By mimicking Nature's way of utilizing multivalent interactions, we introduce in the present work a novel method to improve the strength of cholesterol-based DNA coupling to lipid membranes. The bivalent coupling of DNA was accomplished by hybridization between a 15-mer DNA and a 30-mer DNA, being modified with cholesterol in the 3' and 5' end, respectively. Compared with DNA modified with one cholesterol moiety only, the binding strength to lipid membranes appears to be significantly stronger and even irreversible over the time scale investigated ( approximately 1 hr). First, this means that the bivalent coupling can be used to precisely control the number of DNA per lipid-membrane area. Second, the strong coupling is demonstrated to facilitate DNA-hybridization kinetics studies. Third, exchange of DNA between differently DNA-modified vesicles was demonstrated to be significantly reduced. The latter condition was verified via site-selective and sequence-specific sorting of differently DNA-modified lipid vesicles on a low-density cDNA array. This means of spatially control the location of different types of lipid vesicles is likely to find important applications in relation to the rapid progress currently made in the protein chip technology and the emerging need for efficient ways to develop membrane protein arrays.

Much effort is currently concentrated on research devoted to biofunctional patterned surfaces, which constitute the fundament for the development of microarrays for high-throughput gene and protein analyses. DNA microarrays have proved very successful, [1] and the concept is in the process of being applied to protein arrays. [2] However, in contrast to DNA fragments, proteins are easily denatured in contact with solid supports, and robotic printing of proteins onto chemically reactive glass slides [3] will not necessarily be applicable as a generic protocol for the preparation of protein arrays. Supported phosphatidylcholine lipid bilayers have emerged as interesting candidate substrates for protein chips, since they efficiently reduce nonspecific protein adsorption [4, 5] and, at the same time, allow different strategies for protein immobilization with biospecific water, desalted with a NAP5 column (Amersham Pharmacia, USA) according to manufacturer's protocols, and stored as working stock solutions at À 20 8C until use. Epoxy-derivatized slides were prepared from plain glass slides (Sigma, USA) as previously described. [9] Nhydroxysuccinimide slides were also used to spot the proteins but consistently gave inferior results. Proteins were prepared in NaHCO 3 buffer (0.1 M, pH 9) and arrayed on epoxy slides with a spacing of 180 mm between the spots by using an statistical microarray analysis arrayer (Engineering Services Inc., Ontario, Canada). After a 2-hour incubation period the slides were either used immediately, or stored for future use at 4 8C. The slides, if stored, were typically used within 48 h of printing.Unless otherwise indicated, probing and reactions on slides were performed as follows: Before use, the slides were quenched by treatment with phosphate-buffered saline (PBS) and glycine (0.5 M) on a shaker for 10 min. The slides were blocked with PBS, glycine (0.5 M), and bovine serum albumin (BSA; 1 % w/v) for 20 min, then washed with distilled water and air dried. The labeled probe was then applied: a mixture containing the probe (2 mM) was prepared by adding stock probe solution (0.5 mL, 200 mM) to tris(hydroxymethyl)aminomethane (Tris) buffer (49 mL, 50 mM, pH 8), and BSA (0.5 mL, 1 % w/v). The resulting mixture was applied to each slide by the coverslip method [9] and incubated for 30 min in the dark. The excess probe was washed off after incubation with distilled water, and the slides were subsequently washed with PBS that contained Tween (0.2 % v/v) for 15 minutes on a shaker. The slides were then washed with distilled water, air dried, and scanned with an ArrayWorx microarray scanner (Applied Precision, USA) at 548/595 nm. For the PMSF experiment, each slide was first incubated with freshly prepared PMSF (50 mL, 1 mM in 50 mM Tris, pH 8) for 30 minutes, rinsed extensively with distilled water to remove any free residual PMSF, and screened with FP-Cy3. The inhibition experiments were identical to the probe ± enzyme reactions, except that varying concentrations of trypsin inhibitor (original...

Single-molecule force-spectroscopy methods such as magnetic and optical tweezers have emerged as powerful tools for the detailed study of biomechanical aspects of DNA-enzyme interactions. As typically only a single molecule of DNA is addressed in an individual experiment, these methods suffer from a low data throughput. Here, we report a novel method for targeted, nonrandom immobilization of DNA-tethered magnetic beads in regular arrays through microcontact printing of DNA end-binding labels. We show that the increase in density due to the arrangement of DNA-bead tethers in regular arrays can give rise to a one-order-of-magnitude improvement in data-throughput in magnetic tweezers experiments. We demonstrate the applicability of this technique in tweezers experiments where up to 450 beads are simultaneously tracked in parallel, yielding statistical data on the mechanics of DNA for 357 molecules from a single experimental run. Our technique paves the way for kilo-molecule force spectroscopy experiments, enabling the study of rare events in DNA-protein interactions and the acquisition of large statistical data sets from individual experimental runs.

We present a novel concept for the creation of lipid vesicle microarrays based on a patterning approach termed Molecular Assembly Patterning by Lift-off (MAPL). A homogeneous MAPL-based single-stranded DNA microarray was converted into a vesicle array by the use of vesicles tagged with complementary DNAs, permitting sequence-specific coupling of vesicles to predefined surface regions through complementary DNA hybridization. In the multistep process utilized to fulfill this achievement, active spots consisting of PLL-g-PEGbiotin with a resistant PLL-g-PEG background, as provided by the MAPL process, was converted into a DNA array by addition of complexes of biotin-terminated DNA and NeutrAvidin. This was then followed by addition of POPC vesicles tagged with complementary cholesterol-terminated DNA, thus providing specific coupling of vesicles to the surface through complementary DNA hybridization. Quartz crystal microbalance with dissipation (QCM-D) and optical waveguide lightmode spectroscopy monitoring were used to optimize the multistep surface modification process. It was found that the amount of adsorbed biotinDNA-NeutrAvidin complexes decreases with increasing molar ratio of biotinDNA to NeutrAvidin and decreasing ionic strength of the buffer solution. Modeling of the QCM-D data showed that the shape of the immobilized vesicles depends on the amount of available anchoring groups between the vesicles and the surface. Fluorescent microscopy images confirmed the possibility to create well-defined patterns of DNA-tagged, fluorescently labeled vesicles in the micrometer range.

We present a sensitive and easily regenerated nano-optical sensor based on immobilization of avidincoated colloidal gold particles on a biotin-modified planar lipid bilayer supported on the walls of a quartz cuvette. The so constructed sensing template, being specific for capturing of biotinylated biomacromolecules, is analyzed using optical spectroscopy combined with Mie theory calculations for quantification of the colorimetric changes induced by biorecognition events in the interfacial region of the particles. By further utilizing de Feiter's formalism, which correlates changes in effective refractive index and thickness with adsorbed mass, a good agreement between the Mie theory and experiments is demonstrated. Furthermore, the template is proven sensitive enough to follow the hybridization kinetics of 15-mer fully complementary DNA strands without the introduction of labels or secondary signal amplification.

We have investigated the effect of well-defined nanoscale topography on the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicle adsorption and supported phospholipid bilayer (SPB) formation on SiO2 surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Unilamellar lipid vesicles with two different sizes, 30 and 100 nm, were adsorbed on pitted surfaces with two different pit diameters, 110 and 190 nm, as produced by colloidal lithography, and the behavior was compared to results obtained on flat surfaces. In all cases, complete bilayer formation was observed after a critical coverage of adsorbed vesicles had been reached. However, the kinetics of the vesicle-to-bilayer transformation, including the critical coverage, was significantly altered by surface topography for both vesicle sizes. Surface topography hampered the overall bilayer formation kinetics for the smaller vesicles, but promoted SPB formation for the larger vesicles. Depending on vesicle size, we propose two modifications of the precursor-mediated vesicle-to-bilayer transformation mechanism used to describe supported lipid bilayer formation on the corresponding flat surface. Our results may have important implications for various lipid-membrane-based applications using rough or topographically structured surfaces.

We have investigated the influence of combined nanoscale topography and surface chemistry on lipid vesicle adsorption and supported bilayer formation on well-controlled model surfaces. To this end, we utilized colloidal lithography to nanofabricate pitted Au-SiO(2) surfaces, where the top surface and the walls of the pits consisted of silicon dioxide whereas the bottom of the pits was made of gold. The diameter and height of the pits were fixed at 107 and 25 nm, respectively. Using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique and atomic force microscopy (AFM), we monitored the processes occurring upon exposure of these nanostructured surfaces to a solution of extruded unilamellar 1-palmitolyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles with a nominal diameter of 100 nm. To scrutinize the influence of surface chemistry, we studied two cases: (1) the bare gold surface at the bottom of the pits and (2) the gold passivated by biotinamidocaproyl-labeled bovine serum albumin (BBSA) prior to vesicle exposure. As in our previous work on pitted silicon dioxide surfaces, we found that the pit edges promote bilayer formation on the SiO(2) surface for the vesicle size used here in both cases. Whereas in the first case we observed a slow, continuous adsorption of intact vesicles onto the gold surface at the bottom of the pits, the presence of BBSA in the second case prevented the adsorption of intact vesicles into the pits. Instead, our experimental results, together with free energy calculations for various potential membrane configurations, indicate the formation of a continuous, supported lipid bilayer that spans across the pits. These results are significantly important for various biotechnology applications utilizing patterned lipid bilayers and highlight the power of the combined QCM-D/AFM approach to study the mechanism of lipid bilayer formation on nanostructured surfaces.

We present a new method for quantification of the coupling efficiency between amphiphilic oligonucleotides and suspended small unilamellar lipid vesicles (SUVs). The method employs a supported (phospho)lipid bilayer (SLB)-modified sensor template, which upon exposure to a mixture of SUVs and amphiphilic DNA reacts neither with free SUVs nor with DNA-modified SUVs, but with free DNA only. Using calibration curves obtained by recording the concentration dependence of the initial binding rate of free amphiphilic DNA (in the absence of SUVs), it is demonstrated how concentration determinations of both free and bound DNA in the two-component mixture (amphiphilic DNA and lipid vesicles) can be obtained. The calibration curves and the binding analysis were obtained using a quartz crystal microbalance with dissipation (QCM-D) monitoring. The binding efficiency of DNA coupled to SUVs (Ø approximately 50 nm) with two cholesterol moieties revealed that the bivalent coupling is essentially 100% in the range of approximately 1 to approximately 35 oligonucleotides per vesicle, whereas reversible coupling was confirmed in the case of monovalent coupling. Coupling of DNA via two cholesterol moieties was obtained by prehybridization of two single-stranded DNA strands modified with single cholesterol moieties in their 3' and 5' ends, respectively, and the monovalent coupling was obtained using single-stranded DNA. In the latter case, the analysis of the amount of free DNA at different DNA-SUV ratios also allowed for a determination of the maximum number of available binding sites on the SUVs, shown to be in good agreement with data obtained for DNA coupling on planar surfaces. With the only requirement that the SLB-modified sensor template react with one of the components in the two-component mixture only, as verified through fingerprint analysis of frequency, f, and energy dissipation, D, QCM-D measurements, it is emphasized that the method is generic and offers a fast and reliable method for evaluations of biomolecular modifications of any type of colloidal nanoparticles.