Abstract: We show that optical tweezers are a valuable tool to study the co-translational folding of a nascent polypeptide chain at the ribosome in real-time. The aim of this study was to demonstrate that a stable and intact population of ribosomes can be tethered to polystyrene beads and that specific hook-ups to the nascent polypeptide chain by dsDNA handles, immobilized on a second bead, can be detected. A rupture force of the nascent chain in the range of 10-50pN was measured, which demonstrates that the system is anchored to the surface in a stable and specific way. This will allow in numerous future applications to follow protein folding using much lower forces.
Abstract: The applicability of single-molecule fluorescence assays in liquids is limited by diffusion to concentrations in the low picomolar range. Here, we demonstrate quantitative single-molecule detection at attomolar concentrations within 1 min by excitation and detection of fluorescence through a single-mode optical fiber in presence of turbulent flow. The combination of high detectability and short measurement times promises applications in ultrasensitive assays, sensors, and point-of-care medical diagnostics.
Abstract: Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor--the FhuA receptor of Escherichia coli--is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.
Abstract: The interaction of cationic surfactants with single dsDNA molecules has been studied using force-measuring optical tweezers. For hydrophobic chains of length 12 and greater, pulling experiments show characteristic features (e.g. hysteresis between the pulling and relaxation curves, force-plateau along the force curves), typical of a condensed phase (compaction of a long DNA into a micron-sized particle). Depending on the length of the hydrophobic chain of the surfactant, we observe different mechanical behaviours of the complex (DNA-surfactants), which provide evidence for different binding modes. Taken together, our measurements suggest that short-chain surfactants, which do not induce any condensation, could lie down on the DNA surface and directly interact with the DNA grooves through hydrophobic-hydrophobic interactions. In contrast, long-chain surfactants could have their aliphatic tails pointing away from the DNA surface, which could promote inter-molecular interactions between hydrophobic chains and subsequently favour DNA condensation.
Abstract: The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein-VirE2-enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated-using single-molecule techniques-that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.
Abstract: We describe a method to detect and count transient burstlike signals in the presence of a significant stationary noise. To discriminate a transient signal from the background noise, an optimum threshold is determined using an iterative algorithm that yields the probability distribution of the background noise. Knowledge of the probability distribution of the noise then allows the determination of the number of transient events with a quantifiable error (wrong-positives). We apply the method, which does not rely on the choice of free parameters, to the detection and counting of transient single-molecule fluorescence events in the presence of a strong background noise. The method will be of importance in various ultra sensing applications.
Abstract: 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.
Abstract: Unbinding forces of weak, noncovalent bonds have been measured by scanning force microscopy (1) or biomembrane force probes (2). Initially, these scanning force microscopy measurements focused on feasibility studies to measure single biomolecular interactions (3–5). Recently, however, a few groups showed that these single molecule experiments give a direct link to bulk experiments where thermodynamic data are experimentally acquired (6–9). In contrast with bulk experiments where averaged properties are measured, a single molecular approach gives access to properties that are hidden in the ensemble. These experiments can give insight into the geometry of the energy landscape of a biomolecular bond (7,9–11). Some experiments even showed that intermediate states during unbinding (unfolding) exist which only can be detected by single molecule experiments (12–14).
Abstract: This article describes the design of a dual-beam optical tweezers (OT) instrument which, in contrast to conventional single-beam OT, directly measures the change in light momentum flux when a trapped object experiences a force. Consequently, no local calibration is needed to measure the force acting on a trapped particle. The instrument has a high trapping efficiency and forces up to 200 pN can be measured. In addition, the above-mentioned system operates in conjunction with a three-dimensional steerable single-beam OT. (C) 2002 American Institute of Physics.
Abstract: 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.
Abstract: 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.
Abstract: The chemical long-range ordering in a CoPt alloy film, grown at 800K onto a MgO (001) substrate by molecular beam epitaxy, has been investigated using both X-ray diffraction (XRD) and high-resolution transmission electron microscopy (HRTEM). From HRTEM, we found that this him exhibits a Ll(0)-type order, characterised by a stacking of almost pure cobalt and pure platinum planes along the tetragonal axis parallel to the growth direction. The value of the long-range order parameter deduced from XRD, significantly smaller than 1, is attributed to the existence of antiphase boundaries along the [001] direction. (C) 2001 Acta Materialia Inc. Published by Elsevier Science Ltd. All rights reserved.
Abstract: Angular-dependent x-ray magnetic circular dichroism experiments are performed on Ni/Pt multilayers at the Ni L-2,L-3 edges under magnetic fields up to 50 kG at a temperature of 10 K. By rotating the applied field away from the easy axis a decrease of the ratio of the orbital-to-spin magnetic moment mu(L)/ mu(S) Of Ni is observed for perpendicularly magnetized Ni/Pt layers which show considerable magnetic anisotropy energy (approximate to 20 mu eV/atom). Saturation effects in the recorded spectra, depending on both the thicknesses of Ni and Pt, are shown to lead into erroneous conclusions in the determination of mu(L)/mu(S) and they have to be considered in the analysis. An anisotropy of the Ni-orbital moment Delta mu(L) as large as 22% of mu(L) is determined. Delta mu(L) is demonstrated to be the origin of the magnetic anisotropy energy of 3d ferromagnetic elements.
Abstract: An experimental and theoretical investigation of the structural and micromagnetic properties of the Co,,Pt,, ordered alloy is presented. The Co-3d and Pt-Sd orbital and spin magnetic moments and their anisotropies are determined by means of angle-dependent x-ray magnetic-circular-dichroism (XMCD) measurements. Our calculated spin magnetic moments of both Co and Pt are in good agreement with our experimental results, while the orbital magnetic moments are underestimated. The reduction of the 3d and 5d orbital magnetic moments, observed when the spin magnetic moment is forced out of the easy axis of magnetization, is reproduced by the theory. The calculated XMCD reproduces semiquantitatively the experimental spectra and underestimates the so-called branching ratio. The computed magnetocrystalline anisotropy is of the same order of magnitude as the experimental one with the correct sign.
Abstract: We have investigated by X-ray Magnetic Circular Dichroism (XMCD) the Fe spin and orbital magnetic moments in Fe/Pd(001) multilayers with nominal Fe thicknesses up to 3 Atomic Layers (AL). We find a strong enhancement of both spin and orbital moments (m(spin) approximate to 2.8 mu(B) and m(orb) approximate to 0.3 mu(B)) compared to bulk Fe in excellent agreement with theoretical calculations. For 1 AL of Fe, the magnetic dipole term was deduced using the Stohr and Kong method and found to be non-negligible (20% of m(spin)). We further show that, for 3 AL of Fe, the high spin phase is correlated to a fcc-like crystalline structure with a large atomic volume of Fe (approximate to 12.4 Angstrom(3)).
Abstract: Angle-dependent x-ray magnetic circular dichroism (XMCD) experiments performed at both the Co and Pt L-2,L-3 edges for a cubic CoPt3 thin film, which exhibits growth-induced chemical anisotropy, are used to determine the element specific magnetic anisotropy. The large decrease of the 3d orbital moment, observed when the spins are forced out the easy axis of magnetization by the applied magnetic field, is a consequence of a strong magnetocrystalline anisotropy (MCA). In addition, a weak but systematic variation of the Pt orbital moment indicates that the 5d atoms could play an important role in the MCA. These results correlate the microscopic XMCD evaluation of the MCA and the local structural anisotropy as observed with x-ray absorption fine structure (XAFS). (C) 1998 American Institute of Physics.
Abstract: Angle-dependent x-ray magnetic circular dichroism experiments have been performed at both the Co and Pt L-2,L-3 edges in two epitaxial (111) CoPt3 thin films grown at 690 and 800 K. The analysis of the angular variations of the 3d orbital magnetic moment shows two different magnetic behaviors: a strong perpendicular magnetocrystalline anisotropy (PMA) for the film grown at 690 K and an almost isotropic behavior for the film grown at higher temperature. The same analysis at the Pt L-2,L-3 edges suggests that the 5d electrons play an important role in the PMA. Our results correlate the appearance of PMA with the existence of anisotropic structural effects induced during the codeposition process.
Abstract: We have measured magnetic anisotropy constants and domain structures in arrays of magnetic dots 0.5 mu m wide fabricated out of an epitaxial (0001) hcp cobalt film. Occurrence of a concentric stripe domain pattern for which the magnetization is mostly in-plane with small alternately up and down perpendicular components is found for a 25-nm-thick cobalt dot array. Interestingly, this sample produces a spin reorientation from a quasi in-plane to a fully perpendicularly oriented magnetization when the temperature is lowered, due to an increase of the perpendicular anisotropy at low temperature. (C) 1997 American Institute of Physics. [S0003-6951(97)00145-9].
Abstract: This chapter provides anintroduction to the x-ray magnetic circular dichroism technique.
It discusses the use of this technique for probing themicroscopic origin of the magnetocrystalline anisotropy in transition metal thin
films. Using an appropriate illustrative example, we attempt topresent an up-to-date account of this research field.
Abstract: Early detection of protein aggregation is of great importance in the field of neurodegenerative diseases. The successful detection of the aggregation of the protein α-synuclein in a quantitative, label-free manner by functionalising a microcantilever with α- synuclein monomers and operating it in dynamic mode in the presence of α-synuclein monomers in solution is reported. A total mass of 6 ng of α-synuclein was detected over 9 hours on the surface of the cantilever. The result is compared to conventional fluorescence measurements of α-synuclein aggregation under similar conditions. It is found that the label-free cantilever detection method requires a concentration of protein 50 times smaller than that of the current method and indicated potential for significantly faster response times.
Abstract: Cantilever array-based sensor devices widely utilise the laser-based optical deflection method for measuring static cantilever deflections mostly with home-built devices with individual geometries. In contrast to scanning probe microscopes, cantilever array devices have no additional positioning device like a piezo stage. As the cantilevers are used in more and more sensitive measurements, it is important to have a simple, rapid, and reliable calibration relating the deflection of the cantilever to the change in position measured by the position-sensitive detector. We present here a simple method for calibrating such systems utilising commercially available AFM cantilevers and the equipartition theorem.
Abstract: In diseases such as cancer or during viral infections gene expression is greatly altered. These changes in gene activity can be analysed at different levels of cellular activity, like transcription activation, transcription and translation. Currently, no simple method is available to detect all these biochemical signals simultaneously and rapidly. Micromechanical cantilever sensor array technology is applied, because it has the advantage that sample preconditioning like labelling and amplification is not required. Furthermore, DNA, RNA, protein or combinations thereof could be detected in parallel on a single cantilever array. With such a device, diagnosis and therefore treatment of diseases can be improved. Here we present successive detection of DNA hybridization and antigen using the same micromechanical cantilever sensor array.
Notes: International Conference on Nanoscience and Technology ICN&T 2006
Basel, Switzerland, from July 31 to August 4
Abstract: We used microfabricated cantilever array sensors for an artificial nose setup. Each cantilever is coated on its top surface with a polymer layer. Volatile gaseous analytes are detected by tracking the diffusion process of the molecules into the polymer layers, resulting in swelling of the polymer layers and therewith bending of the cantilevers. From the bending pattern of all cantilevers in the array, a characteristic 'fingerprint' of the analyte is obtained, which is evaluated using principal component analysis. In a flow of dry nitrogen gas, the bending of the cantilevers is reverted to its initial state before exposure to the analyte, which allows reversible and reproducible operation of the sensor. We show examples of detection of solvents, perfume essences and beverage flavors. In a medical application, the setup provides indication of presence of diseases in patient's breath samples.
Notes: International Conference on Nanoscience and Technology ICN&T 2006
Basel, Switzerland, from July 31 to August 4
Abstract: Nanomechanical sensors based on cantilever technology allow the measurement of various physical properties. Here we present a software for the comprehensive analysis of such data. An example for the combined measurement of mass and surface stress is presented.
Notes: International Conference on Nanoscience and Technology ICN&T 2006
Basel, Switzerland, from July 31 to August 4
Abstract: We have investigated the mechanics of individual DNA strands exposed to DNA binding ligands. The interaction of these agents with individual dsDNA strands measured by optical tweezers clearly indicates the ligand-DNA binding mode. As expected, if the compound is intercalating then an increase of contour length is detected. Groove binders affect the overstretching capabilities of the formerly naked dsDNA strand. We interacted SYBR® Green I with naked dsDNA. The binding mode of this compound, which is used for nucleic acid gel staining, is not known. The mechanics of the interaction of SYBR® is revealed by optical tweezers experiments. The force extension curves on single dsDNA fragments show a groove-binding mode, which does not affect the contour length of the molecule but significantly alters the overstretching behaviour of the dsDNA.
Abstract: The development of versatile scanning probe methods such as atomic force microscopy (AFM) makes it today possible to study bio-adhesion on a single molecule level. In this paper, we present AFM-force-spectroscopy experiments on complementary DNA strands. From such experiments, intrinsic thermodynamical properties (energy landscape) of these weak non covalent bonds can be determined.
Abstract: We report a magnetic circular dichroism study at the Ce-2p core level X-ray absorption spectra on the ferromagnet CeRh3B2. The Ce-L-2,L-3 spectra measured on a single crystal are compared to those obtained on CeRu2Ge2 and CeFe2, taken as reference systems. The shape of the circular dichroism signal, that witnesses the 4f hybridisation, supports the delocalised picture of the 4f electrons in CeRh3B2. (C) 1999 Elsevier Science B.V. All rights reserved.
Abstract: Structural and magnetic properties of Co50 +/- X Pt50 -/+ X films 25-50 nm thick, prepared by molecular beam epitaxy onto a Pt buffer grown on MgO (001) substrate have been investigated. A series of 3 samples with different compositions (x = 6, 0, -6) was grown at 800 K on a 10 nm thick Pt buffer and another series of 5 samples of equiatomic composition was prepared at various growth temperatures (390 K less than or equal to T(G)less than or equal to 780 K) On a Pt buffer 4 nm thick. X-ray diffraction and TEM studies show the presence of grains with [111] and [002] orientations, the [002] grains being a mixture of the tetragonal L1(0) ordered phase and of the fee disordered one. Both the thickness of the buffer layer and the deposition temperature are determinant parameters of the structural quality of the films and of the degree of long range order (LRO). An apparent LRO parameter (eta(app)) is deduced from the superstructure and main peak intensity ratio. Its increase with the growth temperature is described through a thermally activated model that yields a small activation energy of 0.28 eV, illustrating the role played by both surface diffusion and surface interactions in building the L1(0) compound in agreement with theoretical predictions. An average uniaxial magnetocrystalline anisotropy energy (K-u(av)) is deduced from the magnetization curves measured by a SQUID. The anisotropy energy of the [002] grains (K-u(002)) is deduced, assuming a linear relationship between the anisotropies and the phase percentages. One observes a continuous but not linear increase of K-u(002) with eta(app).
Notes: 1998 MRS Spring Meeting San Francisco from April 13-17, 1998
Abstract: We report X-ray magnetic circular dichroism experiments performed at the U-M-4.5 edges of an URhAl single crystal. Thanks to the sum rules, we derive an orbital magnetic moment on the uranium atoms equal to (1.63+/-0.14)mu(B). The comparison of the shape of the M-5 dichroic signal with pure J multiplet calculations shows effects due to 5f-hybridization. However, we found that the contribution of the magnetic dipole term [T-Z] to the effective spin moment [S-Z(eff)] = [S-Z] + 3[T-Z] is non negligible. These results indicate an higher localization of the 5f-orbitals with respect to other itinerant ferromagnetic actinides compounds such as UFe2 or UNi2. (C) 1998 Published by Elsevier Science S.A.
Notes: ICFE-3 International Conference on f Elements No3, Paris , FRANCE (14/09/1997)
