Label free analysis of transcription factors using microcantilever arrays.

We report the measurement of protein interaction with double-stranded DNA oligonucleotides using cantilever microarray technology. We investigated two different DNA-binding proteins, the transcription factors SP1 and NF-kappaB, using cantilever arrays as they allow label-free measurement of different biomolecular interactions in parallel. Double-stranded DNA oligonucleotides containing a specific binding site for a transcription factor were sensitized on gold-coated cantilevers. The binding of the transcription factor creates a surface stress, resulting in a bending of the cantilevers. Both transcription factors could be detected independently at concentrations of 80-100 nM. A concentration dependence of the bending signal was measured using concentrations from 100 to 400 nM of NF-kappaB. The experiments show that the recognition sequence of one transcription factor can serve as a reference for the other, highlighting the sequence specificity of transcription factor binding.

Chain-like structure elements in Ni40Ta60 metallic glasses observed by scanning tunneling microscopy.

The structure of metallic glasses is a long-standing question because the lack of long-range order makes diffraction based techniques difficult to be applied. Here, we used scanning tunneling microscopy with large tunneling resistance of 6 GΩ at low temperature in order to minimize forces between probe and sample and reduce thermal fluctuations of metastable structures. Under these extremely gentle conditions, atomic structures of Ni40Ta60 metallic glasses are revealed with unprecedented lateral resolution. In agreement with previous models and experiments, icosahedral-like clusters are observed. The clusters show a high degree of mobility, which explains the need of low temperatures for stable imaging. In addition to icosahedrons, chain-like structures are resolved and comparative density functional theory (DFT) calculations confirm that these structures are meta-stable. The co-existence of icosahedral and chain-like structures might be an key ingredient for the understanding of the mechanical properties of metallic glasses.

Interferon alpha-2a interactions on glass vial surfaces measured by atomic force microscopy.

Atomic force microscopy was used to study adsorption and adhesion peculiarities of interferon alpha-2a on glass and mica surfaces. The specific protein adsorption behavior as a function of the pH value was illustrated on mica by single molecule imaging, while adhesion forces between interferon molecules and inner surfaces of borosilicate glass vials were measured directly under aqueous buffer conditions by force microscopy. We found that the adhesion force on Schott FIOLAX Type I plus was reduced by 40% of the total adhesion force measured on Schott FIOLAX, a standard type I borosilicate glass quality. These results reflect the anticipated superiority of the special "Type I plus" coating over undesired protein adsorption to glass. In addition, this study gives insight into a new method to predict unintended protein adsorption to glass container walls and to characterize the adsorption process by force measurement.

Topological structure and chemical composition of inner surfaces of borosilicate vials.

The use of atomic force microscopy (AFM) and x-ray photoelectron spectroscopy (XPS) is described to characterize the inner surfaces of pharmaceutical vials. The two type I borosilicate glasses included in this study slightly differ in their amounts of alkaline oxides. The topography and chemistry of the inner surfaces of vials are predominantly caused by the forming process. A structural and chemical modification of the inner surface of vials was also observed when exposing the surface to different pH conditions and special treatment like washing and sterilization, which are routine operation steps during galenical manufacturing.

An antibody-sensitized microfabricated cantilever for the growth detection of Aspergillus niger spores.

We demonstrate a new sensitive biosensor for detection of vital fungal spores of Aspergillus niger. The biosensor is based on silicon microfabricated cantilever arrays operated in dynamic mode. The change in resonance frequency of the sensor is a function of mass binding to the cantilever surface. For specific A. niger spore immobilization on the cantilever, each cantilever was individually coated with anti-Aspergillus niger polyclonal antibodies. We demonstrate the detection of single A. niger spores and their subsequent growth on the functionalized cantilever surface by online measurements of resonance frequency shifts. The new biosensor operating in humid air allows quantitative and qualitative detection of A. niger spores as well as detection of vital, functional spores in situ within approximately 4 h. The detection limit of the sensor is 103 CFU mL-1. Mass sensitivity of the cantilever sensor is approximately 53 pg Hz-1.

Measuring site-specific cluster-surface bond formation.

Recent advances in dynamic force microscopy show that it is possible to measure the forces between atomically sharp tips and particular atomic positions on surfaces as a function of distance. However, on most ionic surfaces, the positive and negative ions can so far not be distinguished. In this paper, we use the CaF2(111) surface, where atomic resolution force microscopy has allowed identification of the positions of the Ca2+ and F- ions in the obtained images, to demonstrate that short-range interaction forces can be measured selectively above chemically identified surface sites. Combining experimental and theoretical results allows a quantification of the strength and distance dependence of the interaction of a tip-terminating cluster with particular surface ions and reveals details of cluster and surface relaxation. Further development of this approach will provide new insight into mechanisms of chemical bond formation between clusters, cluster deposition at surfaces, processes in adhesion and tribology, and single atom manipulation with the force microscope.

A label-free immunosensor array using single-chain antibody fragments.

We report a microcantilever-based immunosensor operated in static deflection mode with a performance comparable with surface plasmon resonance, using single-chain Fv (scFv) antibody fragments as receptor molecules. As a model system scFv fragments with specificity to two different antigens were applied. We introduced a cysteine residue at the C terminus of each scFv construct to allow covalent attachment to gold-coated sensor interfaces in directed orientation. Application of an array enabled simultaneous deflection measurements of sensing and reference cantilevers. The differential deflection signal revealed specific antigen binding and was proportional to the antigen concentration in solution. Using small, oriented scFv fragments as receptor molecules we increased the sensitivity of microcantilevers to approximately 1 nM.

Temperature dependence of unbinding forces between complementary DNA strands.

Force probe techniques such as atomic force microscopy can directly measure the force required to rupture single biological ligand receptor bonds. Such forces are related to the energy landscape of these weak, noncovalent biological interactions. We report unbinding force measurements between complementary strands of DNA as a function of temperature. Our measurements emphasize the entropic contributions to the energy landscape of the bond.

Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array.

We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3' and 5' overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.