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J. Phys. Chem. C 2010, 114, 1306–1311
Detection of Hybridization on Nanografted Oligonucleotides Using Scanning Near-Field Infrared Microscopy Ilona Kopf,† Christian Grunwald,‡,§,⊥ Erik Bru¨ndermann,† Loredana Casalis,‡,| Giacinto Scoles,‡,§,| and Martina Havenith*,† Physical Chemistry II, Ruhr-UniVersity Bochum, Bochum, Germany, ELETTRA, Sincrotrone Trieste S.C.p.A., Trieste, Italy, Scuola Internazionale Superiore di Studi AVanzati (SISSA), Trieste, Trieste, Italy, and Italian Institute of Technology (IIT)-SISSA Unit, Trieste, Italy ReceiVed: July 18, 2009; ReVised Manuscript ReceiVed: October 20, 2009
We demonstrate that scattering scanning near-field infrared microscopy (s-SNIM) is a label free analytical method allowing hybridization detection of nanografted DNA patches on a sub-µM scale. On the basis of their distinct dielectric properties in the IR, we can distinguish between single stranded and double stranded DNA. The sensitivity of s-SNIM is found to be increased by 7 orders of magnitude compared to conventional FTIR spectroscopy due to the tip enhanced interaction with the substrate. I. Introduction Solid phase or surface hybridization is the basis of modern microarray and biosensing technologies. These tools function through detection of interactions between nucleic acids immobilized on a solid support (probe) with analyte nucleic acids (target) present in solution. The development of DNA microarrays or biochips revolutionized biological analysis, e.g., enabling simultaneous analysis of an entire genome in a single experiment. In contrast to traditional filter-based assays the miniaturized biochips permit the use of much smaller reaction volumes resulting in low sample consumption and an increased concentration of the nucleic acid reactants resulting in accelerated hybridization kinetics. Additionally, a massive parallelization and faster analysis time became reality. Further miniaturization would allow a reliable detection of weakly expressed genes, PCR-less single cell analysis, or even single molecule DNA detection schemes. Typical spot sizes in current microarrays are in the range of 10-300 µm in diameter. In order to achieve a higher spot density and more complex arrays spot sizes must be reduced. Technologies developed for submicrometer spots are based on scanning probe microscopy (SPM). SPM is able to visualize surface topography with high spatial resolution (reading) but additionally it is capable of manipulating and producing nanostructures on surfaces (writing). For fabrication of nanostructures on surfaces, different scanning probe lithography approaches have been developed: dip-pen nanolithography (DPN),1 meniscus force nanografting,2 nanoshaving, nanopen reader and writer (NPRW), and nanografting.3,4 Following the pioneering work of Liu and co-workers,3 nanografting has now become a well established nanopatterning technique, suitable also for the fabrication of DNA nanostructures.5-9 During scanning under high loading forces, a tip of an * To whom correspondence should be addressed. E-mail:
[email protected]. † Ruhr-University Bochum. ‡ ELETTRA. § SISSA. | IIT-SISSA. ⊥ Present address: Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe University, Frankfurt, Germany.
atomic force microscope (AFM) removes local molecules from a self-assembled monolayer (SAM) and thiolated molecules from the surrounding solution chemisorb onto the freshly exposed area following the scanning track of the AFM tip. Different from spontaneous, unconstrained self-assembly in nanografting, the self-assembly process is tip-induced and spatially confined, and occurs in a high-pressure, high-temperature local environment. Therefore, the kinetics of formation of the molecular SAM produced by nanografting is accelerated with respect to the spontaneous SAM, showing as a consequence a reduced amount of defects.10,11 On the basis of AFM reading, i.e., topographic height and compressibility of the nanopatches, Mirmomtaz et al. have shown in a recent work that DNA nanopatterns produced by nanografting are intrinsically better ordered than spontaneously formed DNA SAMs. The increased order reflects a higher hybridization efficiency, at any probe density.8 Their results envisage the use of such nanodevices for quantitative analysis of PCR free single cell genetic material. However, due to the lack of chemical information and the “poor” lateral resolution, standard AFM cannot give a direct and precise evaluation of probe densities and hybridization efficiencies, if high lateral resolution in liquid environment has not been implemented.12,13 Infrared (IR) spectroscopy is a well-known tool to characterize DNA bases, morphological changes in DNA, and binding of drugs and proteins to DNA in solution assays.14-16 In this paper we investigate the possibilities for label free detection of hybridization using the chemical fingerprint of DNA in the IR frequency range.17,18 Investigation of nanometer sized DNA patches with IR spectroscopy is an experimental challenge, because the inherent diffraction limit is in the range of 5-10 µm for IR wavelength. Scattering scanning near-field infrared microscopy (s-SNIM) is a scanning probe microscopy (SPM) based technique which overcomes the diffraction limit while maintaining its spectroscopic capabilities. Previous studies have shown that s-SNIM can provide information on the material composition of e.g. thin polymer films and allows subsurface imaging of implanted charges in semiconductors. The potential of s-SNIM to characterize biological surfaces was previously demonstrated on SAMs,19,20 lipids,21 and of a single virus22 well below the diffraction limit.
10.1021/jp906813f 2010 American Chemical Society Published on Web 12/28/2009
Hybridization on Nanografted Oligonucleotides
J. Phys. Chem. C, Vol. 114, No. 2, 2010 1307
Our results show that s-SNIM is able to provide a label-free detection of hybridization of only 102-103 DNA molecules. We can demonstrate that the method combines high sensitivity with sub-µm lateral resolution. II. Experimental Methods Sample Preparation. All oligonucleotides were purchased from Biomers in HPLC grade and were used without further purification. We investigated the sequences HS-C6H12-5′-AGA TCA GTG CGT CTG TAC TAG CAC A-3′ for the thiolderivatized single-stranded oligonucleotide (probe DNA) and 5′-TGT GCT AGT ACA GAC GCA CTG ATC T-3′ for the complementary sequence (target DNA). 6-Mercapto-1-hexanol (MCH, g97%) was purchased from Fluka. STE-buffer was composed of the following compounds: 1 M NaCl (Baker), 10 mM Tris (Fluka), and 1 mM EDTA (Fluka) with the pH adjusted to 7.2. Only Milli-Q-water (resistance >18 MΩ/cm) or HPLC water (Baker) was used for buffer preparation. Immediately before use the buffer was additionally purified by a 0.22 µm pore size filter (syringe filters, Carl Roth). Gold coated n(100) silicon (Anfatec Instruments GmbH) was first cleaned with hot piranha solution (3:1 mixture of 96% H2SO4 and 30% H2O2) for approximately 15 min, thoroughly washed with HPLC water and stored in HPLC water. For preparation of ss-DNA SAMs the cleaned substrates were dipped into freshly prepared 1 µM probe DNA in STE-buffer for 1.5 min. Subsequently, the substrates were incubated in 1 mM MCH in STE-buffer for 30 min. Incubating with MCH molecules substitutes unspecific interactions between the oligonucleotide strands and gold surface.23 For FTIR measurements homogeneous MCH SAMs were prepared by incubation of a clean gold substrate for 30 min in 1 mM MCH in STE-buffer and used as reference sample. Ultra flat gold substrates were prepared using the template stripping method.24 Glass slides (BK7, Menzel) were cut into pieces of 5 mm × 15 mm and glued to a gold coated n(100) silicon wafer using an epoxy glue (Epo-tek 377, Epoxy Technology) that was cured 2 h at 150 °C. Immediately prior to use, the gold-silicon interface was opened mechanically and the glass supported template stripped gold (TSG) surface was used for DNA/MCH SAM preparation as described above. All nanografting experiments were carried out with a SolverPro AFM (NT-MDT) using a closed liquid cell in contact mode. For the nanografting process “hard” NSC36C cantilevers (MikroMasch, nominal force constant 0.6 N m-1, tip radius < 10 nm) were employed. Nanografting was performed using either a 0.1 mM MCH solution in STE-buffer or 10 µM probe DNA in a 1:1 mixture (V/V) of STE-buffer and absolute ethanol. Several areas were selected for nanografting. Loading forces from 50 to 80 nN were chosen to locally replace the SAM molecules by molecules from the solution. We used two successive nanografting processes to fabricate a patch-in-a-patch structure. In the first step MCH molecules are nanografted into a probe DNA SAM as described above. Afterward DNA is nanografted into the previously created MCH patches using a concentration of 10 µM probe DNA in a 1:1 mixture (V/V) of STE-buffer and absolute ethanol. After each nanografting process the sample was thoroughly washed with STE-buffer. Double-stranded DNA were obtained after incubating the immobilized probe DNA for 6 h at room temperature in STE-buffer containing 1 µM target DNA. AFM in Liquid. AFM images in liquid were recorded using a SolverPro (NT-MDT) AFM equipped with a closed liquid cell. Characterization of DNA nanostructures in liquid were
Figure 1. Experimental setup of our s-SNIM. fL ) focal length, OM ) off-axis parabolic mirror, M ) mirror, f ) resonance frequency, MCT ) mercury cadmium telluride detector, CaF2 ) calcium fluoride.
carried out in contact mode using a “soft” CSC38B cantilever (MikroMasch, nominal force constant 0.03 N m-1, tip radius < 10 nm) and low loading forces (typically
