Device for filamentous fungi growth monitoring using the multimodal frequency response of cantilevers.

Filamentous fungi cause opportunistic infections in hospital patients. A fast assay to detect viable spores is of great interest. We present a device that is capable of monitoring fungi growth in real time via the dynamic operation of cantilevers in an array. The ability to detect minute frequency shifts for higher order flexural resonance modes is demonstrated using hydrogel functionalised cantilevers. The use of higher order resonance modes sees the sensor dependent mass responsivity enhanced by a factor of 13 in comparison to measurements utilizing the fundamental resonance mode only. As a proof of principle measurement, Aspergillus niger growth is monitored using the first two flexural resonance modes. The detection of single spore growth within 10 h is reported for the first time. The ability to detect and monitor the growth of single spores, within a small time frame, is advantageous in both clinical and industrial settings.

Chladni figures revisited based on nanomechanics.

Chladni patterns based on nanomechanics in the microfluidic environment are presented. In contrast with the macroscopic observations in the gaseous environment, nanoparticles are found to move to the nodes, whereas micron-sized particles move to the antinodes of the vibrating interface. This opens the door to size-based sorting of particles in microfluidic systems, and to highly parallel and controlled assembly of biosensors and nanoelectronic circuits.

Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA.

The availability of entire genome sequences has triggered the development of microarrays for clinical diagnostics that measure the expression levels of specific genes. Methods that involve labelling can achieve picomolar detection sensitivity, but they are costly, labour-intensive and time-consuming. Moreover, target amplification or biochemical labelling can influence the original signal. We have improved the biosensitivity of label-free cantilever-array sensors by orders of magnitude to detect mRNA biomarker candidates in total cellular RNA. Differential gene expression of the gene 1-8U, a potential marker for cancer progression or viral infections, has been observed in a complex background. The measurements provide results within minutes at the picomolar level without target amplification, and are sensitive to base mismatches. This qualifies the technology as a rapid method to validate biomarkers that reveal disease risk, disease progression or therapy response. We foresee cantilever arrays being used as a tool to evaluate treatment response efficacy for personalized medical diagnostics.

Study of the mechanical properties of myomesin proteins using dynamic force spectroscopy.

Myomesin is the most prominent structural component of the sarcomeric M-Band that is expressed in mammalian heart and skeletal muscles. Like titin, this protein is an intracellular member of the Ig-fibronectin superfamily, which has a flexible filamentous structure and which is largely composed of two types of domain that are similar to immunoglobulin (Ig)-like and fibronectin type III (FNIII) domains. Several myomesin isoforms have been identified, and their expression patterns are highly regulated both spatially and temporally. Particularly, alternative splicing in the central part of the molecule gives rise to an isoform, EH (embryonic heart)-myomesin, containing a serine and proline-rich insertion with no well-defined secondary structure, the EH segment. EH-myomesin represents the major myomesin isoform at embryonic stages of mammalian heart and is rapidly down-regulated around birth, but it is re-expressed in the heart of patients suffering from dilated cardio-myopathy. Here, in order to facilitate a better understanding of the physiological, and possibly pathological, functions of myomesin proteins, we explore the mechanical stability, elasticity and force-driven structural changes of human myomesin's sub-molecular segments using single-molecule force spectroscopy and protein engineering. We find that human myomesin molecules are composed of modules (Ig and FNIII), that are designed to withstand force and we demonstrate that the human cardiac EH segment functions like an additional elastic stretch in the middle part of the EH-myomesin and behaves like a random coil. Consequently myomesin isoforms (proteins with or without the EH segment) have different elastic properties, the EH-myomesin being the more compliant one. These findings imply that the compliance of the M-band increases with the amount of EH-myomesin it contains. So, we provide the evidence that not only titin but also other sarcomeric proteins have complicated visco-elastic properties depending on the contractile parameters in different muscle types.

Mechanics and imaging of single DNA molecules.

We review recent experiments that have revealed mechanical properties of single DNA molecules using advanced manipulation and force sensing techniques(scanning force microscopy (SFM), optical or magnetic tweezers, microneedles). From such measurements, intrinsic relevant parameters (persistence length, stretch modulus) as well as their dependence on external parameters (non-physiological conditions, coating with binding agents or proteins) are obtained on a single-molecule level. In addition, imaging of DNA molecules using SFM is presented.

Model energy landscapes and the force-induced dissociation of ligand-receptor bonds.

We discuss models for the force-induced dissociation of a ligand-receptor bond, occurring in the context of cell adhesion or single molecule unbinding force measurements. We consider a bond with a structured energy landscape which is modeled by a network of force dependent transition rates between intermediate states. The behavior of a model with only one intermediate state and a model describing a molecular zipper is studied. We calculate the bond lifetime as a function of an applied force and unbinding forces under an increasing applied load and determine the relationship between both quantities. The dissociation via an intermediate state can lead to distinct functional relations of the bond lifetime on force. One possibility is the occurrence of three force regimes where the lifetime of the bond is determined by different transitions within the energy landscape. This case can be related to recent experimental observations of the force-induced dissociation of single avidin-biotin bonds.

Polymerization and mechanical properties of single RecA-DNA filaments.

The polymerization of individual RecA-DNA filaments, containing either single-stranded or double-stranded DNA, was followed in real time, and their mechanical properties were characterized with force-measuring laser tweezers. It was found that the stretch modulus of a filament is dominated by its (central) DNA component, while its bending rigidity is controlled by its (eccentric) protein component. The longitudinal stiffness of DNA increases 6- to 12-fold when the DNA is contained in the protein helix. Both the stretch modulus and the bending rigidity of a fiber change in the presence of various nucleotide cofactors-e.g., [gamma-thio]ATP, ATP, and ADP-indicating a substantial re-arrangement of spatial relationships between the nucleic acid and the protein scaffold. In particular, when complexed with ATP, a fiber becomes twice as extensible as a [gamma-thio]ATP fiber, suggesting that 32% of the DNA-binding sites have been released in its core. Such release may enable easy rotation of the DNA within the protein helix or slippage of the DNA through the center of the protein helix.

Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy.

We have worked out a procedure for covalent binding of native biomacromolecules on flat gold surfaces for scanning probe microscopy in aqueous buffer solutions and for other nanotechnological applications, such as the direct measurement of interaction forces between immobilized macromolecules, of their elastomechanical properties, etc. It is based on the covalent immobilization of amino group-containing biomolecules (e.g., proteins, phospholipids) onto atomically flat gold surfaces via omega-functionalized self-assembled monolayers. We present the synthesis of the parent compound, dithio-bis(succinimidylundecanoate) (DSU), and a detailed study of the chemical and physical properties of the monolayer it forms spontaneously on Au(111). Scanning tunneling microscopy and atomic force microscopy (AFM) revealed a monolayer arrangement with the well-known depressions that are known to stem from an etch process during the self-assembly. The total density of the omega-N-hydroxysuccinimidyl groups on atomically flat gold was 585 pmol/cm(2), as determined by chemisorption of (14)C-labeled DSU. This corresponded to approximately 75% of the maximum density of the omega-unsubstituted alkanethiol. Measurements of the kinetics of monolayer formation showed a very fast initial phase, with total coverage within 30 S. A subsequent slower rearrangement of the chemisorbed molecules, as indicated by AFM, led to a decrease in the number of monolayer depressions in approximately 60 min. The rate of hydrolysis of the omega-N-hydroxysuccinimide groups at the monolayer/water interface was found to be very slow, even at moderately alkaline pH values. Furthermore, the binding of low-molecular-weight amines and of a model protein was investigated in detail.

Specific antigen/antibody interactions measured by force microscopy.

Molecular recognition between biotinylated bovine serum albumin and polyclonal, biotin-directed IG antibodies has been measured directly under various buffer conditions using an atomic force microscope (AFM). It was found that even highly structured molecules such as IgG antibodies preserve their specific affinity to their antigens when probed with an AFM in the force mode. We could measure the rupture force between individual antibody-antigen complexes. The potential and limitations of this new approach for the measurement of individual antigen/antibody interactions and some possible applications are discussed.

Covalent anchoring of proteins onto gold-directed NHS-terminated self-assembled monolayers in aqueous buffers: SFM images of clathrin cages and triskelia.

N-Hydroxysuccinimide-terminated self-assembled monolayers with linear (CH2)10 chains were prepared on ultraflat Au(111) surfaces from dithiobis(succinimidylundecanoate). These monolayers, which are covalently chemisorbed to gold via thiolate bonds, form a highly reactive amino-group specific carpet at the liquid-solid interface. Proteins bind to it covalently in aqueous buffers under mild conditions; this provides a (general) procedure for protein immobilization for scanning probe microscopy. Using this technique, we have obtained what we believe are the first scanning force microscopy images of clathrin cages and of their in situ disassembly, yielding typical triskelia under non-denaturing conditions.

Immobilizing DNA on gold via thiol modification for atomic force microscopy imaging in buffer solutions.

Thiols, dialkylsulfides, and dialkyldisulfides are known to be chemisorbed with high affinity on gold. We have prepared DNAs of specific length and sequence carrying thiol groups at each end. For this purpose, primers with an HS-(CH2)6-arm at the 5'-end were used to amplify segments of plasmid DNA via the polymerase chain reaction. These thiolated DNAs bind strongly to the large, ultraflat Au surfaces which we have recently described [Hegner, M. et al. (1993) Surface Sci. 291, 39-46], and can be imaged by AFM in liquids (aqueous solutions or propanol). The lengths obtained in the AFM images are consistent with the DNA being in a native B-conformation.

Single amino acid substitutions can convert the uncleaved signal-anchor of sucrase-isomaltase to a cleaved signal sequence.

A hydrophobic segment near the amino terminus (positions 12-32) of rabbit sucrase-isomaltase functions both as a membrane anchor and as a signal sequence for translocation into the endoplasmic reticulum. Unlike most signal sequences, that of sucrase-isomaltase is not cleaved by signal peptidase. Using in vitro transcription and translation systems, we have found that substitution of a single proline, at position 28 or 29, converted the signal-anchor to a cleaved signal sequence, with cleavage occurring after alanine 26 and the introduced proline thereby occupying position +2 or +3 relative to the cleavage site. Two deletions that shorten the transmembrane domain by 8 amino acids were also effective, whereas various other changes upstream and downstream of this domain were without effect. We conclude that susceptibility to mammalian signal peptidase is influenced both by the length of the hydrophobic region and by the secondary structure downstream of the cleavage site.