NANO LETTERS
Single Molecular Imaging and Spectroscopy of Conjugated Polyelectrolytes Decorated on Stretched Aligned DNA
2005 Vol. 5, No. 10 1948-1953
Per Bjo1 rk,*,† Anna Herland,† Ivan G. Scheblykin,‡ and Olle Ingana1 s† Biomolecular and Organic Electronics, Department of Physics, Chemistry and Biology, Linko¨ping UniVersity, SE-581 83 Linko¨ping, Sweden, and Department of Chemical Physics, UniVersity of Lund, P.O. Box 124, SE-22100, Sweden Received July 12, 2005; Revised Manuscript Received September 1, 2005
ABSTRACT DNA is the prototype template for building nanoelectronic devices by self-assembly. The electronic functions are made possible by coordinating electronic polymer chains to DNA. This paper demonstrates two methods for fabrication of aligned and ordered DNA nanowires complexed with conjugated polyelectrolytes (CPEs). The complex can be formed either in solution prior to stretching or after stretching of the bare DNA on a surface. Molecular combing was used to stretch the complexes on surface energy patterned surfaces, and PMMA for the bare DNA. Single molecular spectroscopy, in fluorescence, and microscopy, in atomic force microscopy, give evidence for coordination of the short CPE chains to the aligned DNA.
DNA is one of the most used templates within the field of self-assembly-based biomolecular nanotechnology. The DNA molecule offers a range of interesting properties suitable for such molecular scale constructions, of which one is the very high length to width aspect ratio, that gives the possibilities to form desired nanometer wire geometries. A second very important property is the recognition ability of the DNA chains with its predefined sequences that also gives the opportunity for more complex linear and branched assembling, hence sequence specific adsorption and hybridization.1-4 This fulfills several of the requirements for a template for nanodevice fabrication. However, as intrinsic conduction in DNA does not give the basis for electronic functions,5,6 the DNA template needs to be functionalized with electronic materials in order to assemble functional electronic devices. To obtain such structures, formation of metal clusters on DNA has been widely demonstrated as a possible route for obtaining conducting nanowires. Metal ions have been complexed with DNA chains and reduced to metal particles7,8 and sometimes also with further metallization.3,4,9,10 Another possibility is to decorate DNA with semiconducting11 or surface-functionalized metal nanoparticles.12,13 Alignment and positioning of superconducting nanowires have been demonstrated.14 * Corresponding author. E-mail:
[email protected]. +46 13 282702. Fax: +46 13 28 89 69. † Biomolecular and Organic Electronics. ‡ Department of Chemical Physics. 10.1021/nl051328z CCC: $30.25 Published on Web 09/13/2005
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Keren et al. have shown that a more advanced structure, such as a field effect transistor, can be fabricated by direct assembling of carbon nanotubes to DNA in combination with a metallization technique.15 The combination of inorganic classical metals, semiconductors, and superconductors with the polymeric DNA chain is a combination of soft and hard, which will inevitably lead to mismatch in the material’s properties. Another candidate for electronic functions is conjugated polymers, which can be found in a semiconducting and luminescent state, but which can also be doped into a metallic state.16 While electronic transport properties in these polymers are typically poorer than those found in inorganic metals and semiconductors, the very flexibility of the polymer chain may be an advantage in the assembly of complex three-dimensional structures, using hybridization as an assembly technique. The properties of such conjugated polymers may be adapted to the behavior of biological polyelectrolytes, such as DNA. We have demonstrated how noncovalent complexes between zwitterionic substituted conjugated polymers can be used for detection of hybridization, to the point of allowing single nucleotide mismatch detection.17 Complexation of these water-soluble conjugated polyelectrolytes (CPEs) with biological polyelectrolytes offers an alternative approach for building materials and devices, where the assembly of electronic polymers is controlled by the interactions with macromolecular assemblers in the form of biological poly-
electrolytes such as DNA, RNA, or proteins. CPEs have the advantage of mechanical flexibility and chemical similarity to biomolecules. Complexation does not necessarily suppress further biological recognition through hybridization; this might be a major advantage compared to metallization where recognition may be lost. It is also possible to control the electronic properties by tuning the doping level of the conjugated polymer backbone, which is of importance for more complex structures such as electrochemical transistors.18 The use of CPEs may also give thinner wires, when compared to metallization of DNA through reductive electrodeposition or physical vapor deposition. Metallic forms of conjugated polymers have been grown on DNA templates. Ma et al. have shown that monomers of aniline can be immobilized to stretched DNA and then polymerized to form a conjugated polymer along a DNA chain.19 Starting with polymers or oligomers, we have shown how CPEs may be used to detect hybridization of short oligomers of DNA.17,20-22 With another class of biological polyelectrolytes, we have previously demonstrated how CPEs can report on the misfolding of insulin,23,24 and also to be incorporated in amyloid fibrils during the growth process.25 This gives a well-defined and ordered hybrid material with retained semiconductor properties. In this paper we demonstrate two new principles for nanowire assembly where complexes of λ-DNA (∼48.5 kbp, New England Biolabs) and luminescent CPEs can be aligned and stretched on a substrate and where the complexes are formed in solution prior to the stretching or after the stretching. These nanowires are characterized by single molecule imaging and optical spectroscopy. Two different luminescent conjugated polyelectrolytes, POWT (Figure 1a) and POMT (Figure 1b), both with a thiophene backbone are used. The lengths of the CPEs are 11-17 monomer units.26,27 POWT, with a zwitterionic side chain, has previously been used for biomolecular detection of DNA.17,20-22 POMT, having the same side chain as POWT except for methylation of the carboxyl group, will have a positive net charge up to pH ∼9. In this study we mainly focus on POMT, though the well-documented POWT gives some valuable information. From the detection experiments using POWT, specific hybridization can occur with the CPE complexed with single-stranded DNA.17 Characterization of the complexes in solution was done with fluorescence (experimental details in Supporting Information). The fluorescence shows a slight red shift of the maximum peak both for POMT/DNA and for POWT/DNA, compared to the CPE separate in buffer solution. This is due to planarization of the backbone of the polythiophene on complexation to DNA and is in agreement with previous studies of POWT.17,28 It is interesting to note that no significant sign of polymer aggregation can be detected from these measurements in solution. Aggregations would red shift the maximum peaks to higher wavelength and also decrease the intensity.17,28 This means that if there is any aggregation, it does not depend on polymer-polymer aggregating in solution. The solutions are stable for at least a week, which further supports that there is no major aggregation. Nano Lett., Vol. 5, No. 10, 2005
Figure 1. (a) Poly(3-((S)-5-amino-5-carboxyl-3-oxapentyl)-2, 5-thiophenylene hydrochloride, POWT. (b) Poly(3-[(S)-5-amino5-methoxycarboxyl-3-oxapentyl]-2,5-thiophenylene hydrochloride), POMT. (c) Fluorescent spectra for the CPEs and the CPE/λ-DNA complexes, all in 50 mM 2-morpholinoethanesulfonic acid (MES), pH 5.5 buffer. POWT (b), POWT/λ-DNA (O), POMT (1), and POMT/λ-DNA (3).
Figure 2. The method used for stretching of DNA. (a) The substrate is surface energy modified using a PDMS stamp for 30 min. (b) A droplet is put on the surface containing CPE/DNA complex or bare DNA. (c) The droplet is gently blown of the substrate using a nitrogen gas flow.
Various molecular combing techniques can be used to stretch and align DNA on surfaces.29-34 A schematic drawing of the method used here for stretching the CPE/DNA complexes formed in solution is shown in Figure 2. A solution containing 5 µg/mL λ-DNA and 1.3 µg/mL POMT (monomer MW of 228 g/mol) or POWT (monomer MW of 214 g/mol) is prepared. The buffer used is 50 mM 2-mor1949
Figure 3. Fluorescence image (a) and AFM image (b) of POMT/DNA complexes formed in solution on PDMS-modified surfaces. (c) Height profile of a thicker (1) and a thinner (2) part of the complex in (b), and height profile of a bare DNA chain (3). (d) Fluorescence image of POWT/DNA complexes formed in solution on a PDMS-modified surface. (e) Fluorescence image of DNA decorated with POMT after stretching on PMMA surface. Scale bars: 10 µm (a, d, e) and 500 nm (b).
pholinoethanesulfonic acid (MES), and the pH is set to 5.5. This gives a ratio of little less than 1:1 for polyelectrolyte/ λ-DNA on monomer basis. The ratio is chosen in order to hinder unspecific background staining of free CPE. The substrates used in this study are microscope slides for fluorescence microscope, Si/SiO2 for single molecular spectroscopy, and mica for the AFM studies. All surfaces, except mica, were cleaned by immersion in a 5:1:1 mixture of MilliQ water, H2O2 (30%), and NH3 (25%) for 5 min at 75°. Mica was used freshly cleaved. To be able to stretch DNA on these substrates, the surface energy was modified using a poly(dimethylsiloxane) (PDMS) stamp (see Supporting Information). When PDMS is in close contact with a surface, some of the low molecular weight species in the stamp will be transferred to the surface.35,36 The result is a hydrophobic surface on which DNA can attach, to be elongated along the direction of fluid flow. A 5 µL droplet of CPE/DNA complex was put on the surface and gently blown off, after 1 min of incubation, with nitrogen gas. This resulted in reproducible stretching of the CPE/DNA complexes and for bare DNA. An alternative approach is to decorate the DNA with polyelectrolyte after stretching on a surface. In this case PMMA-coated glass slides (50 mg/mL PMMA dissolved in chloroform, spin coated at 1000 rpm for 1 min) were used 1950
as substrates and the λ-DNA concentration was 0.5 µg/mL (50 mM MES pH 5.5). Droplets of a 5 µg/mL POMT solution (MilliQ water) were used to wash parts of the surface with stretched DNA. Fluorescence imaging was conducted using a Zeiss Axiovert 200M inverted light microscope equipped with an AxioCam HRc CCD. The fluorescent pictures reveal that DNA complexed with POMT or POWT can be stretched into aligned wire-like geometries on PDMS-modified surfaces. As can be seen from parts a and d of Figure 3, the polyelectrolytes often seems to glue many of the DNA chains together to form longer geometries (>26 µm) than possible with one single λ-DNA chain37 (see Figure S1 in Supporting Information for stretched bare DNA on a PMDS-modified surface). From fluorescence microscopy we cannot evaluate the width of the wire, but the complexes do not form any large clusters of aggregates. It is more of a bundle-like structure of the chains. However, it is possible to find stretched complexes that look to be single DNA chains, by imaging with the force microscope (Figure 3b). Dots seen in the fluorescent pictures are most likely nonstretched CPE/ DNA complexes. The complex, formed in solution, of DNA and CPEs is rather stable.20-22 Therefore, the background of free oligoelectrolyte and other nonspecific interactions can be minimized. Also, since the complexes are formed prior Nano Lett., Vol. 5, No. 10, 2005
to stretching, the CPE is not restricted to one side of the DNA which can be an advantage for continuous CPE decoration. AFM images (NanoScope III with a J-scanner head, Digital Instruments, used in tapping mode and in air environment) give information of the distribution of POMT along the DNA chains. Thicker and thinner parts can be observed indicating that the distribution of CPE is not completely homogeneous (Figure 3b). Looking at the height profile of the stretched CPE/DNA complexes, it is possible to say that the thinnest parts of the CPE/DNA complex are higher than those for bare DNA (Figure 3c). The height profiles of the chains show that the thinnest parts are over 0.6 nm and the height then varies up to the thicker parts, which can have heights of more than 1.8 nm. This can be compared to the same value for a bare DNA which is well under 0.5 nm. Larger and thicker dots with heights of several nanometers can sometimes be observed, corresponding to unstretched clusters. The height difference between the thinner parts and a bare DNA is not sufficient to argue that there are CPEs decorated continuously all along the DNA chain. Ma et al. have reported that immobilization of aniline monomers cannot be distinguished from bare DNA in AFM measurements, but after polymerization they have a significant increase of around 0.5 nm.19 In this study, we use CPEs that are around 15 monomers which is between the two cases shown by Ma et al. Thus the height difference observed here can be related to CPE complexed with the DNA, or it might depend on different geometries of the DNA, reflecting changes in the stretching environments compared to bare DNA. However, by use of single molecular spectroscopy (Figure 4), the fluorescence seems to be quite well distributed along the DNA chains. The fluorescence images where brighter parts are alternating with less bright fluorescing parts along the DNA chains also support the AFM measurements. The AFM measurements imply that, compared to metallization,8,9 very thin wire geometries can be created using this method. It is more difficult to stretch individual chains of DNA when complexed with a polyelectrolyte. This is probably due to the polyelectrolyte blocking the normal interaction necessary for DNA stretching, such as hydrophobic, π-π, and electrostatic interactions. In some cases, the CPE may also cross-link between, and within, DNA chains. Optimization of the ionic strength and pH of the buffer as well as using the best possible substrate may compensate for this. There is also an advantage of not having individual CPE/DNA chains.38 The stability is higher and the amount of material is also larger, and characterization might therefore be easier. The alternative method with decoration of the DNA after stretching, gave very well stretched individual CPE/DNA chain complexes. The difficulty with the method was that most of the DNA was detached from the surface on these parts, due to rinsing forces from the water. Using spin-coated PMMA as substrate instead of PDMS-modified surfaces improved the adhesion force of the DNA, but still much of the DNA was detaching. However, when the immersion oil used for the microscope was applied on the rinsed substrate, Nano Lett., Vol. 5, No. 10, 2005
Figure 4. (a) Image of a DNA chain complexed with POMT, scale bar 3 µm. (b) Wavelengths of the fluorescence maxima of some points along the chain in (a). (c) A cartoon showing possible organization of the complexes: (1) a middle size cluster; (2, 3) a small cluster and an individual chain. Objects (2) and (3) can show fluorescence blinking. (d) Spectra of a blinking spot (b), a strong intensity spot (+), and the complex in solution (0). The spectra have been background corrected and normalized for the maximum value for the spectra of the strong intensity spot.
some of the surface-deposited POMT was moved along with the oil to decorate the unaffected DNA just outside where the droplet had rinsed (see Figure 3e, see also Figure S2 in Supporting Information for stretched bare DNA on PMMA surface). As can be seen from Figure 3e, the background is very low. From the fluorescence images the distribution of CPE appears more homogeneous than for the complexes formed in solution. Stronger attachment of the DNA to the surface needs to be obtained for using this method. By use of a wide-field fluorescence microscope designed for single molecule spectroscopy (see Supporting Information), some interesting data are observed (Figure 4). A contrast adjustment of the images confirms the fluorescence to be continuous along the DNA chain without any noticeable gaps. Intensity of bright spots (like, e.g., # 1 in Figure 4a) is often an order of magnitude larger than that of the “continuous” part. Of course, we cannot say that CPE chains cover DNA completely because the length of CPE chain is about 10 nm, which is much less than the spatial resolution of the microscope (500 nm). By diluting the CPE concentration relative to that of DNA, we were able to reach conditions when the fluorescing decoration becomes discontinuous (see Figure S3 in Supplementary Information). Often we could not see any signal 1951
between individual spots situated on one stretched DNA chain. Individual spots then are either small clusters or even single chains of CPE (Figure 4c). Those individual spots showed fluorescence blinking or/and stepwise photobleaching.39 The presence of the blinking effect, demonstrated by time series of fluorescence images (see Figure S3 in Supplementary Information), shows that those polymer clusters should be at most 10 nm in size, where 10 nm is the upper limit of Fo¨rster energy transfer distance toward a fluorescence quencher in conjugated polymers.40-42 The length of the single CPE chain is already about 10 nm. Therefore those “blinking” spots are either individual chains or small aggregates with the chain stacking direction mostly perpendicular to the DNA strand. We were able to measure fluorescence spectra of the individual spots of different brightness. The individual fluorescence spectra seem in most cases to be slightly red shifted compared to the solution spectra, with a maximum peak at around 594 nm, implying that the conformations of the polymer chains are affected either by the stretching procedure or by drying effects. Spectra of the “blinking” spots were found to have maximum of about 590 nm. If the spectra are evaluated more closely along one CPE/ DNA bundle, one can see that the spectra shift some nanometers for different spots (Figure 4a,b). There is often a correlation between the intensity of the spot where the spectra is taken and the shift of the peak maximum. Strong intensity spots tend to be somewhat red shifted, where wavelengths of the maxima around 600 nm are not unusual. The medium and less intense spots have more similarities to the solution spectra. An explanation of this can be that at the brighter spots there are more CPE chains that are packed closer together, making energy transfer possible to lowenergy sites, while in the medium and less bright parts the CPEs are more separated and can adopt more of a solutionlike conformation. Enhancement of a second peak around 640 nm compared to the solution spectra also suggests that the CPE chains are closer together, which further supports this theory. In conclusion, we have demonstrated how a complex of a conjugated polythiophene polyelectrolyte and λ-DNA, formed in solution prior to stretching or after stretching, can be aligned into photoluminescent nanowire arrays by molecular combing techniques on surface energy patterned substrates. Conjugated polyelectrolytes have the advantage that they can form very thin nanowires by complexing with DNA, making this approach promising for nanofabrication. Still, there are improvements needed such as more homogeneous polymer distribution and fully individual stretching of CPE/DNA complexes. Furthermore, targeting positions on the CPE/ DNA complex using the nucleotide sequence and hybridization as address tags and as recognition events may be a possible step for more advanced assembling, also extending from the two-dimensional surface to three-dimensional assembly. Acknowledgment. We acknowledge VINNOVA, the Swedish Research Council, the Knut & Alice Wallenberg 1952
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