Christoph Wälti

Synthesis of core-shell gold coated magnetic nanoparticles and their interaction with thiolated DNA

Core-shell magnetic nanoparticles have received significant attention recently and are actively investigated owing to their large potential for a variety of applications. Here, the synthesis and characterization of bimetallic nanoparticles containing a magnetic core and a gold shell are discussed. The gold shell facilitates, for example, the conjugation of thiolated biological molecules to the surface of the nanoparticles. The composite nanoparticles were produced by the reduction of a gold salt on the surface of pre-formed cobalt or magnetite nanoparticles. The synthesized nanoparticles were characterized using ultraviolet-visible absorption spectroscopy, transmission electron microscopy, energy dispersion X-ray spectroscopy, X-ray diffraction and super-conducting quantum interference device magnetometry. The spectrographic data revealed the simultaneous presence of cobalt and gold in 5.6±0.8 nm alloy nanoparticles, and demonstrated the presence of distinct magnetite and gold phases in 9.2±1.3 nm core-shell magnetic nanoparticles. The cobalt-gold nanoparticles were of similar size to the cobalt seed, while the magnetite-gold nanoparticles were significantly larger than the magnetic seeds, indicating that different processes are responsible for the addition of the gold shell. The effect on the magnetic properties by adding a layer of gold to the cobalt and magnetite nanoparticles was studied. The functionalization of the magnetic nanoparticles is demonstrated through the conjugation of thiolated DNA to the gold shell.

Formation and manipulation of two-dimensional arrays of micron-scale particles in microfluidic systems by surface acoustic waves

The two-dimensional concentration and manipulation of micron-scale particles by orthogonal, surface acoustic, standing waves is demonstrated. The particles are organized by liquid pressure waves in a microfluidic system over a piezoelectric substrate and form a uniform two-dimensional array with a spacing governed by the mechanical nodes of the two orthogonal, surface acoustic, standing waves. The nodal spacing can be controlled in each orthogonal direction independently by adjustment of the radio frequency applied to the separate acoustic wave transducers. This technique could be used to enhance the particle concentrations at sensing locations in DNA or protein array detectors.

Surface-Immobilized Peptide Aptamers as Probe Molecules for Protein Detection

We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.

Selective dielectrophoretic manipulation of surface-immobilized DNA molecules

The fabrication of nanoscale molecular devices is becoming increasingly important and research into their fabrication has intensified over the last few years. In particular, the attachment of molecular objects onto various surfaces has attracted considerable attention. Here, we report a multistep surface immobilization procedure, which allows the specific and controlled attachment of very long DNA molecules onto gold electrodes. Further, we report the effect of dielectrophoresis on these surface-bound DNA molecules with respect to amplitude and frequency, and we show that selected surface-immobilized DNA molecules can be manipulated by dielectrophoresis. Finally, we investigated the use of dielectrophoresis in conjunction with the multistep surface immobilization of fluorescently labelled, surface-bound λ-DNA in a basic data-storage device.

Electrical protein detection in cell lysates using high-density peptide-aptamer microarrays

Background The dissection of biological pathways and of the molecular basis of disease requires devices to analyze simultaneously a staggering number of protein isoforms in a given cell under given conditions. Such devices face significant challenges, including the identification of probe molecules specific for each protein isoform, protein immobilization techniques with micrometer or submicrometer resolution, and the development of a sensing mechanism capable of very high-density, highly multiplexed detection. Results We present a novel strategy that offers practical solutions to these challenges, featuring peptide aptamers as artificial protein detectors arrayed on gold electrodes with feature sizes one order of magnitude smaller than existing formats. We describe a method to immobilize specific peptide aptamers on individual electrodes at the micrometer scale, together with a robust and label-free electronic sensing system. As a proving proof of principle experiment, we demonstrate the specific recognition of cyclin-dependent protein kinases in whole-cell lysates using arrays of ten electrodes functionalized with individual peptide aptamers, with no measurable cross-talk between electrodes. The sensitivity is within the clinically relevant range and can detect proteins against the high, whole-cell lysate background. Conclusion The use of peptide aptamers selected in vivo to recognize specific protein isoforms, the ability to functionalize each microelectrode individually, the electronic nature and scalability of the label-free detection and the scalability of the array fabrication combine to yield the potential for highly multiplexed devices with increasingly small detection areas and higher sensitivities that may ultimately allow the simultaneous monitoring of tens or hundreds of thousands of protein isoforms.

Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity

Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture protein based on a cystatin scaffold that displays high affinity for human IL-8 with a KD of 35±10 nM and excellent ligand specificity. The change in the phase of the electrochemical impedance from the serum baseline, ∆θ(ƒ), measured at 0.1 Hz, was used as the measure for quantifying IL-8 concentration in the fluid. Optimal sensor signal was observed after 15 min incubation, and the sensor exhibited a linear response versus logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. A detection limit of around 90 fg/ml, which is significantly lower than the basal clinical levels of 5–10 pg/ml, was observed. Our results are significant for the development of point-of-care and early diagnostics where high sensitivity and short time-to-results are essential.

Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating.

Controlling Liquid Crystal Alignment Using Photocleavable Cyanobiphenyl Self-Assembled Monolayers

We report on the development of novel cyano-biphenyl-based thiolate self-assembled monolayers designed to promote homeotropic alignment of calamitic liquid crystals. The molecules developed contain an ortho-nitrobenzyl protected carboxylic acid group that on irradiation by soft UV (365 nm) is cleaved to yield carboxylic acid groups exposed at the surface that promote planar alignment. Using a combination of wetting, X-ray photoelectron spectroscopy, Fourier transform-infrared reflection absorption spectroscopy, and ellipsometry we show that high photolysis yields (>90%) can be achieved and that the patterned SAMs are suitable for the controlled alignment of calamitic liquid crystals. This study further shows that such photo-patterned SAMs can be used to control the formation of focal conic domains (FCDs) in the smectic-A phase in terms of positioning and size confinement on surfaces.

Acousto-microfluidics: Transporting microbubble and microparticle arrays in acoustic traps using surface acoustic waves

We demonstrate that aqueous suspensions of microbubbles, formed into arrays using standing surface acoustic waves (SSAWs), can be transported by controlled modulation of the SSAW frequency. The array is repeatedly captured at a sequence of spatial positions along the acoustic beam path and long-range transportation is achieved by periodic cycling of the applied frequency across the transducer bandwidth. We also demonstrate that controllable alignment and transport can be achieved in a detachable microfluidic device, where the microfluidic channel, in which particle transport occurs, is separated from the piezoelectric substrate by an acoustic coupling gel. Proof-of-concept transport is first discussed using a test system of latex particles before the non-invasive manipulation technique is applied to arrays of microbubbles. We explore the role of acoustic radiation forces in the spatial control of particles by analysing the dynamics of particle manipulation by SSAWs. Our results highlight the exquisite control which we have over the position and transport of particles and we anticipate that this technique could find wide applications for the accurate and programmable, non-invasive ordering and transport of biological samples in microfluidic systems.

Influence of alternating current electrokinetic forces and torque on the elongation of immobilized DNA

The authors investigate the elongation and orientation of different-sized deoxyribose nucleic acid (DNA) molecules, tethered onto gold electrodes via a terminal thiol, under the influence of high frequency ac electric fields. The DNA molecules are elongated from a random coil into an extended conformation and orientated along the electric field lines as a result of the forces acting on the molecules during the application of the ac electric fields. Elongation was observed in the frequency range 100 kHz-1 MHz, with field strengths of 0.06-1.0 MV/m. Maximum elongation for all DNA fragments tested, irrespective of size, was found for frequencies between 200 and 300 kHz. The torque acting on the induced dipole in the DNA molecules, complemented by a directional bias force, opposite in direction to the dielectrophoretic force, provides the main contribution to the elongation process. The length of elongation is limited to either half the distance between opposing electrodes or to the contour length of the DNA, whichever is shorter. Further, the authors show that the normalized length of the elongated DNA molecules is independent of the contour length of the DNA