DNA Chips with Conjugated Polyelectrolytes in Resonance Energy

Dec 8, 2009 - Jens A. Wigenius*†, Karin Magnusson†, Per Björk†, Olof Andersson‡ and Olle Inganäs*†. † Biomolecular and Organic Electroni...
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DNA Chips with Conjugated Polyelectrolytes in Resonance Energy Transfer Mode Jens A. Wigenius,*,†,§ Karin Magnusson,†,§ Per Bj€ork,† Olof Andersson,‡ and Olle Ingan€as*,† †

Biomolecular and Organic Electronics, Department of Physics, Chemistry and Biology (IFM), Link€ oping University, SE-581 83 Link€ oping, Sweden and ‡Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Link€ oping University, SE-581 83 Link€ oping, Sweden. § These authors contributed equally to this work. Received August 20, 2009. Revised Manuscript Received November 13, 2009 We show how to use well-defined conjugated polyelectrolytes (CPEs) combined with surface energy patterning to fabricate DNA chips utilizing fluorescence signal amplification. Cholesterol-modified DNA strands in complex with a CPE are adsorbed to a surface energy pattern, formed by printing with soft elastomer stamps. Hybridization of the surface bound DNA strands with a short complementary strand from solution is monitored using both fluorescence microscopy and imaging surface plasmon resonance. The CPEs act as antennas, enhancing resonance energy transfer to the dye-labeled DNA when complementary hybridization of the double strand occurs.

1. Introduction Microarray techniques have become considerably important tools for the advancement of genomics and proteomics. Large-scale array sensors with parallelized detection are expected to play important roles in point-of-care diagnosis or in screening for biomarkers. Different types of costly and time-consuming biomolecular labeling, e.g., fluorescence, has traditionally been the method of choice for signal detection in microarrays. In this work, luminescent conjugated polyelectrolytes (CPEs) are employed as a means of detection of hybridization in DNA microarrays. CPEs, consisting of a carbon backbone with alternating single and double bonds, make up an interesting and rapidly developing class of organic polymers. The alternating bonds form a πconjugated system defining the photophysical and semiconducting properties of the CPE. Changes in the geometry and organization of the CPE lead to shifts in the absorption and emission spectra.1 It has been shown that CPEs may interact with biomacromolecules such as DNA or proteins, both in solution and on surfaces, and that the interactions can be easily followed with spectrophotometry.2-7 This enables the use of CPEs as tools for research and medical diagnostics. For instance, Leclerc et al. reported “super lightning” or fluorescence signal amplification, where the collective response from a large number of CPEs in an aggregated cluster of dye-labeled single DNA strands (ssDNA) renders an enhanced fluorescence resonance energy transfer *Corresponding authors. E-mail: [email protected] (O.I.), [email protected] (J.A.W.). (1) Nilsson, K. P. R.; Andersson, M. R.; Inganas, O. J. Phys.: Condens. Matter 2002, 14, 10011–10020. (2) Charych, D. H.; Nagy, J. O.; Spevak, W.; Bednarski, M. D. Science 1993, 261, 585–588. (3) Nilsson, K. P. R.; Inganas, O. Nat. Mater. 2003, 2, 419–U10. (4) Ho, H.-A.; Boisssinot, M.; Bergeron, M. G.; Corbeil, G.; Dore, K.; Boudreau, D.; Leclerc, M. Angew. Chem., int. Ed. 2002, 41, 1548–1551. (5) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339–1386. (6) Herland, A.; Inganas, O. Macromol. Rapid Commun. 2007, 28, 1703–1713. (7) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 896– 900. (8) Ho, H.-A.; Dore, K.; Boissinot, M.; Bergeron, M. G.; Tanguay, R. M.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2005, 12673–12676.

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(FRET) between the CPE and the dye after hybridization of a few double strands (dsDNA).8,9 There are many techniques used to fabricate DNA arrays. Recently, Erkan et al. demonstrated a method utilizing hydrophobic interactions between cholesterol-modified DNA and a photolithographically manufactured pattern of SU-8, a standard photo resist.10 A faster and cheaper method is soft lithography, e.g., microcontact printing (μCP), where a relief patterned soft rubber stamp is coated with the DNA strand to be printed on the substrate of choice.11 The substrate is usually functionalized to achieve better adhesion of the DNA strand. The most commonly employed stamp material, poly(dimethylsiloxane) (PDMS) contains small amounts of low molecular residuals that can be transferred to the substrate in contact printing,12 leading to a pattern with different surface energy properties.13,14 Where the stamp has been in contact, transfer of the low molecular residuals renders a hydrophilic substrate more hydrophobic. We refer to this method of patterning as PDMS staining, or stain for short. DNA can be attached to stained areas, and DNA stretched by molecular combing can be located between such areas.15,16 Hybridization to stretched ssDNA has not yet been shown, suggesting steric restraints imposed by the hydrophobic interactions with the surface. For this reason, alternative attachment geometries are desired. In the present work, we show that a cholesterol tag covalently attached to the end of the DNA molecule is adsorbed to the PDMS stain, rendering a thin layer of attached ssDNA with the bases free for hybridization (Figure 1). DNA immobilization and (9) Dore, K.; Leclerc, M.; Boudreau, D. J. Fluoresc. 2006, 16, 259–265. (10) Erkan, Y.; Czolkos, I.; Jesorka, A.; Wilhelmsson, L. M.; Orwar, O. Langmuir 2007, 23, 5259–5263. (11) Lange, S. A.; Benes, V.; Kern, D. P.; Horber, J. K. H.; Bernard, A. Anal. Chem. 2004, 76, 1641–1647. (12) Glasmastar, K.; Gold, J.; Andersson, A. S.; Sutherland, D. S.; Kasemo, B. Langmuir 2003, 19, 5475–5483. (13) Wang, X. J.; Ostblom, M.; Johansson, T.; Inganas, O. Thin Solid Films 2004, 449, 125–132. (14) Asberg, P.; Nilsson, K. P. R.; Inganas, O. Langmuir 2006, 22, 2205–2211. (15) Bjork, P.; Holmstrom, S.; Inganas, O. Small 2006, 2, 1068–1074. (16) Bjork, P.; Herland, A.; Scheblykin, I. G.; Inganas, O. Nano Lett. 2005, 5, 1948–1953.

Published on Web 12/08/2009

DOI: 10.1021/la903101v

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Figure 1. (a) DNA X and DNA B-Cy5 are mixed in equal concentration to form the chol-DNA XB-Cy5 (b), with two cholesterol molecules and a 15 bp sticky end where the complementary strand could be hybridized. (c) A relief patterned PDMS stamp (black) is placed in conformal contact with a clean substrate, leaving behind a hydrophobic pattern upon removal. (d) The cholesterol-tagged DNA strand is adsorbed to the hydrophobic pattern. (e) After incubation with tPOMT (green) to this pattern, FRET could be observed with fluorescence microscopy, and (f) modified after hybridization with DNA B0 .

hybridization are demonstrated both with imaging surface plasmon resonance (iSPR) and fluorescence microscopy, using the commercially available dyes Cy5 and Alexa350 as well as the CPE tPOMT,17 which has been shown to interact with dsDNA along the DNA axis.18 tPOMT is a regioregular and well-defined CPE with a chain length of 12 thiophene rings, and therefore has better defined physical and chemical properties compared to POWT, used in earlier DNA hybridization studies, which has a chain length from 11 to 22 thiophenes.19 We also demonstrate the possibility of fluorescence signal amplification between the CPE and Cy5 by designing the FRET conditions.

2. Experimental Section All experiments were performed in a clean room environment with controlled temperature, humidity, and subdued light in order to minimize photobleaching. All chemicals were used as provided by Sigma Aldrich (Sweden), unless otherwise noted. 2.1. Buffers. All buffers were prepared on the day of use, in doubly distilled deionized water (18 MΩ, Milli-Q, Millipore) (mqH2O), phosphate-buffered saline (PBS) pH 7.4 (10 mM Na2HPO4, 10 mM NaH2PO4, 150 mM NaCl),and sodium acetate (NaAc) pH 5.5 (NaAc 50 mM). During the SPR measurements, HEPES-buffered saline (HBS-N) was used as provided by GE Healthcare, Uppsala, Sweden. 2.2. Conjugated Polyelectrolyte. Poly(3-[(S)-5-amino-5methoxycarboxyl-3-oxapentyl]-2,5-thiophenylene hydrochloride) (17) Nilsson, K. P. R.; Aslund, A.; Berg, I.; Nystrom, S.; Konradsson, P.; Herland, A.; Inganas, O.; Stabo-Eeg, F.; Lindgren, M.; Westermark, G. T.; Lannfelt, L.; Nilssonl, L. N. G.; Hammarstrom, P. ACS Chem. Biol. 2007, 2, 553–560. (18) Bjork, P.; Thomsson, D.; Mirzov, O.; Wigenius, J.; Inganas, O.; Scheblykin, I. G. Small 2009, 5, 96–103. (19) Nilsson, K. P. R.; Olsson, J. D. M.; Konradsson, P.; Inganas, O. Macromolecules 2004, 37, 6316–6321.

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(tPOMT) (in-house synthesis, Chart 1) was dissolved in mqH2O and stored as stock solutions (1 mg mL-1) at -20 °C protected from light. Sample solutions of tPOMT were further diluted prior to use. 2.3. DNA. ssDNA (Chart 1) was delivered (Eurogentec S.A., Belgium) as lyophilized powder and restored with mqH2O to stock solutions (50 μM) in aliquots, to avoid cycles of freezing and thawing, and stored at -20 °C protected from light. Triethyleneglycol (TEG) cholesterol was covalently attached (labeled cholDNA) to DNA X (15 bp), DNA A (30 bp), DNA B (30 bp), and DNA C (30 bp). One set of chol-DNA B was labeled with the dye Cy5 (chol-DNA B-Cy5) (absorption max 646 nm, emission max 662 nm). Chol-DNA C was labeled with the dye Alexa 350 (cholDNA C-Alexa350) (absorption max 343 nm, emission max 443 nm), in both cases at the 50 end. Complementary strands matching DNA A and DNA B from the 50 end (DNA A0 and DNA B0 ) were also purchased, two sets labeled with fluorescent dyes Alexa 350 (DNA B0 -Alexa 350) and Cy5 (DNA A0 -Cy5) and two unlabeled sets. The chol-DNA was used either as ssDNA or dsDNA with two TEG cholesterol molecules at one end and a 15 bp sticky end at the opposite end. They were formed through hybridization for 1 h in stoichiometric ratio (10 μM) of the 30 bp long DNA A, B, or C with the 15 bp long DNA X complementary to the 30 end of DNA A, B, or C. 2.4. Master and Stamp Fabrication. PDMS stamps were molded on a relief master, fabricated with standard photolithography on a silicon wafer with SU-8 (Micro Chem Corp.) as photoresist. The master contained two areas of circular hollow disks, with a radius of either 20 or 40 μm, separated by 40 or 80 μm, respectively. To avoid adhesion between the master and the stamp, silanization was necessary. By submerging the master in a solution of 50 mL xylene and 300 μL dimethyldichlorosilane for 5 min, followed by extensive rinsing with xylene and ultrasonication for 5 min in mqH2O, a thin antiadhesion layer is achieved on the master. PDMS is prepared by mixing the two-component Langmuir 2010, 26(5), 3753–3759

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Article Chart 1. DNA Sequences with Modifications and Chemical Structure of tPOMT

silicon elastomer, Sylgard 184 (Dow Corning Corp., USA), curing agent, and base with a mass ratio of 1:10. To avoid trapping of air bubbles in the stamp, the liquid elastomer was degassed before being cast onto the master, and cured in a convection oven for 45 min in 85 °C. The stamps were gently peeled off from the master after cooling and used immediately.

2.5. Adsorption of Chol-DNA to Stained Substrates. Standard microscope glass slides, or silicon wafers with a native oxide layer (∼1.5 nm), were used as substrates in the fluorescence experiments and imaging null ellipsometry measurements, respectively. All substrates were cleaned by immersion in a 5:1:1 mixture of mqH2O, H2O2 (30%), and NH3 (25%) for 10 min at 85 °C (TL1 wash), followed by extensive rinsing in mqH2O and dried in a stream of nitrogen. The substrates were then treated in an oxygen radio frequency (RF) plasma chamber (Pico-RF, Diener Electronic, Germany) for 60 s (175 W, 0.05 Torr). A fresh PDMS stamp was put into conformal contact with the substrate for 2 min to create the optimal PDMS pattern for chol-DNA adsorption (Figure 1). The stained surface was incubated with 20-70 μL of the desired chol-DNA (1 μM) to be adsorbed for 15 min, followed by careful rinsing in NaAc buffer. A drop of tPOMT (20-50 μL, 1 μg mL-1) was then incubated onto the pattern for 15 min, if tPOMT were to be complexed to the adsorbed chol-DNA. It is also possible to form the complex by mixing chol-DNA (10 μM) with tPOMT (10 μg mL-1) for 15 min, and further diluting the solution five times with PBS prior to incubation. For hybridization with complementary DNA (cDNA) to the adsorbed cholDNA, the cDNA strand (0.1 μM in PBS) was incubated as a drop onto the adsorbed chol-DNA pattern for 15 min. The samples were always dried gently in a stream of nitrogen before examination. 2.6. Characterization with Imaging Ellipsometry. Imaging null ellipsometry, EP3 (Accurion GmbH, Germany), was used to analyze chol-DNA adsorption to the stained substrate. The EP3 is an ordinary null ellipsometer equipped with an X,Y,Z translation stage and a charge-coupled device (CCD) camera as the detector. This makes it possible to calculate the nulling conditions in each pixel individually using the provided software (EP3 View V2.05) and display a map of the ellipsometer parameters, Δ and Ψ. By building a model system of the sample and addressing known refractive indices (n = 1.5 commonly used for biomacromolecules, n = 1.44 PDMS20) and thicknesses for the different layers in the sample, it is also possible to calculate and display a map of the layer thickness. The lateral resolution of the system is determined by the objective of the CCD camera. Under optimal conditions, this is 1 μm, using a 10 magnifying objective, and the thickness resolution is 0.1 nm. The imaging null ellipsometer measures the film thickness as an average over a defined region of interest (ROI). All thickness measurements have been calculated as an average of at least five ROIs measured on five different chol-DNA chips. The samples were characterized directly after patterning. A nonpatterned clean area of the substrate was used to determine the SiO2 thickness of each substrate and (20) Persson, N. K.; Inganas, O. Sol. Energy Mater. 2006, 90, 3491–3507.

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used in the model. The average thickness and standard deviation was calculated from at least 10 measurements. 2.7. Characterization with iSPR. iSPR measurements were performed using an in-house custom-built instrument based on spectral interrogation in the Kretschmann attenuated total reflection configuration. Surface plasmons were excited by means of a 25 mm equilateral prism (BK7 glass, Melles Griot) onto which the substrates were optically coupled using a small drop of refractive index matching oil (Cargille, Inc.). Light from a monochromator (SpectraPro 300i, Acton Research Corp.) was collimated and polarized before being guided toward the prism by means of a mirror configuration. A CCD detector (Retiga Exi, Qimaging Corp., 12 bit 1 MP without IR-filter) with imaging optics was used to measure the intensity of the reflected light. During all measurements, the angle of incidence was fixed at 74.5°. At this angle, one pixel of the CCD detector depicted a 2.2 μm  7.0 μm area of the substrate. SPR images were acquired by scanning the wavelength of the incident light in the interval 650-800 nm and subsequently determining the wavelength that gave a minimum in the reflectivity of transverse magnetic (TM)-polarized light, λSPR, for each pixel of the image. The reflectivity of transverse electric (TE)polarized light was used for normalization. A small-volume poly(methyl methacrylate) (PMMA) flow cell was tightly sealed to the substrate, and samples were delivered at a flow rate of 30 μL min-1 by means of a syringe pump. In between sample injections, a continuous flow of buffer was maintained. During the sample injections, the reflectivity of TM-polarized light was monitored as a function of time. The substrates used in the iSPR measurements consisted of 12 mm  12 mm glass slides coated with a thin layer of gold (GE Healthcare, Uppsala, Sweden). These surfaces were cleaned in a 1:1:5 mixture of 30% hydrogen peroxide (Merck KGaA, Germany), 25% ammonia (Merck, KGaA), and mqH2O (Milli-Q, Millipore). In addition, some SPR gold slides were modified to resemble the fluorescence substrates by evaporation of a 1.5 nm adhesion layer of Ti and a 20 nm thin film of SiO2 on top of the gold. These substrates were cleaned in an ultrasonic bath for 5 min followed by a 1 min exposure to oxygen plasma prior to use. 2.8. Fluorescence Microscopic Evaluation. Fluorescence evaluation of the chips was done with a Zeiss Axiovert 200 M inverted light microscope, with a mercury lamp (HBO 100) as the light source and equipped with an AxioCam HRc CCD camera. All samples were immediately analyzed after preparation and illuminated as little as possible in the fluorescence microscope to minimize photobleaching. The microscope was focused on the sample at the edge of the pattern and moved to an unexposed area before capturing of an image. Microscopic photoluminescence pictures were recorded in reflection mode using either 470 nm/40 or 546 nm/12 nm band-pass excitation and corresponding long pass emission filters at 515 or 590 nm. The exposure time was kept constant in each experiment to make it possible for comparison of fluorescence intensity. The images were analyzed using Zeiss AxioVision PC software. By using Adobe Photoshop, the intensity and color content of the individual channels (red, green, and blue) could be compared. The average color channel (red, green DOI: 10.1021/la903101v

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Figure 2. Fluorescence microscopy and sketch of DNA strands adsorbed in each panel: (a) Chol-DNA XC-Alexa 350 adsorbed to a PDMS surface energy pattern; inset shows a non-patterned substrate after incubation with chol-DNA XC-Alexa 350 (excitation with 365 nm, exposure time 1.22 s). (b) DNA B0 -Alexa 350 adsorbed to a PDMS surface energy pattern; inset shows DNA B0 -Alexa 350 incubated to an adsorbed chol-DNA XA pattern (excitation with 365 nm, exposure time 3.00 s). (c) Hybridization of cDNA B0 -Alexa 350 to a chol-DNA XB chip after 96 h of storage in dry conditions exposed to ambient atmosphere (excitation with 365 nm, exposure time 1.25 s). and blue) intensity was divided by the luminosity, and standard deviation was calculated from 12 different spots measured over 125 000 pixels. Gold is a strong quencher for fluorescence in a detection system and, hence, a slightly risky substrate since unspecific adsorption to the gold surface will not be visible through fluorescence. Another complication is that a gold film rapidly regains its hydrophobicity when exposed to air, making evaluation of hydrophobic contrast patterns difficult.

3. Results 3.1. Adsorption of Chol-DNA to a PDMS-Patterned Substrate. The adsorption of chol-DNA to PDMS was studied by incubating a patterned glass substrate with a solution of cholDNA XC-Alexa350. The surface was examined with fluorescence microscopy, after rinsing with NaAc and careful drying under a stream of nitrogen. It was determined that a 2 min contact time between the stamp and the substrate resulted in optimal sharpness of the adsorbed chol-DNA pattern. Incubation of the chol-DNA solution on the substrate for 15 min was sufficient to achieve a clear and distinct fluorescence pattern. This fluorescence pattern is nowhere to be found on an identical sample without the pattern (Figure 2a and inset). Three glass substrates were patterned with PDMS, in order to study hybridization of a cDNA strand to a chol-DNA chip. CholDNA B0 -Alexa 350 was adsorbed on the first, chol-DNA XA was adsorbed on the second, and chol-DNA XB was adsorbed on the third. A drop of DNA B0 -Alexa 350 was added to the adsorbed chol-DNA XA and chol-DNA XB pattern for an additional 15 min. The substrates were examined with fluorescence microscopy after rinsing and drying. A weak blue fluorescent pattern could be detected on the substrate where only DNA B0 -Alexa 350 was incubated (Figure 2b), confirming that DNA could adsorb to the PDMS pattern without the cholesterol tag, as has been shown earlier.16 When comparing Figure 2b with the distinct blue 3756 DOI: 10.1021/la903101v

fluorescence visible on a substrate patterned with DNA XB (Figure 2c), it is clear that adsorption mediated through interaction between the PDMS residuals and the cholesterol tag on the DNA is more effective. Even more interesting is that there is no observable blue fluorescence when non-cDNA B0 -Alexa350 was incubated onto the chol-DNA XA pattern (Figure 2b inset). When DNA B0 -Alexa 350 was incubated to the complementary chol-DNA XB pattern, a clear blue fluorescent pattern was visible, as seen in Figure 2c. By storing the chol-DNA chip in ambient room temperature and humidity, it was observed that the chip retains its ability to hybridize with the complementary strand after 96 h of storage (Figure 2c). 3.2. Characterization with iSPR. The adsorption of cholDNA to PDMS patterned substrates was also monitored in situ using iSPR, to confirm the results from the fluorescence measurements. To this end, a PDMS pattern consisting of 300 μm wide stripes separated by 100 μm was printed on thoroughly cleaned iSPR substrates. The patterned surfaces were docked into the iSPR instrument, and chol-DNA (1 μM in PBS) was introduced for 5 min by means of a fluidic system. For the PDMS-patterned gold surfaces, a uniform increase could be seen, with very little difference in the SPR response over the different regions of the substrate (data not shown). The chol-DNA adsorbed in nearly equal amounts to both the PDMS patterned and nonpatterned regions of the gold surface. The silicon dioxide substrates, however, did not adsorb the chol-DNA. Figure 3a shows the adsorption of chol-DNA XB and two DNA strands, A0 and B0 (0.1 μM), to a PDMS-patterned SiO2 surface. There is a substantial shift in the SPR response over the residual PDMS areas (upper curve) upon exposure to chol-DNA XB, which can not be seen over the bare SiO2 (lower curve). Upon introduction of the non-cDNA A0 , a weak unspecific adsorption to both the PDMS and the SiO2 areas can be observed. During the injection of the cDNA B0 , however, the response is slightly larger over the PDMS areas to which DNA XB has bound. The rapid negative shifts in SPR response upon introduction of the different DNA strands is caused by bulk refractive index changes due to the fact that the samples were dissolved in a different buffer. Through subtraction of the curves from the two regions, the effect of the buffer change is canceled out. In the resulting curve (Figure 3b), the difference in binding between the A0 and B0 strands is also more clearly visible. Figure 3c shows SPR wavelength maps of the patterned SiO2 surface, acquired before (1) and after (2) adsorption of chol-DNA XB. The shift in λSPR can be seen even more clearly in the line profiles in Figure 3d, taken along the x-axes of the maps in Figure 3c. The response upon addition of the complementary strand, B0 , is quite low. Because the B0 strand is only about a quarter the size of the chol-DNA XB, the SPR response to the binding of B0 is expected to be at most 25% of the XB response, under the assumption that each bound chol-DNA XB molecule is accessible to the B0 strand. In the present case, however, under conditions of a continuous flow, and with the B0 concentration used in the SPR study, the amount of bound B0 can barely be resolved in the SPR wavelength maps. However, the most important result from the iSPR data is the resistance of the SiO2 regions to adsorption of the chol-DNA as well as the A0 and B0 strands. 3.3. Hybridization with Complementary ssDNA and Investigation of Fluorescence Signal Amplification. The Cy5labeled chol-DNA B-Cy5 was hybridized with chol-DNA X, forming doubly cholesterol-tagged DNA with a 15 bp long sticky end of DNA XB-Cy5, and incubated as a drop onto a patterned glass substrate. A second substrate was incubated with unlabeled chol-DNA XA, followed by incubation with tPOMT. The Langmuir 2010, 26(5), 3753–3759

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Figure 3. iSPR response during injection of chol-DNA XB, non-complementary A0 , and complementary B0 from a region of the substrate patterned with PDMS residuals and on bare SiO2 (a), and after subtraction of the two curves (b). iSPR wavelength maps before (1) and after (2) adsorption of chol-DNA XB (c), and the appurtenant profile (d) of PDMS on SiO2 (blue) and after chol-DNA XB (green) and DNA B0 (red) injection.

Figure 4. Fluorescence images of DNA/tPOMT complex adsorbed to a PDMS patterned glass surface. The adjacent sketches illustrates the complex components: DNA (black), cholesterol (yellow), Cy5 dye (red), tPOMT chain (green), and FRET direction (yellow arrow); (a) cholDNA XA/tPOMT, (b) chol-DNA XA/tPOMTþA0 -Cy5, (c) chol-DNA XB-Cy5/tPOMT, (d) chol-DNA XB-Cy5/tPOMTþ B0 . (e) Bar plot of the relative color information for the red and green channel of individual panels; the blue channel intensity was zero in all panels.

resulting chol-DNA XA/tPOMT pattern fluoresced in bright green under 470 nm illumination (Figure 4a). The fluorescence from the sample with DNA XB-Cy5/tPOMT leans instead more toward the red (Figure 4c). Since Cy5 on its own is not visible when illuminated at 470 nm, the red fluorescence must derive from FRET between the excited tPOMT and the Cy5 dye. The substrates were again illuminated with 470 nm in the fluorescence microscope, after incubation with the cDNA B0 onto the cholDNA XB-Cy5/tPOMT pattern and DNA A0 -Cy5 to the cholDNA XA/tPOMT pattern. The exposure time was kept constant with the previous fluorescence picture to allow for comparison of Langmuir 2010, 26(5), 3753–3759

the intensities. The red fluorescence from the chol-DNA XB-Cy5/ tPOMTþ B0 sample was slightly increased, rather than decreased, which was expected after the repeated rinsing steps (Figure 4d). From the sample containing chol-DNA XA/tPOMTþ A0 -Cy5, a weak yellow/red fluorescence was detected (Figure 4b). Again the red fluorescence must derive from FRET between the tPOMT and the Cy5 dye, showing that hybridization of the A0 -Cy5 with the adsorbed chol-DNA XA/tPOMT has occurred. The weaker FRET signal in Figure 4b compared to Figure 4d could depend on longer distances between the donor (tPOMT) and the acceptor (Cy5) when bound to different strands (chol-DNA XA/tPOMT DOI: 10.1021/la903101v

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Table 1. Film Thickness Measurement from Imaging Ellipsometry Measurements layer

thickness (nm)

PDMS DNA B DNA B-Cy5 DNA B/tPOMT

1.4 ( 0.1 2.4 ( 0.2 3.3 ( 0.2 6.5 ( 1.1

þA0 -Cy5, Figure 4b, instead of the same strand as in Figure 4d chol-DNA XB-Cy5/tPOMT þB0 ). To further analyze and quantify the fluorescence picture, we extracted the average red, green, and blue color content of each picture (Figure 4e). The red color increases in intensity while the green channel decreases when the DNA B0 is hybridized to the chol-DNA XB-Cy5/tPOMT. The total luminosity at the same time increased with 20%. Intensity from the blue channel was zero in all samples, and for the Cy5 fluorophore alone was zero in all channels, as no excitation occurred. Red/green ratios are 0.58, 0.83, 1.55, and 2.12 in Figure 4a-d, indicating a considerable modification of conditions for FRET. Chol-DNA chips with single-stranded chol-DNA B was, as expected, slightly more sensitive to rinsing, resulting in less adsorbed material and longer exposure time (data not shown), compared to double-stranded chol-DNA containing two cholesterol tags. A single cholesterol anchor has a weaker interaction with the surface, but retains the ability to bind the cDNA B0 . Film thickness measurements were performed with imaging ellipsometry on the chol-ssDNA samples (Table 1). Chol-DNA B has an average film thickness of 2.4 ( 0.2 nm after rinsing, compared to chol-DNA B in complex with tPOMT with an average film thickness of 6.5 ( 1.1 nm. A dsDNA strand 30 bp long is expected to be ∼10 nm (3.4 A˚ bp-1), and the tPOMT alone is expected to be around 2-3 nm. Our interpretation is that cholDNA B is packed in a spaghetti like stack, while the tPOMT stabilizes the ssDNA forming a complex stretched out from the PDMS patterned substrate, resulting in a thicker film. We have previously shown that tPOMT in solution with a 20 bp dsDNA forms clusters, 8-18 nm in hydrodynamic diameter, containing a few DNA strands and tPOMT molecules.18 The imaging ellipsometry measurements could indicate that the DNA/tPOMT clusters are rearranged to individually attached duplexes.

4. Discussion The adsorption of DNA to PDMS-patterned substrates is due predominately to interactions between the cholesterol tags and the PDMS residuals, as shown by fluorescence microscopy (Figure 2a). It is also clear (Figure 2b) that untagged DNA interacts with the hydrophobic PDMS pattern, albeit to a much less degree (Figure 2a). Thus adsorbed DNA strands are easily washed away, as compared to the chol-DNA. Chol-DNA can form layers on PDMS that are dense enough to prevent further adsorption of, for example, non-cDNA to the underlying hydrophobic regions (inset in Figure 2b). No adsorption to the more hydrophilic glass or SiO2 surface between the PDMS domains could be detected with fluorescence microscopy. This was further supported by the iSPR measurements where no response was seen from the areas lacking the PDMS stain, neither for the chol-DNA or for the complementary strands. The adsorbed chol-DNA could hybridize with its complementary strand, seen both with fluorescence microscopy and iSPR. tPOMT can most probably bind along the full length of the adsorbed chol-DNA strand. However, the distance and orientation between Cy5 and tPOMT will influence the degree of FRET. 3758 DOI: 10.1021/la903101v

Since the chips with ssDNA exhibited a slightly weaker fluorescence and also were more sensitive to rinsing, we decided to focus on doubly anchored chol-DNA. Modification with more than one cholesterol tag per ssDNA molecule might be a means of increasing the interaction with the surface. However, in experiments with a set of chol-DNA X with a mirrored sequence shift, where the hybridized dsDNA had cholesterol in the middle of the chol-DNA XB strand, hybridization with DNA B0 to immobilized chol-DNA was not seen. Therefore we conclude that end modification is preferable. We have seen that a PDMS-anchored chol-DNA chip can be stored (for several days) with retained hybridization capacity, an important feature for a DNA chip. Another essential feature for a useful DNA array is the possibility for adsorption of more than one chol-DNA strand to the array. This could be easily achieved using, for example, a multichannel flow system or by means of piezodispensation. Another essential feature is the ability to detect species present at low concentrations. We have previously determined the binding constants of DNA oligomers to another CPE, to fall in the range 10-100 nM.21,22 The DNA affinity, however, is not yet known for tPOMT, but is presumably similar. The weaker red fluorescence detected from the sample containing chol-DNA XA/ tPOMT þ A0 -Cy5 in Figure 4b, where the Cy5 dye was conjugated onto the complementary strand, compared to DNA XBCy5/tPOMTþ B0 , could be due to the longer distance between the donor tPOMT and the acceptor Cy5. The increased intensity in Figure 4d, in comparison with Figure 4c, is a possible indication of fluorescence signal amplification or super lightning, previously reported by Leclerc and co-workers. This effect was reported to improve the detection limit of hybridization down to the zepto molar range.8 In their work, a similar CPE forms large clusters with a dye-labeled ssDNA, ∼100 nm in hydrodynamic radius. When a small amount of cDNA strands are hybridized with the aggregate (one complementary ssDNA to one ssDNA/CPE aggregate), a collective and amplified FRET response occurs. The short distance between the large amounts of acceptors (the dye) in these aggregates is probably one reason for the amplification. These clusters could be covalently attached to functionalized glass substrates, and, upon hybridization with a small amount of the complementary strand, fluorescence signal amplification was again detected.23 In our system, however, a much thinner layer is adsorbed on the patterned surface, as has been shown both from iSPR and imaging ellipsometry (∼ 7 nm thick). A close-packed system is a prerequisite for fluorescence amplification to occur. This could be the case in Figure 4d, but in our system the molecules are closely packed in a dense surface film rather than as aggregates in solution.

5. Conclusions Both single-stranded and double-stranded DNA, tagged with one or two cholesterol molecules, adsorb to the hydrophobic regions on a surface energy patterned substrate. The adsorbed DNA retains its ability to specifically hybridize with its cDNA strand. The CPE tPOMT can be used to detect adsorption of the surface-bound DNA. tPOMT can also be used to detect hybridization with the complementary strands, both single-handedly and via FRET with a dye-labeled DNA strand. The FRET (21) Bjork, P.; Persson, N. K.; Peter, K.; Nilsson, R.; Asberg, P.; Inganas, O. Biosens. Bioelectron. 2005, 20, 1764–1771. (22) Karlsson, K. F.; Asberg, P.; Nilsson, K. P. R.; Inganas, O. Chem. Mater. 2005, 17, 4204–4211. (23) Najari, A.; Ho, H. A.; Gravel, J. F.; Nobert, P.; Boudreau, D.; Leclerc, M. Anal. Chem. 2006, 78, 7896–7899.

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measurements indicate that fluorescence signal amplification may occur in the present system, thus lowering the detection limit of DNA hybridization. The DNA chip retains its ability for hybridization with its complementary strand for at least 4 days. Acknowledgment. We thank Andreas A˚ slund, Peter Konradsson, and Peter Nilsson, IFM, Link€oping University, for synthesizing tPOMT. We also thank Ida Hederstr€om, Mikael Karlsson, Louise Carlsson, Erik Nelsson, Noomi Altg€arde, and

Langmuir 2010, 26(5), 3753–3759

Article

Andreas Skallberg for the preliminary study of cholesterol-tagged DNA adsorption to PDMS surface energy pattern. Funding from the Swedish Strategic Research Foundation SSF through OBOE, the Organic Bioelectronics Research Center, is likewise acknowledged. Instrument grants from the Knut and Alice Wallenberg Foundation made experiments possible. The Swedish Research Council (VR) is acknowledged for financial support to the construction of the iSPR instrument. The content of this work is the sole responsibility of the authors.

DOI: 10.1021/la903101v

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