Niepelt et al. Nanoscale Research Letters 2011, 6:511 http://www.nanoscalereslett.com/content/6/1/511 nanowire-based protein sensors could detect streptavidin binding down to a concentration of 2.5 nmol, underlin- ing the capability of ZnO nanowires for the electrical detection of biomolecules. Hereby the nanowire has the character of an active channel in a field effect transistor whose conductivity is governed by the field effect induced by the assembly of charged target molecules on the semiconductor surface. In this study, we will extend the successful biofunctio- nalization of ZnO nanowire surfaces to DNA using the organosilane glycidyloxypropyltrimethoxysilane (GOPS) [14-16]. DNA molecules are negatively charged and therefore applicable for the electrical detection with bio- functionalized nanowires. In contrast to the detection of streptavidin in [10], where the current increases when streptavidin is attached, the current through the intrinsi- cally n-type ZnO nanowire should decrease as the nega- tively charged DNA target molecules induce a depletion zone inside the nanowire. Experimental The ZnO nanowires have been grown via vapor-liquid- solid mechanism in a horizontal tube furnace [17] at a pressure of 150 mbar with a growth temperature of 1,050°C. The nanowires evolve in a typical spaghetti-like structure with wire diameters around 100 nm and wire lengths up to some tens of micrometers. To remove adsorbed species from the nanowires and supply a clean and reactive ZnO surface, the samples have been oxy- gen-plasma-treated for 30 min at 5 Pa oxygen pressure with a plasma energy of 50 W. As this treatment is known to enhance the dissociative adsorption of water on the ZnO surface [18], the samples have been stored in a cabinet dryer at 100°C for at least 5 min before the silane treatment to remove water residues from the surface. Subsequent silanization took place in 10 mM GOPS in a water-free toluene solution (for details on chemicals resource, see Additional file 1). The silanization mechanism is displayed in Figure 1a. The samples have been incubated stirring constantly for at least 6 h at 70° C. The samples have been rinsed three times subse- quently with pure toluene for 5 min in each case and then dried with compressed air. The 5’-amino-modified oligonucleotides were dissolved in 5 × PBS buffer (pH 7.4) with a concentration of 10 μmol for the biofunctionalization. The single-stranded DNA sequences of the applied oligonucleotides consist- ing of 27 bases are shown in Table 1. The fluorescence markers that are linked to the 3’-end and enable the detection of the molecules with fluorescence microscopy are also listed in Table 1. The DNA solution was pipetted onto the nanowire samples and incubated over- night under wet conditions to immobilize the Page 2 of 7 nucleotides on the GOPS surface via the 5’-amino linker (see Figure 1b). Afterward, the samples were rinsed for 10 min in 15 mmol Tris-HCl buffer (pH 8) in order to get rid of unbound DNA and then dried with com- pressed air. To ensure covalent binding following the scheme in Figure 1 takes place, control samples have been pre- pared both leaving out the GOPS silanization step and with DNA that does not have an amino linker. The bio- functionalized nanowires were mechanically transferred to silicon or glass substrates by a gentle pressure imprint of the growth substrate. The as-grown nanowires have been characterized with a JEOL JSM-6490 scanning electron microscope. Subse- quently to the biofunctionalization, the samples were investigated with a Zeiss Axio Imager 2 fluorescence microscopy setup. Fluorescence microscopy is known as an adequate tool for the analysis of DNA monolayers on thin film surfaces as well as on nanowires [9,19]. The microscope makes use of a filter cube to detect marker- specific fluorescence. The samples marked with cyanine 3 (Cy3) dyes turned out to be more photo stable than the fluorescein isothiocyanate (FITC)-marked samples. Thus, we will present here mainly Cy3-marked samples. Results and discussion Dark field and fluorescence images of biofunctionalized ZnO nanowires are shown in Figure 2a,b,c,d. One can clearly see that every nanowire that can be identified in the dark field image is showing a fluorescence signal. However, the luminescence intensities differ extremely for the investigated wires. This can be explained by the fact of different diameters of the nanowires and thus dif- ferent nanowire surface areas. The intensity of the emitted light is also appearing non-uniformly along the nanowire axis, as clearly visible on the wire highlighted in Figure 2d. These intensity fluctuations might be owing to only fragmentary DNA coverage or variations of the nanowire thickness. In Figure 3b, the fluorescence intensity along the nanowire axis (dotted line) in Figure 3a is shown. It is clearly visible that the fluorescence intensity is not only fluctuating but even disappearing in some regions of the wire. This leads to the assumption that the functionalized nanowire might not be comple- tely covered with DNA. In Figure 3c,d, detailed scanning electron microscope (SEM) pictures of a typical ZnO wire used in this study are shown. One can see that the wire itself has a non- uniform thickness, varying up to a factor of 2. The detailed surface morphology of the functionalized wires is not clearly resolvable within an SEM; however, a uni- form coverage of the nanowire is noticed. A respective SEM image can be found in the supplementary informa- tion (Additional file 1). Thus, we can also trace back a