λmax of photoluminescence emission spectrum versus time of PTAA, PD31, PD11, and PD13 solutions.
of λmax of UV-vis spectra in the POMA-DNA system.6-9 So, it may be surmised that conformational change of PTAA chains anchored to the DNA periphery occurs simultaneously in a cooperative fashion. In Figure 7d, the λmax versus time plots are compared for PTAA, PD31, PD11, and PD13 hybrids. With addition of DNA, the blue shift of the emission peak increases significantly and also makes it faster. A possible reason is that the DNA-bound PTAA molecules have reduced the translational degree of freedom due to large mass. Consequently, the rotations of chain segments are very effective compared to that of unbound PTAA chains, forming shorter conjugated chains and resulting in increased band gap causing a blue shift of the emission peak. So from these UV-vis and PL results, it may be concluded that PTAA chains are bound to the DNA surface and the bound PTAA chains exhibit different PL property from that of pure PTAA solution. So PTAA may also be used as an optical probe for DNA detection and hybridization as in cationic substituted polythiophenes.13,34 However, the detection time in this case is somewhat longer than that reported by Leclerc and co-workers due to the weaker interaction (H-bonding) than the ionic interaction used by them. However, its importance is that the cell property may remain unchanged due to the absence of an ionic field in the PTAA during the sensing process. In the PL spectra of PTAA (Figure 7a), we have not observed any sharp isosbestic point. A probable reason is the formation of a duplex and triplex. In pure PTAA, the spectra 1-5 correspond to almost a diffuse isosbestic point at ∼475 nm. However, the spectra 5-7 characterizing triplex formation do not produce a common isosbestic point with the former. The diffuse nature of the isosbestic point may be due to the different aggregated lengths Langmuir 2010, 26(13), 11025–11034
of PTAA chains produced from the conformational transitions. In the hybrids, the aggregate structure is highly polydisperse, so there is an absence of the isosbestic point. The emission spectra by exciting with different wavelengths (370, 380, and 390 nm) are presented in Figure 8 and also in Supporting Information Figure 9. There is a red shift in the emission peak with increasing excitation wavelength. This may be attributed to the different equilibrium excited states produced after excitation with different wavelengths. With increasing excitation wavelength, the excited polymer molecules are vulnerable for larger conformational changes, producing a more stable equilibrium excited state with lower energy. As a result, the radiative emission that occurs during the transition from the stable equilibrium excited state to the ground state is of lower energy, causing a red shift of the emission peak. With increasing aging time (207 h), the formation of the complex with DNA proceeds toward completion. As a result, the conformational change becomes more difficult, causing a lower red shift of the emission peak with increasing excitation wavelength. Also, with increasing DNA concentration in the blend, PTAA binds more tightly because of the larger availability of phosphate groups for H-bonding. Hence, the conformational transition of PTAA will be more difficult, causing almost a negligible red shift in the emission spectra. Thus, the aggregation in both the pure and blend state can be well understood from the excitation wavelength dependency of emission spectra. The lower the red shift of the emission peak with an increase of excitation wavelength, the higher the aggregated (duplex/triplex) state formation. The PL intensity decay curve is shown in Supporting Information Figure 10. The fluorescence lifetime serves as a sensitive parameter for exploring the aggregation around a fluorophore. DOI: 10.1021/la101215v
11031
Article
Mukherjee et al. Table 2. Lifetimes of PTAA and Its Blends in Aqueous Solutions after 7 Days of Aging Time system
τ1 (ns)
τ2 (ns)
a1
a2
χ2
τf
PTAA PD31 PD11 PD13
0.57 0.66 0.56 0.56
2.79 2.68 2.8 2.56
0.84 0.69 0.79 0.76
0.16 0.31 0.21 0.24
1.13 1.16 1.15 1.2
0.9279 1.285 1.024 1.03
Figure 9. Plot of effective particle diameter (obtained from DLS measurements) versus aging time of PD31, PD11, and PD13 hybrid solutions and pure PTAA solution having the same concentration as that in PD11 hybrid.
Figure 8. PL emission spectra by exciting at different wavelengths of (a) pure PTAA solution of the same concentration as that in PD11 after 3 h of aging, (b) PD11 hybrid solution after 3 h of aging, and (c) PD11 hybrid solution after 207 h of aging. Table 1. Lifetimes of PTAA and Its Blends in Aqueous Solutions after 24 h of Aging Time system
τ1 (ns)
τ2 (ns)
a1
a2
χ2
τf
PTAA PD31 PD11 PD13
0.04 0.573 0.58 0.54
0.81 2.6 2.73 2.68
0.95 0.95 0.97 0.97
0.05 0.05 0.03 0.03
1.056 1.15 0.9 1.12
0.0747 0.67435 0.6519 0.6069
Typical biexponential decay profiles of PTAA and its hybrids are evident from the curves. The lifetime values are presented in Tables 1 and 2. It is apparent from the tables that the average lifetime value increases with increasing aging time and it also increases with increasing DNA concentration in the hybrid. 11032 DOI: 10.1021/la101215v
A probable reason is that with time aggregation of PTAA chains to form a duplex/triplex occurs, resulting in the stacking of thiophene rings which stabilize the excitions, yielding a higher lifetime. In the case of hybrids, the stabilization of excitions also occurs through the base pairs of DNA increasing the lifetime. 3.6. DLS Study. To verify whether the unique changes in PL properties are due to settling of PTAA aggregates, we have conducted dynamic light scattering (DLS) studies of both pure polymer and its blends at different aging times. The DLS size distribution curves of pure PTAA and its hybrids of varying compositions and aging times are presented in Supporting Information Figure 11. All the curves are Gaussian type, yielding effective particle diameters which are plotted with aging time in Figure 9. From the figure, unchanged particle diameter after an initial slow decrease in both pure PTAA and in the hybrids is observed. The initial decrease indicates settling of bigger particles, but after 75 h of aging the particle size remains invariant and the same particle diameter indicates no further complexation with another PTAA chain. However, PL spectra indicate aggregation with time, which is only possible if some new H-bonding of the complementary groups within the complex (already produced) occurs. This additional intra-H-bonding of the complex can produce conformational changes, causing a blue shift in the PL spectra. Also, it is interesting to note that the particle size increases with an increase in DNA concentration, giving definite proof of hybrid formation of the polymer with DNA. Further, with increasing DNA concentration, the bigger sized hybrid particles do not settle down, indicating that the gravitational force of particles is balanced by the polar interaction of larger DNA contents with water. In the PL spectra of Figure 7a, there is an initial decrease of PL intensity followed by an increase for pure PTAA while in the case of PTAA-DNA hybrid there is a continuous rise of PL intensity (Figure.-7b). In no case can the change in PL intensity be correlated with the DLS data (small decrease of particle size up to 70 h of aging), because the slight precipitation is not practically responsible for the large PL intensity enhancement in the hybrid system. So it may be concluded that the PL intensity change with Langmuir 2010, 26(13), 11025–11034
Mukherjee et al.
Article
Table 3. Conductivity Data (S/cm) of PTAA-DNA System at 30 °C sample
undoped state
I2-doped state
PTAA PD31 PD11 PD13 Na-DNA
1.02 10-8 7.27 10-10 3.15 10-10 8.17 10-10 1.3 10-10
4.0 10-7 3.1 10-7 1.37 10-7 2.24 10-7
Figure 10. I-V characteristic curves of PTAA, Na-DNA, and PD11 hybrid.
time is therefore due to the duplex and triplex formation of PTAA and of the hybrid. 3.7. Conductivity. The dc conductivities of the hybrids are presented in Table 3. On addition of DNA, the dc conductivities of PTAA decrease by 2 orders in the hybrids and become equal to that of Na-DNA in the undoped state. The decrease of conductivity of PTAA in the hybrids supports the twisting of the PTAA chains decreasing conjugation length. It is of interest to see how much we can increase the conductivity of the hybrid with external doping. When doped with iodine, the conductivity of the hybrid increases by 3 orders and remains the same as that in the pure PTAA (doped) system possibly due to the large contribution from extrinsic charge carriers produced on doping. In Figure 10, the I-V characteristic curves are compared for Na-DNA, PTAA, and PD11 hybrid, indicating that Na-DNA is more semiconducting than PTAA and the PD11 hybrid shows an intermediate semiconducting behavior. The semiconducting behavior of other hybrids is evident from Supporting Information Figure 12, and they are also behaving as a semiconductor with intermediate properties of Na-DNA and PTAA. PTAA is an electronic semiconductor, whereas in Na-DNA the absence of electronic conductivity is probable for the absence of conjugated π-bonds. In the I-V curves, at a particular voltage, the higher current in Na-DNA than that of PTAA and the PD hybrids may be due to conduction by the Naþ ions. In the blends, apart from the inherent conductivity of PTAA chains, there may be some additional charge transfer between the PTAA chains and base pairs of DNA through π-π interaction. Also, the Naþ ions of Na-DNA may be transferred through PTAA chains by exchanging with Hþ (PTAA = PTAA- þ Hþ) of PTAA. The probable directions of charge transfer in the hybrid are indicated by arrows in Figure 2. All these factors yield an increase in the current of blends compared to that of PTAA in the I-V curves.
helpful for understanding the mechanism of polyplex formation. From the FTIR spectra, it is certain that H-bonding interaction between the phosphate group of DNA and the carboxylic acid group of PTAA is responsible for the polyplex formation between the components. The H-bond formation is a slow process because conformational change of PTAA chains is required for bringing the H-bonding sites of the components nearer. Such a conformational transition causes twisting of the PTAA chains. Hence, the conjugation length of PTAA chains decreases, showing a blue shift of the absorption peak in the UV-vis spectra with time. The change in PL spectra of the hybrids with aging time is also interesting. Here, the emission peak also shows a blue shift with aging time (i.e., with hybrid formation), and this has been attributed to the shortening of conjugation length increasing the exciton energy level. Most important is the enhancement of PL intensity with polyplex formation. The formation of the polyplex stabilizes the excitons as evidenced from the increase of average lifetime values compared to that of pure PTAA (Tables 1 and 2). The stability of excitons may arise for the delocalization of PTAA excitons on DNA base pairs in the polyplex. Also, the aggregation of PTAA with DNA prevents energy transfer with other PTAA chains, decreasing the nonradiative decay paths of the excitons. Thus, due to the polyplex formation, PL intensity increased significantly. The polyplex produces a stable equilibrium excited state, causing a lower red shift of the emission peak with increasing excitation wavelength. It is due to the difficulty in conformational change of bound PTAA causing a small change in the equilibrium excited state and thus lowering the red shift. Now, we would discuss the morphology and structure of the polyplexes both in solution and in the solid state. From the CD spectra of the polyplex solution, it is evident that the double helical structure of DNA is retained having a B helix structure. The DLS study has been used to understand the state of hybrid formation in solution. The diameters of the polyplexes are greater than that of PTAA, and it also increases with an increase in DNA concentration. These results support polyplex formation in the solution state for the three compositions studied here. In the solid state, both the DNA and the polylplexes exhibit fibrillar morphology as is evident from TEM micrographs (Figure 1) Polyplexes take the shape of DNA fibrils because DNA is a much bigger molecule than PTAA (having no characteristic morphology), which becomes adsorbed on the DNA periphery. Probably this adsorption of PTAA on DNA periphery dictates the polyplexes to have fibrilar morphology. Due to this polyplex formation, the diameter of the DNA fibril has increased, and from the increment of fibrillar diameter an approximate model of nanostructured self-assembly of DNA and PTAA of the polyplex has been proposed (Figure 2). The polyplex formation through the adsorption of PTAA chains on the DNA periphery is further supported from the spreading of green fluorescence through the whole DNA fibrillar network. The polyplex formation is finally supported from the conductivity data. In the undoped state, the same conductivity values of polyplexes as that of DNA and in the doped state the similar conductivity values of polyplexes as that of doped PTAA suggest that the conductivity is contributed from both the components of the polyplexes. The intermediate I-V curves of the polyplexes with those of the components also support the polyplex formation.
4. Discussion
5. Conclusion
The forces of polyplex formation of negatively charged DNA with the weak acid PTAA would be discussed here. Certainly, the new properties developed due to the polyplex formation would be
So it may be concluded that PTAA produces good semiconducting polyplexes with DNA when the mixtures of aqueous solutions of the components are aged for 15 days. In the polyplexes,
Langmuir 2010, 26(13), 11025–11034
DOI: 10.1021/la101215v
11033
Article
DNA remains double stranded and the interaction between DNA and PTAA is H-bonding type. A conformational transition causing twisting of the PTAA chains for bringing the H-bonding sites of the components nearer occurs during the polyplex formation, causing a blue shift in the UV-vis and photoluminescence spectra with time. The PL enhancement in the polyplex formation and the semiconducting nature of the polyplexes may be useful toward the design of optoelectronic devices. Acknowledgment. P.M. acknowledges the Council of Scientific and Industrial Research, New Delhi for providing the fellowship. We also acknowledge the Department of Science and Technology, New Delhi for financial support. The help
11034 DOI: 10.1021/la101215v
Mukherjee et al.
extended by Dr. Arun Bandyopadhyay of Indian Institute of Chemical Biology, Kolkata for confocal fluorescence microscopy and Dr. Nitin Chattopadhyay of Jadavpur University, Kolkata for fluorescence lifetime measurement is gratefully acknowledged. Supporting Information Available: Energy minimized model of PTAA, TEM micrographs, Confocal micrographs, FT-IR spectra, UV-vis spectra, PL spectra, Emission intensity vs time plots, Emission spectra by exciting at different wavelengths, Time-resolved fluorescence decay curves, DLS-histogram and I-V curves of PTAA and PTAA-DNA hybrids. This material is available free of charge via the Internet at http://pubs.acs.org.
Langmuir 2010, 26(13), 11025–11034
