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Martin Hegner

Publications and source records attributed to Martin Hegner.

18 recordsLinked to original sources
PubMed2007

Investigating the molecular mechanisms of in-plane mechanochemistry on cantilever arrays.

Free-standing cantilevers, which directly translate specific biochemical reactions into micromechanical motion, have recently attracted much attention as label-free biosensors and micro/nano robotic devices. To exploit this mechanochemical sensing technology, it is essential to develop a fundamental understanding of the origins of surface stress. Here we report a detailed study into the molecular basis of stress generation in aqueous environments focusing on the pH titration of model mercaptohexadecanoic acid self-assembled monolayers (SAMs), using in situ reference cantilevers coated with nonionizable hexadecanethiol SAMs. Semiautomated data analysis and a statistical model were developed to quantify cyclic deprotonation/protonation reactions on multiple arrays. In-plane force titrations were found to have the sensitivity to detect ionic hydrogen bond formation between protonated and nonprotonated carboxylic acid groups in the proximity of the surface pK1/2, which generated a mean tensile differential surface stress of +1.2 +/- 0.3 mN/m at pH 6.0, corresponding to 1 pN attractive force between two adjacent MHA molecules. Conversely, the magnitude of compressive differential surface stress was found to increase progressively with pH >/= 7.0, reaching a maximum of -14.5 +/- 0.5 mN/m at pH 9.0, attributed to enhanced electrostatic repulsion between deprotonated carboxylic acid groups. However, striking differences were observed in the micromechanical responses to different ionic strength and ion species present in the aqueous environment, highlighting the critical role of counter- and co-ions on surface stress. Our findings provide fundamental insights into the molecular mechanisms of in-plane mechanochemistry, which may be exploited for biosensing and nanoactuation applications.

Biosensing Techniques↗
PubMed2006

Conformational change of bacteriorhodopsin quantitatively monitored by microcantilever sensors.

Bacteriorhodopsin proteoliposomes were used as a model system to explore the applicability of micromechanical cantilever arrays to detect conformational changes in membrane protein patches. The three main results of our study concern: 1), reliable functionalization of micromechanical cantilever arrays with proteoliposomes using ink jet spotting; 2), successful detection of the prosthetic retinal removal (bleaching) from the bacteriorhodopsin protein by measuring the induced nanomechanical surface stress change; and 3), the quantitative response thereof, which depends linearly on the amount of removed retinal. Our results show this technique to be a potential tool to measure membrane protein-based receptor-ligand interactions and conformational changes.

Bacteriorhodopsins↗
PubMed2005

Micromechanical cantilever array sensors for selective fungal immobilization and fast growth detection.

We demonstrate the use of micromechanical cantilever arrays for selective immobilization and fast quantitative detection of vital fungal spores. Micro-fabricated uncoated as well as gold-coated silicon cantilevers were functionalized with concanavalin A, fibronectin or immunoglobulin G. In our experiments two major morphological fungal forms were used--the mycelial form Aspergillus niger and the unicellular yeast form Saccharomyces cerevisiae, as models to explore a new method for growth detection of eukaryotic organisms using cantilever arrays. We exploited the specific biomolecular interactions of surface grafted proteins with the molecular structures on the fungal cell surface. It was found that these proteins have different affinities and efficiencies to bind the spores. Maximum spore immobilization, germination and mycelium growth was observed on the immunoglobulin G functionalized cantilever surfaces. We show that spore immobilization and germination of the mycelial fungus A. niger and yeast S. cerevisiae led to shifts in resonance frequency within a few hours as measured by dynamically operated cantilever arrays, whereas conventional techniques would require several days. The biosensor could detect the target fungi in a range of 10(3) - 10(6) CFUml(-1). The measured shift is proportional to the mass of single fungal spores and can be used to evaluate spore contamination levels. Applications lie in the field of medical and agricultural diagnostics, food- and water-quality monitoring.

Aspergillus niger↗
PubMed2005

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.

Antigens↗
PubMed2005

Micromechanical mass sensors for biomolecular detection in a physiological environment.

Micromechanical cantilever arrays are used to measure time-resolved adsorption of tiny masses based on protein-ligand interactions. Here, streptavidin-biotin interactions are investigated in a physiological environment. A measurement method is introduced using higher flexural modes of a silicon cantilever in order to enhance the sensitivity of mass detection. Modeling the cantilever vibration in liquid allows the measurement of absolute mass changes. We show time-resolved mass adsorption of final 7+/-0.7 ng biotinylated latex beads. The sensitivity obtained is about 2.5 pg/Hz measuring at a center frequency of 750 kHz.

Biomechanical Phenomena↗
PubMed2005

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.

DNA↗
PubMed2005

Enantioselective recognition of phenylalanine by a chiral amphiphilic macrocycle at the air-water interface: a copper-mediated mechanism.

The synthesis of a new chiral amphiphilic calix[4]resorcinarene, tetrakis(N-methylprolyl)tetraundecylcalix[4]resorcinarene (L-RA-Pro), bearing four L-prolyl moieties at the macrocyclic upper rim and four undecyl chains at the lower rim is described. This synthesis has been carried out via a Mannich-type reaction of L-proline and formaldehyde. It has been shown by means of Langmuir balance technique that L-RA-Pro self-assemble as well-defined monomolecular layers at the air-water interface. The effect of various cations on the stability of these monolayers has been studied. The experiments reveal that while there is a slight stabilization effect of K+, Cd2+, Co2+, Mg2+, and Ni2+, there is a high decrease in the collapse pressure in the presence of Cu(II) cation, showing that monolayers of L-RA-Pro, formed at the air-water interface, have a certain selectivity for copper(II) ions with regard to other cations tested. This supramolecular complex exhibits enantioselective recognition properties vs phenylalanine; the mechanism of this interaction is discussed.

Air↗
PubMed2005

Myomesin is a molecular spring with adaptable elasticity.

The M-band is a transverse structure in the center of the sarcomere, which is thought to stabilize the thick filament lattice. It was shown recently that the constitutive vertebrate M-band component myomesin can form antiparallel dimers, which might cross-link the neighboring thick filaments. Myomesin consists mainly of immunoglobulin-like (Ig) and fibronectin type III (Fn) domains, while several muscle types express the EH-myomesin splice isoform, generated by the inclusion of the unique EH-segment of about 100 amino acid residues (aa) in the center of the molecule. Here we use atomic force microscopy (AFM), transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy for the biophysical characterization of myomesin. The AFM identifies the "mechanical fingerprints" of the modules constituting the myomesin molecule. Stretching of homomeric polyproteins, constructed of Ig and Fn domains of human myomesin, produces a typical saw-tooth pattern in the force-extension curve. The domains readily refold after relaxation. In contrast, stretching of a heterogeneous polyprotein, containing several repeats of the My6-EH fragment reveals a long initial plateau corresponding to the sum of EH-segment contour lengths, followed by several My6 unfolding peaks. According to this, the EH-segment is characterized as an entropic chain with a persistence length of about 0.3nm. In TEM pictures, the EH-domain appears as a gap in the molecule, indicating a random coil conformation similar to the PEVK region of titin. CD spectroscopy measurements support this result, demonstrating a mostly non-folded conformation for the EH-segment. We suggest that similarly to titin, myomesin is a molecular spring, whose elasticity is modulated by alternative splicing. The Ig and Fn domains might function as reversible "shock absorbers" by sequential unfolding in the case of extremely high or long sustained stretching forces. These complex visco-elastic properties of myomesin might be crucial for the stability of the sarcomere.

Circular Dichroism↗
PubMed2005

Rapid biosensor for detection of antibiotic-selective growth of Escherichia coli.

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to an approximate sensitivity of approximately 100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, approximately 1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.

Anti-Bacterial Agents↗
PubMed2004

Micromechanical oscillators as rapid biosensor for the detection of active growth of Escherichia coli.

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated by common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The sensor is able to detect active growth of Escherichia coli cells within 1 h which is significantly faster than any conventional plating method which requires at least 24 h. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of active bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing by adding antibiotics to the nutritive layer.

Biosensing Techniques↗
PubMed2004

Inkjet deposition of alkanethiolate monolayers and DNA oligonucleotides on gold: evaluation of spot uniformity by wet etching.

Inkjet printing allows localized, contact-free deposition of liquids onto arbitrary substrates. In this article we demonstrate the fast formation of high-quality self-assembled monolayers (SAMs) on gold surfaces. Using a selective etch process, we verify the uniformity of the deposited spots. A direct comparison with microcontact-printed SAMs on Au revealed similar resist quality as inkjet-deposited alkanethiolate SAMs. Likewise, inkjet printing of thiol-functionalized and non-thiolated single-stranded DNA oligomers formed molecular layers protecting Au from etchants. For all compounds used, we achieved etched patterns that were homogeneous and free of defects. These results indicate that an inkjet is a convenient tool for surface functionalization and the direct writing of molecular films and resists.

Alkanes↗
PubMed2002

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.

Base Sequence↗
PubMed2002

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.

Base Sequence↗
PubMedDate not supplied

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.

Adsorption↗
PubMedDate not supplied

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.

Boron Compounds↗