In case of a two spin system we have first experimentally studied and measured connectedness and separation defined by the EQP (Extended Quantum Particle) model. We have prepared an EPR state of the labelled chloroform 1H and 13C nuclear spins and measured the related density matrix by NMR quantum state tomography. We discuss some consequences of these experimental findings for quantum computing and molecular recognition processes.

[1] I. L. Chuang et al., Proc. R. Soc. Lond. A 1998 454, 447-467
[2] Z. Árvay, presentation on EUCOST conference and Scientific Report COST Action MP0802, 2009

M-DNA is a type of metalated DNA obtained from the complexation of base pairs with metal ions. Experiments carried-out on Zn-DNA suggested a metallic behavior, with an insulator-metal transition from the natural DNA. We inspect if and how the possible electronic coupling between the metal and the nucleic acid bases may be responsible for such a transition [1]. The effect of other metals such as Cu and Ag [1] is also taken into account to inspect which metal elements are most suitable to perturb the electronic structure of DNA in a desired way. An alternative way to go beyond the limits of native-DNA for nanotechnology impact is to design and syntesize more conductive structures by chemical modifications of the nucleobases. One particular approach relies on the expansion of each natural base with a benzene ring that is covalently bonded to the base and co-planar with it: xDNA [2]. Recent findings reported enhanced stacking interactions in xDNA duplexes and suggested they might be suitable molecular-wires. Here we investigate possible ways of tailoring the DNA electronic structure by combining the metal-doping and aromatic insertion, namely M-DNA and M-xDNA (Figure 1). DFT calculations of the electronic structure are performed, with several localized basis sets, different exchange-correlation functionals, with and without implicit solvent. We analyze the stability properties of salient selected structures. We show [1,3,4] efficient hybridization between the metal and the natural (or expanded) bases, with the possibility of gap modulations (shrinking or enlargement) by using different metal species, which may be the key to electrical non-linearities toward DNA-based nano-circuits. This line of research can also complement the experimental investigation of M-DNA via electron tunneling spectroscopy [4].

Fig 1. DNA, M-DNA, xDNA, M-xDNA (from left to right) schemes and optimized structures

[1] G. Brancolini and R. Di Felice, “Electronic properties of metal-modified DNA base pairs”. J. Phys. Chem. B 112, 14281 (2008).
[2] J. Gao, H. Liu, E. T. Kool, “Assembly of the Complete Eight-Base Artificial Genetic Helix, xDNA, and Its Interaction with the Natural Genetic System” , Angew. Chem. Int. Ed. 44, 3118 (2005).
[3] G. Brancolini and R. Di Felice, “Combined effects of metal complexation and size expansion in the electronic structure of DNA base pairs”, (manuscript in preparation).
[4] (a) G. Brancolini, E. Shapir, A. B. Kotlyar, R. Di Felice, D. Porath, “Evidence of gap modulation in DNA by complexation with metal ions”, (manuscript in preparation). (b) E. Shapir, A. Calzolari, C. Cavazzoni, D. Ryndyk, G. Cuniberti, A. B. Kotlyar R. Di Felice, and D. Porath, “Electronic structure of single DNA molecules resolved by scanning tunneling spectroscopy”, Nature Materials 7, 68 (2008). 9

DNA adsorption on electrode surfaces is of fundamental interest for the development of DNA-based biosensors. The adsorption and the redox behaviour of thrombine binding aptamer (TBA) and extended TBA (eTBA) were studied using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite and glassy carbon. The different adsorption patterns and degree of surface coverage were correlated with the sequence base composition, presence/absence of K+ and voltammetric behaviour of TBA and eTBA. In the presence of K+ only a few single-stranded sequences present adsorption, while the majority of the molecules form stable and rigid quadruplexes with no adsorption. Both TBA and eTBA are oxidized and the only anodic peak corresponds to guanine oxidation. Upon addition of K+ ions TBA and eTBA fold into quadruplexes, observed by the decreasing of guanine oxidation peak and occurrence of a new peak at higher potential values due to the oxidation of G-quartets. The higher oxidation potential of G-quartets is due to the difficulty for electrons transfer from the inside of the quadruplex to the electrode surface than from the more flexible single-strands.

Atomic force microscopy (AFM) is conceptually simple, yet very powerful microscopy technique, which is essential for studying programmable self-assembly process of DNA and RNA nanostructures. Although originally developed for based on tunneling effects in near contact of semiconductors it is now mostly used to image non-conducting samples particularly organic as well as inorganic specimens at the atomic and molecular scale by measuring the forces between the tip and the sample. Since AFM can produce high- resolution topographical images at the nanometer level without altering the sample with stains, it bypasses the problem faced by other techniques such as the scanning electron microscopy. Here we will discuss using atomic force microscopy for various biologically relevant applications. Particularly it becomes an essential analysis in rationally design of artificial DNA and RNA 2D scaffolds. Additionally we will present imaging of protein crystals, surfactants as well as living organisms where AFM serves as a platform for biomaterials surface analysis. Very important is to identify challenges and advantages of AFM over other compatible methods and how important is understanding of your molecule to molecule and sample to surface interactions.

Raman spectroscopy is an important analytical tool as it allows the highly specific detection and identification of molecules and even biological particles. However, the Raman effect is intrinsic weak, but Raman signals get efficiently enhanced by the interaction of molecules with a nanostructured metallic surface (for example metal nanoparticles, roughened metallic electrodes or metal island films) yielding in the so-called surface enhanced Raman spectroscopy (SERS). [1] Through the combination of the SERS effect with scanning probe microscopy (SPM) the so-called tip enhanced Raman spectroscopy (TERS) is created. This technique combines the spatial resolution of various SPM techniques (such as atomic force microscopy – AFM or scanning tunneling microscopy – STM) with the highly specific spectroscopic information of Raman spectroscopy at a nanometer scale. [2]
Within this contribution the application of TERS for the scanning and identification of biological (sub-µm) compartments is presented. First investigations within our group of this technique towards the investigation of complex biomolecules were performed on single bacterial cells, illustrating the great capability for nanobiological applications. However, spectral fluctuations in band position and intensity are interpreted as indication of fluctuations of molecules within the outer cell membrane. [3] A further milestone towards powerful nanobioanalytics are our TERS investigations of single virus particles, being a promising tool for single virus identification in future work. [4] Moreover the TERS technique was applied for investigations of supported lipid structures and human cells. [5]

[1] Hering, K.; Cialla, D.; Ackermann, K.; Doerfer, T.; Moeller, R.; Schneidewind, H.; Mattheis, R.; Fritzsche, W.; Roesch, P.; Popp, J. Analytical and Bioanalytical Chemistry 2008, 390, 113-124
[2] Deckert-Gaudig, T.; Bailo, E.; Deckert, V. Journal of Biophotonics 2008, 1, 377-389.
[3] Neugebauer, U.; Roesch, P.; Schmitt, M.; Popp, J.; Julien, C.; Rasmussen, A.; Budich, C.; Deckert, V. ChemPhysChem 2006, 7, 1428- 1430.
[4] Cialla, D.; Deckert-Gaudig, T.; Budich, C.; Laue, M.; Moeller, R.; Naumann, D.; Deckert, V.; Popp, J. Journal of Raman Spectroscopy 2009, 40, 240-243
[5] Böhme, R.; Richter, M.; Cialla, D.; Rösch, P.; Deckert, V.; Popp, J. Journal of Raman Spectroscopy 2009, 40, 1452-1457.

Direct digital counting (DDC) of biomolecules offers sizable advantages over existing analog technologies in terms of accuracy, sensitivity, robustness and cost-effectiveness. Nanobarcodes are naometer-sized structures with high combinatorial diversity that can be used for DDC to compile of virtual biomolecule inventories from biological samples. Such direct barcoding approach opens unique new opportunities for biomedical applications. Fluorescent nanobarcodes rely on conventional microscopic technologies for their optical detection and identification. These fluorescent structures, or nanostrings, are already finding commercial applications and outperforming existing bioanalytical techniques. Electronic nanosensors with dimensions comparative to those of individual biomolecules are becoming feasible at the latest nodes of CMOS fabrication. Nanobarcodes for electronic detection with such electronic nanosensors would further advance miniaturization, cost-efficiency and thoroughput, however, they require fundamentally different technological approaches. One specific technological difference is the requirement to dynamically control the passage of the nanobarcodes through an electronic nanodetector. To address this challenge, model dumbbell structures were developed and characterized and their precise dynamic control was achieved and characterized via magnetophoresis.

Biography
Dr Dimitrov is a Group Leader within the University of Queensland’s, Australian Institute for Bioengineering and Nanotechnology (AIBN) a position he has held since 2006. Prior to this he was the Founder and CTO of NanoString Technologies in Seattle (2003-2006), a company he founded to commercialize his invention of fluorescent nanobarcodes for single molecules. Prior to NanoString, Dr. Dimitrov was the Director of the DNA Microarray Laboratory at the Institute for Systems Biology in Seattle (2000-2003), and played a significant role in the formation and early growth of the Institute. During his research career Dr. Dimitrov has made many significant research discoveries. Most importantly he invented and pioneered the barcodes for single-molecule detection, which are currently on the market and finding diverse applications in biomedical research, diagnostics, biosurveillance. Dr. Dimitrov's current focus is on a new generation of nanobarcodes that combine magnetophoresis and electronic detection and hold the promise to be significantly smaller, less costly, and more user-friendly than the NanoString product. Dr. Dimitrov holds a Ph.D. in Biochemistry from Baylor College of Medicine

An electronic microarray has been used to carry out directed self-assembly of higher order 3D structures from biotin/streptavidin, DNA and enzyme derivatized nanoparticles. Structures with up to fifty layers of alternating biotin and streptavidin, DNA and enzyme nanoparticles were fabricated using a 400 site CMOS microarray system. In this process, reconfigurable electric fields produced by the microarray were used to rapidly transport, concentrate and accelerate the binding of 40 and/or 200nm nanometer bio-derivatized nanoparticles to selected sites on the microarray. The nanoparticle layering process takes less than one minute per layer. The nanoparticle addressing/binding process was monitored by changes in fluorescence intensity as each nanoparticle layer was deposited. The final multilayered 3-D structures are about two microns in thickness and 50 microns in diameter. More recently we have successfully fabricated enzyme- nanoparticle layers with streptavidin-alkaline phosphatase, glucose oxidase-avidin, and streptavidin-HRP. Up to 47 layers were addressed with 200nm nanoparticles and enzyme activity was retained in the assembled structure. Most recently, we have used AC dielectrophoretic techniques on a 100 site microarray device to carry out the manipulation and positioning of DNA derivatized nanopartilces. This work represents a unique example of combining “top-down” and “bottom-up” technologies into a novel nanofabrication process. Such processes will be useful for the hierarchal assembly of 3D nano, micro, and macrostructures for a variety of electronic/photonic, nanomaterials, energy and biosensor applications.

Michael J. Heller received his Ph.D. in Biochemistry from Colorado State University in 1973. He was an NIH Postdoctoral Fellow at Northwestern University from 1973 to 1976. Dr. Heller was supervisor of the DNA Technology Group at Amoco Corporation from 1976 to 1984, and then the Director of Molecular Biology at Molecular Biosystems, Inc., from 1984 to 1987. He then went on to Integrated DNA Technologies, where he served as President and Chief Operating Officer from 1987 to 1989. He was the Chief Technical Officer at Nanogen, Inc., located in San Diego, California form 1993 to 2001. Dr. Heller is now a professor in the departments of Bioengineering and Nanongineering at the University California San Diego. He also serves as a consultant to Nanogen. Dr. Heller has extensive industrial experience in biotechnology; with particular expertise in the areas of DNA probe diagnostics, DNA synthesis, and fluorescent-based detection technologies. He has been the founder of several high technology companies. Nanogen Inc., the most recently formed is directed at the development of novel microelectronic DNA array technology. He has numerous patents and publications related to his work in biotechnology, on DNA arrays and in the medical diagnostics areas. Dr. Heller has also served on several review panels for the National Nanotechnology Initiative.

The mechanical properties and the deformation behaviour of biological cells are mainly determined by the cytoskeleton. These properties are closely linked to many biophysical and physiological events in the cells, and may, thus, be exploited as potent biomarkers.
We propose an advantageous approach to characterize cellular mechanical properties: through the application of dielectrophoretic forces, different types of cells are stretched between microelectrodes. We first apply our newly developed dielectrophoretic stretcher to compare cell lines that have been derived from malignant and non-malignant tissues. Small numbers of human cells of cancerous origin and from related non-cancerous tissue were sufficient to obtain data that allowed for unambiguously distinguishing these cells. By applying our technique to malignant and non-malignant mouse fibroblasts, we observed a deformation response to the electric field that confirmed the results obtained from the human cells. In both cases, non-malignant cells turned out to be softer than their malignant counterparts. A detailed analysis of the dielectric properties of the human cells using dielectrophoretic spectroscopy showed that the differences in the stretching response are due to cell-specific mechanical properties and do not originate from differences in the polarisability. In order to elucidate which parts of the cytoskeleton of the two cell types are mainly responsible for the different stretching behaviour, an actin- and a microtubule-specific toxin were added to the cancerous and non-cancerous cells. Specific microtubular structures of the two cell types were identified as the major cause for the behaviour observed.
The heating induced by the electric field was analysed through measuring the temperature distribution on the chip by infrared thermography. It showed that our system produces a negligible temperature increment in the voltage and buffer electric conductivity range chosen for the experiments. The technique is, thus, suitable for working with living cells.
Finally, the integration of our dielectrophoretic stretcher into a microfluidic system was realised and is described.
Our approach shows enormous potential, making it attractive for numerous biomedical diagnostic purposes.

Specific interaction with phosphorothioate residues (PS) was used to connect two silver nanoparticles to opposite ends of the double stranded DNA. The double stranded poly(dA)-poly(dT) and poly(dG)-poly(dC) containing 5 PS residues at both 5' ends of these double stranded DNA was synthesized using enzymatic method developed by us earlier (1). The length of the DNA produced using this method can be varied from several nanometers up to microns. We have demonstrated that incubation of 100 bp (PS)5-(dG)100- (dC)100(PS)5 with 15 nm Ag-NPs yielded a mixture of particle monomers, dimers, trimers etc. In the conjugates each pair of Ag-NPs is bridged by a single DNA molecule. The relative ratio between conjugates containing different number of particles depends on a particle to DNA molar ratio and can be varied widely. The individual conjugates can be efficiently separated into discrete bands by the gel (see Fig. 1). The fractions extracted from the gel slices were further purified by size-exclusion HPLC and visualized by TEM. A vast majority of molecules in bands 1, 2 and 3 (see Fig. 1 A) correspond to particle monomers, dimers and timers correspondingly (Fig. 1B, C and D). We performed experiments with shorter poly(dG)-poly(dC), (PS)5- (dG)30-(dC)30(PS)5 and with poly(dA)-poly(dT) of different lengths and obtained similar results. We have also demonstrated in preliminary experiments that the particles can also be connected by end-functionalized parallel stranded G4-DNA structures.
Conductivity measurements of the DNA-NP conjugates are on the way. The conjugates upon the formation of a bridge between the two metal electrodes will connect them electrically. This will hopefully enable us to directly measure electrical current trough various DNA structures and to address the issue of charge transfer in DNA.

[1] Borovok N, Molotsky T, Ghabboun J, Porath D, Kotlyar A. Efficient procedure of preparation and properties of long uniform G4- DNA nanowires. 2008 Anal Biochem. 374, 71-78.

Based on the i-motif structure, a DNA quadruplex which is formed by DNA containing stretches of cytosine under slight acidic conditions, we have demonstrated a DNA nanomotor driven by pH-change and shown its working ability. We have also employed this DNA nanmotor to fabricate functional devices such as: pH trigged DNA nanocompartment, enthalpy-driven three-State switching of superhydrophilic/ superhydrophobic surface, photo-pH dually modulated fluorescence switch and controllable assembly of gold nanoparticles.¹

In this talk, we will present our recent studies on DNA motors and smart materials based on i-motif structures. As shown in Figure above (left), using an untraditional three electrodes electrochemical devices, the DNA motor could also be actuated by electric signals. This result will benefit to the integration of DNA motors into conventional silicon based nanodevices for future applications.² As illustrated in Figure above(right), we have prepared a pH responsive hydrogels entirely made of DNAs. This hydrogel is formed by cross-linking a single type DNA Y-unit building block through the formation of inter-unit i-motif structures. The fast transformation of i-motif has enabled such DNA gel to respond quickly to environmental pH changes to controllably trap and release cargos (i.e. GNPs here) in a pH-dependent manner. In principle, other materials, such as nanoparticles, therapeutic proteins, polymers and even protein producing system could be incorporated into such DNA gel system, allowing the development of functional, responsive biomaterials that have applications in a wide range of disciplines, biosensing, tissue engineering, nano- mechanical devices, and drug delivery etc.³

[1] [1] Liu, H.; Liu, D.; Chem. Commun., 2009, 2625-2636.
[2] Yang, Y.; Fan, C.; Liu, D. et al. Nano Letters 2010, in press.
[3] Enjun Cheng, Dejian Zhou, Dongsheng Liu, et al. Angew. Chem., Int. Ed. 2009, 48, 7660-7663.

We have developed a series of encapsulated SERS-active labels (SERS nanotags) and several different assay formats that are designed to be used with small or handheld Raman readers for chemical and biochemical sensing applications. We will describe the basic nanomaterials-driven compositions, highlighting the challenges and successes in synthesis, characterization, and scale-up. We will then highlight current applications, ranging from in vivo imaging to ultrasensitive detection of proteins and DNA to detection of low molecular weight species such as aflatoxin and even TNT.

DNA has a valence band composed of nucleobases HOMO orbitals and a conduction band composed of nucleobases LUMO orbitals. They are separated by an energy gap of ~ 4 eV causing the macromolecule to be an insulator. In order to improve conducting properties of DNA one should add either additional electrons in the conducting band or additional holes in the valence band. A possibility of electron doping of DNA emerged with discovery of M-DNA, a complex which DNA spontaneously forms with transition metal cations, Co2+, Ni2+ and Zn2+, in buffered water solutions at high pH (= 8.5). The M-DNA complex forms when divalent metal cations, M2+ replace imino protons, H+ from the hydrogen bonds between complementary nucleobases in the core of the double helix and bring a net positive charge on the DNA molecule. When the complex is dried this positive charge cannot be simply neutralized with negative ions on the outer surface of the double helix and free electrons from the frozen salt matrix must penetrate the double helix to ensure local charge neutrality.
Here we present spectroscopic measurements on the first example of a chemically electron-doped metal- DNA complex. We show that a doping level as high as the stoichiometric limit of one electron per base pair can be achieved in a solid metal-DNA complex Zn-DNA extracted with a freeze-drying method. The doped unpaired electrons occupy LUMO levels of the nucleobases as detected with Electron Spin Resonance (ESR) and X-ray Absorption Near Edge Structure (XANES) measurements. Delocalization and strong correlations between the unpaired electrons are evident from a temperature-independent spin susceptibility and a microwave (MW) conductivity above 100 K. Below 50 K the unpaired electrons start to localize which is evident from a sharp decrease in the MW conductivity and a Curie-like increase in the spin susceptibility. Obviously, the realization of high electron density in the conduction band of DNA by electron doping in the metal-DNA complex, which is essential for any future application of M-DNA in nanoelectronics, also introduces the physics of strongly correlated systems into doped DNA systems.

DNA templated nanowires of pentynyl-modified poly2-(2-thienyl)-pyrrole undergo functionlisation via metallisation and “click chemistry“. The nanowires retain their 1D-nanostructure and conductive properties after modification and show promise for incorporation into micro-electrode devices.

Conductive 1D structures, so-called nanowires, are particularly important due to their potential use in photodetectors, waveguides and chemical sensors. In the last of these devices advantage is taken of the highly sensitive nature of the electrical conduction to external pertubations, making them ideal as transducing elements. A variety of methods for preparing such structures have been developed, including the use of double helical DNA as a template. This approach has proved quite general and has enabled the preparation of conducting 1D nanostructures of metals, binary inorganics as well as molecular-based materials.This is possible because as individual molecules, the strands exhibit extensive non-covalent interactions with a wide variety of precurors, e.g. metal ions, small molecules, oligomers etc. This ability to “dope” the biopolymer with these precursors controls the resulting material formation. However, for use as sensing elements it is not sufficient to produce conducting structures; these must also be capable of functionalisation with appropriate receptors so as to prime them for capture of a target. Here we report the preparation of a conducting DNA-based nanowire system that can be readily functionalised. The approach uses individual strands of DNA to template the growth of a conducting polymer, pentynyl-modified poly-2-(2-thienyl)-pyrrole. The resulting, supramolecular conductive polymer/DNA hybrid material is formed as nanowires (step (a) in figure above) which can undergo functionalisation using “click” chemistry, demonstrated here with a variety of molecular groups (step (b) in figure above). In addition the terminal alkyne groups strongly influence the subsequent templating reaction of Tollens’ reagent to produce uniform conductive nanowires comprised of many connected Ag clusters. We confirm that the nanowires are electrically conducting before and after these processes using a range of probe microscopy techniques.

STM and STS of poly(G)-poly(C) DNA. (a) Topography image of the DNA on Gold (b). 3D presentation of the same image (c) STS on the molecule.

DNA has been in the center of the scientific research for decades. In particular, DNA was considered as one of the attractive candidates for molecular electronics. Attempts to resolve the energy level structure of single DNA molecules span over the last two decades, thanks to the unique ability of scanning tunneling spectroscopy (STS) to probe the local density of states of deposited objects. Nevertheless, success was hindered by extreme technical difficulties in stable deposition and reproducibility. By measuring STS on DNA at cryogenic temperature, for the first time we disclosed the energy spectrum of poly(G)-poly(C) DNA and G4-DNA molecules deposited on gold. The tunneling current-voltage (I-V) characteristics and their derivatives (dI/dV-V) exhibit a clear gap and a peak structure around the gap. Fluctuations in the I-V curves are observed and statistically characterized. The character of the observed dI/dV-V peaks is assigned to orbitals originating from the different molecular components, namely the nucleobases, the backbone and the counterions, by means of ab initio Density Functional Theory calculations.
As time allows, I will show a selection of the following topics from our research: (i) Progress in electrical transport measurements. (ii) Comparison of polarizability between G4-DNA variants.

[1] “Direct Measurement of Electrical Transport Through Single DNA Molecules of Complex Sequence”, Hezy Cohen, Claude Nogues, Ron Naaman and Danny Porath, PNAS 102, 11589 (2005).
[2] "Long Monomolecular Guanine-Based Nanowires." Kotlyar, A. B., Borovok, N., Molotsky, T., Cohen, H., Shapir, E. & Porath, D. Adv. Mater. 17, 1901 (2005).
[3] “Electrical characterization of self-assembled single- and double-stranded DNA monolayers using conductive AFM”, Hezy Cohen et al., Faraday Discussions 131, 367 (2006).
[4] “Long Monomolecular G4-DNA Nanowires”, Alexander Kotlyar, Nataly Borovok, Tatiana Molotsky, Hezy Cohen, Errez Shapir and Danny Porath, Advanced Materials 17, 1901 (2005).
[5] "Polarizability of G4-DNA Observed by Electrostatic Force Microscopy Measurements", Hezy Cohen Hezy Cohen et al., Nano Letters 7(4), 981 (2007).
[6] “High-Resolution STM Imaging of Novel Poly(G)-Poly(C)DNA Molecules”, Errez Shapir, Hezy Cohen, Natalia Borovok, Alexander B. Kotlyar and Danny Porath, J. Phys. Chem. B 110, 4430 (2006).

Recently we developed several nano-biotechnological approaches aimed at the realization of arrays of biomolecules (arrays of DNA, proteins, antibodies) with micro- or nanoscale spatial resolution. Surface chemistry procedures were combined with lithographic techniques for the realization of nanostructured substrates with biorecognition capability, and the various processes were optimized in order to obtain highly uniform and functional immobilized biomolecules (for instance, retention of the native folding of fluorescent proteins). Moreover, based on the covalent immobilization of stable and versatile SAMs of programmable chemical reactivity, this approach constitutes a general platform for the parallel site-specific deposition of a wide range of molecules, such as organic fluorophores and water soluble colloidal nanocrystals. Notably, we developed versatile strategies compatible with the use of a wide range of different materials (e.g., plastic materials, glass, quartz, silica) of great interest for the realization of biochips for analytical and diagnostic applications. In this talk I will present some possible approaches for the implementation of biosensors and Lab-on-chip devices, based on polymeric materials, exploiting membrane receptors, hybrid systems, colloidal semiconductor nanocrystals (NCs), novel materials and interfaces and different fluorescence or electrical sensing schemes, for highly sensitive real-time analyses of biomolecules. I will also show some recent strategies for the development of cell chips for diagnostic and drug screening based on different detection methods for real-time monitoring of morphological changes and/or of specific cellular processes.

There is an increasing demand for multiplex techniques enabling the simultaneous assessment of multiple biomolecules in one biological sample in research and clinical diagnostics. Due to their high flexibility microbead-based multiplex detection systems close a gap between single and high-throughput detection methods.
Within the framework of the InnoProfile project “Real-time-PCR-Array“ we developed a microbead-based multiplex antibody and DNA detection technology. Main pillars for this technology are A) Fluorescent microbead populations which are defined by varying ratios of two fluorophores and B) The multi-fluorescent color detection platform VideoScan with novel pattern-recognition algorithms.
A) Microbeads: Proteins for the detection of antibodies or oligonucleotide capture probes for the detection of PCR products are coupled on microbeads. Specific antibodies bind to microbead-coupled antigens and are detected by a fluorescence-labeled secondary antibody. PCR products are fluorescence-labeled during the PCR reaction and hybridize to microbead-coupled capture probes. Binding of fluorescence-labeled secondary antibodies or PCR products results in a specific fluorescence corona around a microbead. The intensity of a fluorescence signal correlates with quantities of bound antibodies or PCR products. Fluorescence corona and affiliation of one microbead to one of 18 microbead populations are automatically analyzed by the VideoScan platform. Size, surface, thermostability and degree of carboxylation of microbeads as well as photostability of fluorophores determine efficiency and robustness of the interpretation process.
B) The VideoScan platform is a flexible imaging technology with a broad spectrum of applications such as I) Protein and DNA quantification due to fluorescence signals on microbead surfaces, II) Cell- and tissue- based immunofluorescence assays, III) Bacterial adhesion and invasion assays by the detection and quantification of fluorescent bacteria. The VideoScan platform is based on automatic capturing and processing of two-dimensional images and consists of the following main components: motorized inverse microscope with three fluorescence channels, motorized scan-stage, digital grey-scale camera and computer running software for controlling and processing.
The VideoScan is a flexible and automatic multi-parametric analysis system with a great potential for further diagnostic and biochemical applications.

Branched DNA can be used to create structures with feature sizes significantly smaller than are accessible via photolithography. There is great potential utility in learning how to attach functional guests onto DNA nanostructures in such a manner that the guests can be organized with a level of structural precision comparable to that of the DNA scaffold. Gold Nano-Particles (AuNPs), have been the prototypical targets for this undertaking, largely because their electrical and optical properties are strongly dependent on their diameter. Unfortunately, branched DNA nanostructures will generally only form in a buffer solution that has approximately a 10 mM concentration of divalent counter ions – typically Mg2+. AuNPs, on the other hand, tend to aggregate rapidly in such solutions, which limits their availability for attaching to DNA scaffolds. We have found that AuNPs that have both DNA strands and zwitterions attached to their surfaces can resist aggregation in the typical buffers used for DNA nanostructure assembly; further, the DNA on the NPs retains its availability for hybridization. We have built a two dimensional scaffold of triangular units that does not form in the absence of Mg2+ ions. The scaffold has spaced triplets of sticky ends that serve as attachment points for AuNPs. The connections between the scaffold and the AuNPs include both flexible, single- stranded DNA and relatively rigid double helices. With the proper lengths of flexible and rigid linker segments, triplets of binding sites on a single triangle are occupied by single AuNPs, as can be observed via scanning electron microscopy.

Contrast enhanced scanning electron micrographs of 5 nm diameter gold nanoparticles. A) The AuNPs in the absence of the DNA scaffold, organized by capillary forces during drying. The average center-to-center particle spacing is ~12 nm. B) Similar particles arranged on a DNA scaffold (not visible). The average spacing is approximately the expected 28 nm.

I will present a general method for solving a key challenge for nanotechnology: programmable self- assembly of complex, three-dimensional nanostructures. Previously, scaffolded DNA origami had been used to build arbitrary flat shapes 100 nm in diameter and almost twice the mass of a ribosome. We have succeeded in building custom three-dimensional structures that can be conceived as stacks of nearly flat layers of DNA. Successful extension from two-dimensions to three-dimensions in this way depended critically on calibration of folding conditions. We also have explored how targeted insertions and deletions of base pairs can cause our DNA bundles to develop twist of either handedness or to curve. The degree of curvature could be quantitatively controlled, and a radius of curvature as tight as 6 nanometers was achieved. This general capability for building complex, three-dimensional nanostructures will pave the way for the manufacture of sophisticated devices bearing features on the nanometer scale.

Surface enhanced Raman spectroscopy (SERS) is an emerging technology in the field of analytics. Due to the high sensitivity in connection with specific molecular fingerprint information SERS can be used in a variety of analytical, bioanalytical, and biosensing applications. SERS could also be used for a chip based detection of biologically active molecules and low molecular substances. However, for the SERS effect, substrates with metal nanostructures are needed. The broad application of this technology is greatly hampered by the lack of reliable and reproducible substrates. To use SERS as standard analytical tool cheap and reproducible substrates are required.
Here we highlight an alternative approach to produce low cost and reproducible substrates for SERS applications, which allows a simple, economical and highly space-resolved production. The fabrication process is based on an enzyme induced deposition of silver nanoparticles. The special structural feature of the enzymatically generated silver nanoparticles allows their application for a variety of excitation frequencies in the visible and NIR region. Furthermore, the exceptional morphology of the SERS substrate enables a qualitative and semi quantitative statement of the SERS activity due to a simple measurement of the DC conductivity measurement.
These novel SERS substrates of enzymatically generated silver nanoparticles were used for the qualitative and quantitative chip based detection of low molecular substances like dyes and antibiotics. Furthermore, due to the combination of SERS and magnetic nanoparticles, a sequence specific detection of DNA of epizootic disease causing pathogens was realized. Here we will present our first results on this bioanalytical system. In first experiments we investigate the applicability of our system with short synthetic oligonucleotides and evaluate the specificity. Furthermore we transfer this system to DNA strands that were amplified via PCR, which is closer to reality than short synthetic oligonucleotides. As PCR targets sequences of the bacterium Mycoplasma mycoides subspecies mycoides small colony type (MmmSC), which causes the epizootic disease contagious bovine pleuropneumonia (CBPP) were used. In the end we will display the potential of this bioanalytical system for the analysis of PCR product and native ssDNA.

Acknowledgement Funding of research projects “Jenaer Biochip Initiative (JBCI)” within the framework “Unternehmen Region – Inno Profile” and ATLAS from the Federal Ministry of Education and Research, Germany (BMBF) is gratefully acknowledged. K. Weber gratefully acknowledges financially support by “Carl-Zeiss-Stiftung”.

Controlled positioning and immobilization of DNA molecules and structures on the chip is a crucial open challenge for the realization of the full potential of the bottom-up DNA-nanotechnology, and to further study such systems. Dielectrophoresis (DEP) can be used to trap DNA molecules and self-assembled structures, such as DNA origami [1] which is a striking example of an efficient DNA bottom-up technique. The optimized DEP-method gives a high yield of single structure trapping between nanoelectrodes, and thus provides controlled positioning of the structures on a chip and means of bridging bottom-up and top-down fabrication approaches.

In this study we have utilised DEP to trap DNA origami structures, and after the immobilization of an individual DNA structure between the nanoelectrodes, the electrical conductivity of the structure was characterized. We have fully analyzed both DC and AC characteristics (AC impedance spectroscopy) of single trapped rectangular DNA origami structures within environments with different humidity levels, and also defined the detailed equivalent circuit model describing the conductivity mechanism of the structure [2]. The results revealed that the nature of the conductivity is not purely Ohmic but that it is a combination of an ionic diffusion and electronic conductivity. Also other kinds of selfassembled DNA structures are being measured and analyzed with the same method.

Figure. Inset: AFM Image of a rectangular DNA origami trapped and immobilized between nanoelectrodes. Graph: AC impedance spectroscopy data measured from the structure (circles) and the response of a fitted equivalent circuit (line).

[1] A. Kuzyk, B. Yurke, J.J. Toppari, V. Linko, and P. Törmä, Small 4(4) (2008) 447. [2] V. Linko, S.-T. Paasonen, A. Kuzyk, P. Törmä, and J.J. Toppari, Small 5(21) (2009) 2382.

We consider two different topics. The first one is related to the interaction of organic molecules with surface plasmon polaritons (SPP), i.e. electro-magnetic fields confined within nanoscale dimensions near a metal surface. We present the observation of a large room-temperature vacuum Rabi splitting [1] for SPP and dye molecules, paving the way for the use of strong coupling phenomena in a variety of molecular plasmonics systems.

The second topic concerns DNA origami, a method of creating complicated nanoscale shapes out of DNA using its self-assembly properties. We show how DNA origami structures can be positioned on chip using dielectrophoresis [2], and how they can be used as a nanobreadboard to assemble complicated patterns with below 10 nm resolution [3]. We also discuss recent work on combining our carbon nanotube [4] and DNA-related research.

[1] T.K. Hakala, J.J. Toppari, A. Kuzyk, M. Pettersson, H. Tikkanen, H. Kunttu, and P. Törmä, Vacuum Rabi splitting and strong coupling dynamics for surface plasmon polaritons and Rhodamine 6G molecules, Phys. Rev. Lett. 103, 053602 (2009).
[2] A. Kuzyk, B. Yurke, J.J. Toppari, V. Linko, and P. Törmä, Dielectrophoretic trapping of DNA origami, Small 4, 447 (2008).
[3] A. Kuzyk, K.T. Laitinen, and P. Törmä, DNA origami as a nanoscale template for protein assembly, Nanotechnology 20, 235305
(2009).
[4] M. Rinkiö, A. Johansson, G-S. Paraoanu, and P. Törmä, High speed memory from carbon nanotube field-effect transistors with high-? gate dielectric, Nano Lett. 9, 643 (2009).

The information encoded in the base sequence of the nucleic acids provides instructive information for the self-assembly of supramolecular nanostructures exhibiting programmable topological properties, recognition and catalytic functions, and molecular machinery operations. These different topics will be addressed with the following examples:

- Nucleic acids exhibiting tailored complementarities or carrying appropriate chemical functionalities self-assemble into catenated DNA chains, hexagonic strips, graphitic-type nanotubes, and covalently linked nanotubes. The nanostructures enable the programmed addressing of proteins and the activation of enzyme cascades, and they act as nano-containers for metal/semiconductor nanoparticles.

- Different DNA machines that function as a "bipedal walker", "gear", molecular "seesaw", molecular "crane", and three coherent "tweezers" will be introduced. The significance of these structures as nano- transporters, controlled release systems and biomolecular computing will be discussed.

- The self-assembly of nucleic acids into supramolecular structures exhibiting tailored recognition properties (aptamers) or catalytic functions (DNAzymes) will be presented. The use of the systems for amplified sensing, and particularly, as isothermal self-replicating systems, will be introduced.

- The functionalization of semiconductor quantum dots (QDs) with metal-porphyrin/G-quadruplex structures will be described, and the utilization of the systems for optical sensing and photocatalysis will be introduced.

The manipulation of biological molecules at the nanometer scale by means of ultrashort laser pulses shows great promise for applications in molecular medicine. Because of their unique optical properties, like the plasmon resonance, noble metal nanoparticles can act as laser light antennas and allow a very local energy conversion of laser pulses from the nanoparticle into the surrounding medium [1, 2]. In this talk, we present experiments of the energy conversion from metal nanoparticles into double stranded DNA molecules induced by laser light. After the silver nanoparticles were bound to DNA, the DNA was immobilized on a thermo- and electron sensitive layer (polymethylmethacrylate, PMMA) and irradiated by laser light at a wavelength of 800 nm. The influences of the excited nanoparticles on the DNA-molecules were characterized by atomic force microscopy.

[1] Garwe, F., Bauerschafer, U., Csaki, A., Steinbruck, A., Ritter, K., Bochmann, A., Bergmann, J., Weise, A., Akimov, D., Maubach, G., Konig, K., Huttmann, G., Paa, W., Popp, J. and Fritzsche, W. (2008) Optically controlled thermal management on the nanometer length scale. Nanotechnology, 055207
[2] Csaki, A., Garwe, F., Steinbruck, A., Maubach, G., Festag, G., Weise, A., Riemann, I., Konig, K. and Fritzsche, W. (2007) A Parallel Approach for Subwavelength Molecular Surgery Using Gene-Specific Positioned Metal Nanoparticles as Laser Light Antennas. Nano Lett. 7, 247 - 253

We are utilizing DNA to form nanometer-scale structures¹ with potential for application as integrated circuit templates. Using DNA origami² we have constructed nanoscale objects having a range of sizes and shapes.³ Importantly, our DNA nanostructures contain multiple branching points, have asymmetric junctions, and feature linewidths of about 10 nm (see Figure 1), all desirable properties for forming nanoelectronic components. We are optimizing procedures for selectively seeding and plating DNA origami. Our present efforts focus on the formation of three-dimensional DNA origami constructs for nanoelectronics applications and the controlled assembly of DNA origami into periodic arrays. This work showcases the considerable capabilities of DNA for bottom-up self-assembly of nanoscale features.

Figure 1. Atomic force microscopy characterization of thin, branched DNA origami structures. (A) “B” shape. (B) “Y” shape. (C) “U” shape. Scale bars are 200 nm.

[1] Becerril, H.A.; Woolley, A.T. DNA-Templated Nanofabrication. Chem. Soc. Rev. 38, 329-337 (2009).
[2] Rothemund, P.W.K. Folding DNA to Create Nanoscale Shapes and Patterns. Nature 440 297-302 (2006).
[3] Pound, E.; Ashton, J.R.; Becerril, H.A.; Woolley, A.T. PCR-based Scaffold Preparation for the Production of Thin, Branched DNA Origami Nanostructures of Arbitrary Sizes. Nano Lett. 9, 4302-4305 (2009).

The emerging field of nanophotonics has created a diverse set of novel tools and opportunities for biological sensing, optical imaging and photonics-based therapy (e.g., photodynamic therapy and photothermal therapy). Compared with their well documented impact on in vitro clinical diagnosis and biomarker discovery, nanophotonics’ translation into clinical viable tools for in vivo imaging and therapy is slowed by many formidable hurdles (e.g., delivery to the correct cells/tissues at a level that is safe for patients). Three lipid-based nanophotonics platform technologies will be introduced here with the goal of addressing some of these translation hurdles. First, we created a metal free phototransducing lipid nanovesicle that combines the powerful phototransucing properties (e.g., photothermal, photoacoustic) of gold nanoparticles and the natural biocompatibility and superior drug carrying capability of liposomes. Such ‘phototransducing liposomes’ could transform traditional organic drug nancarriers into versatile biophotonic tools. Secondly, by mimicking the assembly and functions of natural lipoproteins, we created several classes of lipid nanoparticles suitable for the systematic targeted delivery of photodiagnostic and therapeutic agents to cancer cells. These spherical and small (<40nm) nanostructures are designed for optimizing the nanoparticle tumor penetration and the intracellular probe delivery. In the third platform, we integrated the classic hairpin molecular beacon concept into lipid nanostructures and created a powerful nucleic acid sequence- controlled lipid nanoparticle aggregation strategy that has a number of potential utilities for in vivo biosensing and imaging.

We studied surface organization of lipophilic guanosine derivatives with one, two and three alkanoyl tails in thin films on water surface and in Langmuir-Blodgett (LB) films transferred onto freshly cleaved mica substrate. The derivative with one alkanoyl group exhibits irreversible pressure-area isotherms and ribbon- ike LB film structures. The derivatives with two and three side groups show reversible isotherms with similar critical surface pressures, whilst their LB film structures are quite different. The derivative with two tails forms films with flat terraces of micrometer size, while the derivative with three tails exhibits irregular thread-shaped assemblies. These observations demonstrate that molecular assembly of LB films of guanosine derivatives can be effectively manipulated by altering the number of attached lipophilic groups.

From the point of view of nanotechnology, the material with two tails (dG(C10)2) seems to be the most promising for applications requiring homogeneous films with well defined and tunable thickness. Our preliminary investigations of multilayer LB films of this derivative showed that regular terrace-like morphology is preserved also in multilayer structures. The derivative with three tails (G(C10)3) is likely unsuitable for applications, as its LB film structures are very irregular and practically cannot be controlled. The ribbon-like LB film structure of the derivative with one tail (GACE(C10)) is promising for applications requiring 1D building blocks. Its properties might be further improved by developing appropriate methods leading to alignment of the ribbons along a predetermined direction. In addition to this, intermolecular hydrogen bonding, which might occur in Langmuir films of this derivative, provides additional possibility for tuning the properties of its LB film structures. The tuning can be achieved either via adding assembly stimulating ions to the water subphase or by dilution of the material with some other lipophilic molecules. The arrangements of the molecules on surface can be studied by a Langer-Blodgett technique, Atomic force microscopy and Sum-frequency generation nonlinear optical method.

Figure 1: Surface pressure versus area isotherms of Langmuir films of GACE(C10), dG(C10)2 and G(C10)3 on water subphase.

Figure 2: AFM images of LB films of GACE(C10) (left), dG(C10)2 (middle), and G(C10)3 (right) deposited on muscovite mica. Vertical cross sections along the marked lines are reported below each image. The values displayed at the bottom are vertical distances between the markers of the same colour.

[1] Devetak et al., Surface structure of Langmuir-Blodgett films of lipophilic guanosine derivatives, Applied Surface Science 256, 7, (2010) 2038-2043.
[2] Kunstelj et al., Self-organization of guanosine 5’-monophosphate on mica, Coll.Surf. B: Biointerfaces 59 (2007) 120-127.

While self-assembly has proven its usefulness for the generation of various nano- and mesoscopic structures, there remains a fundamental obstacle in creating integrated, highly ordered multicomponent nanodevices: the precise spatial control over each of the device components has proven to be exceedingly difficult. Here, a high-precision, high-throughput method for pattern transfer of nano-objects is demonstrated by producing arrays of quantum dots [1]. The method, field-induced nanolithography (FINAL), utilizes dielectrophoresis for trapping and it can be extended to arranging carbon nanotubes, biomolecules, and other objects into desired configurations. The trapping as well as the subsequent deposition from the master stamp to the target plate are made in parallel fashion, enabling large area printing and mass production. Furthermore, the master stamp is i) reusable, and ii) more importantly and in contrast to conventional methods such as microcontact printing and nanoimprint lithography, universal: one master stamp having a 2D matrix of electrodes could be used to transfer any desired pattern to the target plate. Due to its robustness, versatility, and universality, the method has remarkable application potential in nanotechnology.

[1] T. K. Hakala, V. Linko, A.-P. Eskelinen, J. J. Toppari, A. Kuzyk, and P. Törmä, Small 5 (2009) 2683-2686.

DNA has a superior self-assembly properties, which have been utilized in many nanoscience applications Maybe the most prominent examples are the different variations of origami structures constructed from DNA, including 2D structures of arbitrary shapes1,2 and even 3D structures with functionality3,4. We would like to use the DNA origami as a template for structured molecule attachment for self-assembled electronics.

[1] Rothemund, P.W.K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297-302 (2006).
[2] Andersen, E.S. et al. DNA Origami Design of Dolphin-Shaped Structures with Flexible Tails. ACS Nano 2, 1213-1218 (2008).
[3] Andersen, E.S. et al. Self-assembly of a nanoscale DNA box with a controllable lid. Nature 459, 73-76 (2009).
[4] Douglas, S.M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414-418 (2009).

We investigate the dynamics of electrically actuated DNA layers which are attached to a gold surface. The DNA conformation on the surface is modulated (switched) by electric fields due to the intrinsic charge of the DNA backbone. The fluorescence signal of a dye at the distal end of the DNA strand is used to monitor the conformation change, as the apparent quantum yield depends strongly on the distance between the dye and the metal surface. The modulation efficiency of our layers is affected by the salt concentration in buffer solution due to counter ion screening at the metal/solution interface and the molecular dynamics of the DNA switching are found to depend on the charging properties of this electrochemical double layer. A novel method to perform time resolved measurements allows us to monitor the individual upward and downward motions of the electrically driven DNA motion on a sub?microsecond timescale. We determine the dynamic properties of the system by evaluation of the characteristic rise and fall times. Employing the time resolved method, influences of the DNA surface density and the DNA length on these time constants are studied. The introduced methods are then applied to monitor the binding of proteins (target) to DNA layers (probe) and to study the dynamics of electrically actuated DNA?protein conjugates. IgG antibodies and antibody fragments are bound to the DNA layer and the influence of the attached proteins on the DNA dynamics are studied. As the bound proteins increase the hydrodynamic drag and thus the rise time of the DNA probe, we can infer the size of the proteins from an analysis of the switching dynamics of the DNA?protein conjugate.

The electrical properties of nanowires are influenced by minor perturbations to the surrounding environmen because of their high surface-to-volume ratio and small size.[1] When functionalized with appropriat ‘capture probes’ and integrated into electronic devices, nanowires can act as transducers for label-fre electrical detection of a variety of chemical and biological species.[2,3] DNA templating provides a practica and reproducible route to nanowire formation that has been shown to be compatible with a wide variety o materials.[4] Current work focusing on DNA-based nanowires of thienyl-pyrrole derivatives with alkyny groups is presented. FTIR data demonstrates the availability of the alkyne group for subsequent nanowir functionalisation using ‘click’ chemistry. Atomic Force Microscopy (AFM) shows well aligned DNA-template nanowires on SiO2 substrates (see Figure), while electrical characterization using methods such as Scanne Conductance Microscopy (SCM) confirm the conducting nature of these nanomaterials.

Figure: Black arrow: DNA/pentnyl-TP ~5-6 nm in height stretched on silanized silicon (111) surface, the scale bar = 3 µm and the height scale is 10 nm.

[1] Lutz, Jean-Francois. et al. (2008), Prog. Polym., Sci. 33, 1-39.
[2] Yogeswaran, U. (2008), Sensors, 8, 290-313.
[3] Lange, U. et al. (2008), Analytica Chimica acta, 614, 1-26.
[4] Houlton et al. (2009), Chem. Comm., 1797-1806.

Phytophthora [1] is a genera of fungal-like plant parasitic pathogens that causes commercially severe plant losses worldwide. The most famous example is the Great Famine in Ireland mid of the nineteenth century. It was caused by P. infestans the causal agent of the late blight of potato. Today the most dangerous Phytophthora species are regulated, some as quarantine organisms [www.eppo.org] like P. ramorum [2] the causal agent of Sudden Oak Death in the natural habitats along the Californian coast side in the USA [www.suddenoakdeath.org]. To prevent the spread of these pathogens, plants and plant products must be examined before they are sold or exported. Furthermore there are strict regulations for plant control at the boarders. Therefore the plant protection services and the plant inspectors ask for highly specific, sensitive and easy to handle diagnostic methods, which give results within short time.
We demonstrate a lab-on-a-chip system for rapid nucleic acid-based analysis within approx. 3 hours that can be applied for diagnosis of Phytophthora. This microchip system integrates a PCR-chamber for amplification and labelling of nucleic acids and microarray technology for sequence specific analysis within a single platform (fig. 1). All necessary reaction steps can be performed consecutively within a single chip with integrated microchannels and microchambers. The microarray is read out by a simple electrical measurement. To enable the electrical readout all spots are placed in electrode gaps. In case of successful hybridisation, elementary silver can be deposited by an enzymatically catalysed reaction. If so the gaps will be closed and electrical conductivity can be measured [3]. Temperature management of PCR- and hybridisation-zone is realised by two independent Peltier elements. The chip will be integrated in a compact portable system with a pump and power supply so the development of a point of care device is possible (fig. 2).
Because of its universal character this lab-on-a-chip system is open for other applications such as qualitative diagnosis of infectious or hereditary diseases based on specific nucleic acid sequences.

Funding by the Federal Agency for Agriculture and Food (BLE) is acknowledged (FKZ: 28-1-42.028-06).

[1] Erwin D. C. and O.K. Ribeiro, Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, Minnesota, 1996, pp. 562
[2] Werres, S., et al., Phytophthora ramorum sp. nov., a new pathogen on Rhododendron and Viburnum. Mycol Res, 2001. 105(10): p. 1155-1165.
[3] Möller, R., et al., Electrical DNA-chip-based identification of different species of the genus Kitasatospora. Appl Microbiol Biotechnol, 2008. 77(5): p. 1181-1188.

Due to its exceptional self-assembly properties, DNA could become a key player in bottom-up fabrication of nanoscale systems.¹ A striking example of a DNA self-assembly technique is “DNA origami“ which involves folding of long single-stranded DNA with the help of short oligonucleotides.² DNA molecules and DNA self- assembled structures can be used as a template for attachment of various materials (proteins, carbon nanotubes, nanoparticles etc). In case of DNA origami each short oligo can serve as a pixel. Therefore, origami structures can be decorated with complex patterns with below 10 nm resolution. Ability to control relative position of molecules with nanometer precision is of extreme importance for understanding fundamental biomolecular interactions, e.g. of enzymes. Recently we have demonstrated that DNA origami structures can be used for assembly of complex proteins patterns.³ Similar approach can be used for assembly of enzymes and carbon nanotubes. To realize the full potential of DNA origami as a template, we have developed dielectrophoresis based method for controlled trapping of origami structures between nanoelectrodes.⁴Such trapping is the first demonstration of the dielectrophoretic manipulation of complex self-assembled structures and is an example of successful combination of bottom-up and top-down fabrication approaches.

[1] N. C. Seeman, Nature 421 (2003) 427.
T. H. LaBean and H. Li, Nano Today 2 (2007) 26.
[2] P. W. K. Rothemund, Nature 440 (2006) 297
[3] A. Kuzyk, K. T. Laitinen, P.Törmä, Nanotechnology 20 (2009) 235305.
[4] A. Kuzyk, B. Yurke, J. J. Toppari, V. Linko, P. Törmä, Small 4 (2008) 447.

Dielectrophoresis (DEP) is a suitable method for the microintegration of bottom-up nanoscale structures in an existing top-down microelectronic environment. This technique is based on the moving of polarizable objects under the influence of an AC-field. In combination with real-time microscopy the characterization and investigation of the DEP parameters for nanoscale objects can be achieved. By ajusting of different process parameters for every nanostructure or biospecies it is possible to influence orientation and integration geometry between microelectrodes. This integration principle offers a great potential for nanophotonics, nanoelectronics and on-chip applications. It was possible to direct cells, form conductive nanoscale bridges between two electrodes and stretch DNA-based structures between electrodes. Furthermore, integration of nanosized structures e.g. nanoparticles and nanowires could be also demonstrated. Current developments are focused on the investigation of the sensoric potential of defined integrated nanostuctures for bioanalytical applications.

Figure 1) Microintegrated nanoparticles (top, left), nanowires (top, middle), defined nanoparticle bridge (top, right) and sensoric measurement on nanoparticle bridge (bottom).

It is known that guanines, which have the lowest ionization potential among DNA bases, promoting charge migration through the DNA. These properties make G-quadruplexes very promising for nanoelectronic. Experimental studies showed that that G-quadruplexes are characterized by a clear polarizability, which is indicative of possible electrical conductivity.
Direct measurement of the DNA conductivity is a very challenging task due to multiple reasons. One of them is the inability to efficiently transfer current through G4-DNA molecules deposited in-between metal electrodes with poor electrical contact between the DNA and the electrode surface. In order to overcome this problem and to promote an electric communication between the G4-DNA and the electrodes we decided to use G4-DNA-AgNP conjugates schematically presented in Figure 1A. As seen in the figure the two particles are connected to one another though a parallel stranded G-quadruplex molecule. The G4-DNA structures were prepared using a 5'-aaaaa(20G)aaaaa-3' oligonucleotide, containing 20G in the middle of the strand and 5 phosporithioate A bases (a) at its 3' and 5'-ends and 15 nm silver nanoparticles (AgNP). The strands were assembled at high oligonucleotide concentrations (>0.5 mM) in the absence of K and Na. Incubation of the strands in these conditions yields individual G4 molecules and not long G-wires composed of a large number of annealed oligonucleotides. We have shown that the CD spectrum of the structures is similar to that reported for four stranded G-quadruplexes. We have also demonstrated that the structures are uniform and migrate as a single band during electrophoresis. The phosporithioate fragments on both side of the G-quadruplex bind covalently to the surface of silver particles linking them together into a dimer (see Fig. 1A). Incubation of AgNPs with three-four molar excess of the end-functionalized G-quadruplexes lead to the formation of short chains of the particles (see Fig 1B) in which the two neighboring particles are connected by the G-quadruplex. Electrical measurements of these structures, we believe, will provide new insights into the mechanism of DNA conductivity.

Figure1. TEM images of AgNP dimers (A) and short chains of AgNP (B) connected by G4-DNA

DNA-origami is prepared using long single-stranded DNA molecules folded into arbitrary 2D-planar nanostructures utilizing various short helper strands as clamps. It represents a self-assembling of 90 x 90 nm objects with roughly 6 nm resolution from the 7 kilobase single-stranded genomic DNA of M13mp18 (Fig. 1) [1,2]. We can carry out different methods of functionalizations of gold, silver, core-shell, silica, and magnetic nanoparticles, and would like to combine these particles with the DNA super structure. A result is shown in (Fig. 2). This image demonstrates that the integration of gold nanoparticles functionalized with DNA to the surface of the “smiley” origami structure could be accomplished using gold nanoparticles of different sizes. We compared different conjugations with 5, 12 and 30 nm gold nanoparticles. They are stable under hybridization up to 60°C. Finally, we showed that the addition of specific attachment gold nanoparticles conjugated smiley’s to the electrodes during dielectrophoresis (Fig. 3).
Possible applications of this technology for the precise combination of metal nanoparticles with DNA superstructures are expected in nanoelectronics and nanophotonics.

[1] N. C. Seeman, Nature 421 (2003) 427-431.
T. H. LaBean and H. Li, Nano Today 2 (2007) 26-35.
[2] P. W. K. Rothemund, Nature 440 (2006) 297-302.
[3] S. Tuukkanen, A. Kuzyk, J. J. Toppari, V. P. Hytönen, T. Ihalainen, and P. Törmä, Appl. Phys. Lett. 87 (2005) 183102.
S. Tuukkanen, A. Kuzyk, J. J. Toppari, H. Häkkinen, V. P. Hytönen, E. Niskanen, M. Rinkiö, and P.Törmä, Nanotechnology 18 (2007) 295204.
S. Tuukkanen, J. J. Toppari, A. Kuzyk, L. Hirviniemi, V. P. Hytonen, T. Ihalainen, and P. Törmä, Nano Lett. 6 (2006) 1339-1343.
[4] A. Kuzyk et al., Nanotechnology 20, 235305 (2009).

Nanoparticles conjugated with biological linkers are promising materials for the controlled bottom-up assembly of nanostructures.
In this work both, streptavidin and DNA were investigated as linker molecules to connect 20 nm gold nanoparticles. In a first step, the nanoparticle surface was modified by multiple ligand exchange reactions. We prepared biotinylated gold nanoparticles and activated a controlled streptavidin-mediated aggregation process by carefully adjusting the nanoparticle:streptavidin stoichiometry. Using an alternative approach, we exploited specific Watson Crick base pairing to form gold nanoparticle aggregates from nanoparticles which were functionalized with single DNA strands. To this end, single stranded DNA was modified with a 2xDithiol-Phosphoramidite linker at the 5’ end to allow for an attachment to the gold nanoparticle surface. To purify mono-conjugated from un-conjugated and multi-conjugated nanoparticles, Anion-Exchange High Performance Liquid Chromatography was used. Nanoparticle structures of high order were then formed by DNA programmed self-assembly.
The nanoparticle structures were characterized by a variety of methods. Optical extinction spectroscopy was performed to monitor a shift in the surface plasmon resonance peak upon nanoparticle aggregation and to study the kinectics of the aggregation process. Dynamic Light Scattering was used for size analysis of the nanoparticle assemblies in aqueous solutions; moreover, the nanoparticle assemblies were imaged on surfaces by Transmission Electron Microscopy.

The excitation of localized surface plasmons in noble metal nanoparticles by electromagnetic irradiation results in strong light scattering and a characteristic plasmon band. The intensity and position of this band depends on different parameters like size, shape, material and material composition [1]. For sensoric applications, the change of the local refractive index by binding of biomolecules to the particle surface is an important factor, causing a detectable shift of the plasmon band. In this work we want to demonstrate the detection of DNA hybridization by single particle spectroscopy. Therefore, metal particles were immobilized on a glass substrate via silane chemistry [2]. A capture oligonucleotide, modified with a thiol group, was attached to the particle surface [3] and hybridizied with a target oligonucleotide with complementary sequence. After each step the scattering spectra of the particle was collected and compared with each other. Caused by refractive index changes in the local environment of the nanoparticle, the maximum of the detected plasmon band shifts slightly to higher wavelengths (see Fig.1).

Fig.1: black – immobilized gold nanoparticle (80 nm); blue – immobilized capture oligonucleotide; purple – hybridized target oligonucleotide; a slightly red shift after each step was detected

The project was financially supported by the German Research Foundation DFG (FR 1348/12-1)

[1] E. Hutter, and J. H. Fendler, Adv. Mater. 16, 1685 (2004).
[2] R. Möller et al., NAR 80, e91 (2000).
[3] A. Csáki et al., NAR 29, e81 (2001).

Figure 1: Scheme of the hybridization chain reaction in solution

With the goal of determining the presence of pathogens when present in very small amount, such as for the screening of pathogens in drinking water, an analytical method of very high sensitivity must be implemented. As often this type of determinations should be performed at the point-of-care, with simple, automatic and inexpensive hardware, a chemical (or nanotechnological) signal-amplification step is required prior to biosensor read-out.
The hybridization chain reaction (HCR), originally proposed by Dirks and Pierce (2004), is an isothermal, enzyme-free process where a hybridization event triggers the polymerization of oligonucleotides into a long albeit nicked double-stranded nucleic acid molecule. The two kinds of co-monomer oligonucleotides are present in solution as closed hairpins, are opened and added to the growing polymer only after the initial triggering hybridization event.
We have designed HCR oligonucleotide sets to target DNA sequences of relevance for the protection from bioterrorism acts towards water systems. In this communication, we presents our results of the HCR amplifications for different DNA targets of C. parvum, G. lamblia and Hepatitis E virus that have been performed both in solution and in a microarray format, to make it more amenable of use in diagnostic or point-of-interest assays. Our need of detecting natural occurring sequences forcedly limits our possibility of sequence optimization, reducing it to the selection of a target sequence with low secondary structure within a consensus sequence defined by bioinformatics tools. The design of the HCR hairpins was the performed on the defined target sequences with the aid of NANEV (Goodman, 2005).
Our results seem to point out that there is a general sensitivity advantage in a biosensor read-out of the presence of naturally occurring nucleic acids sequences when the hybridization chain reaction is used as a signal-amplification step. This strategy could result of general interest for the analytical determination of nucleic acids.

[1] Goodman, P. (2005). "NANEV: a program employing evolutionary methods for the design of nucleic acid nanostructures" BioTechniques, 38(4): 548–550
[2] Goodman, P. (2005). "NANEV: a program employing evolutionary methods for the design of nucleic acid nanostructures" BioTechniques, 38(4): 548–550

The use of DNA as a template to control and direct metal deposition for the preparation of one-dimensional, metallic nanostructures is well-established.[1] The formation of copper nanostructures by such methods may be of particular interest due to their potential application as interconnects in future generations of nanoelectronic devices. Atomic force microscopy studies of copper nanostructures prepared using such a “DNA templating” strategy has previously been reported, whereby Cu2+ ions are first associated with “template” DNA molecules and subsequently reduced to Cu0 with ascorbic acid.[2] Here, extensive characterization of the chemical, structural, and electrical properties of copper nanostructures, prepared using this “DNA templating” method, is described.[3] Determination of the chemical composition of the nanostructured material (considered important due to the possibility for the chemical reduction of the Cu2+ ions to result in the preferential formation of Cu2O, over metallic Cu0) has been established using X-ray photoelectron spectroscopy and X-ray diffraction techniques. From these studies conclusive evidence is found that chemical reduction of the Cu2+ ions does result in the formation of the desired metallic Cu0 structures. Atomic force microscopy reveals the Cu0 structures to be 3.0 - 5.5 nm in height, and to exhibit a granular, “beads-on-a-string” morphology, see figure 1. Finally, scanned conductance microscopy has been employed as a means of probing the electrical properties of the Cu0 structures. Scanned conductance phase images of individual Cu0 nanostructures suggest that despite their metallic nature, significant levels of electrical resistance are experienced along the structures. This is thought to be a consequence of their “beads-on-a-string” morphology, and small lateral dimensions (sub-10 nm) resulting in significant electron scattering at the structure surface, as well as the presence of significant tunneling barriers at the Cu0 particle-particle junctions throughout the metal structures.

Figure 1. Tapping Mode AFM image of a Lambda-DNA-templated copper nanostructure, immobilized upon a Si/SiO2 substrate modified with a trimethylsilane self-assembled monolayer.

[1] (a) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G. Nature, 1998, 391, 775 (b) Keren, K.; Krueger, M.; Gilad, R.; Ben-Yoseph, G.; Sivan, U.; Braun, E. Science, 2002, 297, 72 (c) Richter, J.; Seidel, R.; Kirsch, R.; Mertig, M.; Pompe, W.; Plaschke, J.; Schackert, H. K.; Adv. Mater., 2002, 12, 507
[2] Monson, C. F; Woolley, A. T. Nano Lett. 2003, 3, 359
[3] Watson, S. M. D.; Wright, N. G.; Horrocks, B. R.; Houlton, A. Langmuir, 2010, 26, 2068

Surface enhanced Raman scattering (SERS) is an emerging tool for biological assays. It can provide detection limits, which are at least as good as those of fluorescence and enable multiplex detection capabilities due to the highly specific Raman fingerprint of each molecule. Due to the application of a microfluidic system the advantages of microfluidics concerning small sample volumes as well as an automated sample preparation and detection can be utilized.
Here we present the first results of a microfluidic system for chip-based detection using SERS. On the one hand we used the microfluidic platform for chip-based sequence specific DNA detection using SERS. In many cases when using microfluidics for the identification of defined DNA sequences a differentiation between positive and negative binding events is hard to achieve since unbound labelled oligonucleotides can not be removed by washing and therefore might be detected just as the hybridised labelled DNA targets. This disadvantage can be circumvented by applying a chip substrate, which can be placed within a microfluidic chamber. Due to the spatially defined alignment of different target DNA strands a sequence specific multiplex detection is possible.
This approach can also be adapted for the detection of low molecular substances.

Acknowledgement The Funding of the Carl-Zeiss-Stiftung as well as the research project “Jenaer Biochip Initiative (JBCI within the framework “InnoProfile - Unternehmen Region” from the Federal Ministry of Education and Research (BMBF) Germany is gratefully acknowledged.