Christof Gutsche

Optical tweezers with 2.5 kHz bandwidth video detection for single-colloid electrophoresis.

We developed an optical tweezers setup to study the electrophoretic motion of colloids in an external electric field. The setup is based on standard components for illumination and video detection. Our video based optical tracking of the colloid motion has a time resolution of 0.2 ms, resulting in a bandwidth of 2.5 kHz. This enables calibration of the optical tweezers by Brownian motion without applying a quadrant photodetector. We demonstrate that our system has a spatial resolution of 0.5 nm and a force sensitivity of 20 fN using a Fourier algorithm to detect periodic oscillations of the trapped colloid caused by an external ac field. The electrophoretic mobility and zeta potential of a single colloid can be extracted in aqueous solution avoiding screening effects common for usual bulk measurements.

Polyelectrolyte-compression forces between spherical DNA brushes.

Optical tweezers are employed to measure the forces of interaction within a single pair of DNA-grafted colloids, dependent on the molecular weight of the DNA chains, and the concentration and valence of the surrounding ionic medium. The resulting forces are short range and set in as the surface-to-surface distance between the colloidal cores reaches the value of the brush height. The measured force-distance relation is analyzed by means of a theoretical treatment that quantitatively describes the effects of compression of the chains on the surface of the opposite-lying colloid. Quantitative agreement with the experiment is obtained for all parameter combinations.

Colloids dragged through a polymer solution: Experiment, theory, and simulation

We present microrheological measurements of the drag force on colloids pulled through a solution of lambda-DNA (used here as a monodisperse model polymer) with an optical tweezer. The experiments show a drag force that is larger than expected from the Stokes formula and the independently measured viscosity of the DNA solution. We attribute this to the accumulation of DNA in front of the colloid and the reduced DNA density behind the colloid. This hypothesis is corroborated by a simple drift-diffusion model for the DNA molecules, which reproduces the experimental data surprisingly well, as well as by corresponding Brownian dynamics simulations.

Micro-rheology on (polymer-grafted) colloids using optical tweezers

Optical tweezers are experimental tools with extraordinary resolution in positioning (± 1 nm) a micron-sized colloid and in the measurement of forces (± 50 fN) acting on it-without any mechanical contact. This enables one to carry out a multitude of novel experiments in nano- and microfluidics, of which the following will be presented in this review: (i) forces within single pairs of colloids in media of varying concentration and valency of the surrounding ionic solution, (ii) measurements of the electrophoretic mobility of single colloids in different solvents (concentration, valency of the ionic solution and pH), (iii) similar experiments as in (i) with DNA-grafted colloids, (iv) the nonlinear response of single DNA-grafted colloids in shear flow and (v) the drag force on single colloids pulled through a polymer solution. The experiments will be described in detail and their analysis discussed.

Forces between blank surfaces as measured by the colloidal probe technique and by optical tweezers--a comparison.

The well-established atomic force microscopy (AFM)-based colloidal probe technique (CPT) and optical tweezers (OT) are combined to measure the interaction forces between blank SiO(2) surfaces in aqueous ionic solutions (CaCl(2)) of varying concentration at pH 7. Spherical colloids (SiO(2), diameter approximately 4.63 +/- 0.05 microm) taken out of the same batch are used by both methods. In the case of CPT, a single colloid is glued to a cantilever, and the interaction forces with a plain SiO(2) surface are determined in dependence on the concentration of the surrounding medium. For the OT studies, two colloids (one fixed to a micropipet by capillary action, the other held with the optical trap) are approached to each other in nanometer steps, and the resulting forces are measured for the same media as in the CPT experiment. Both techniques fit well to each other and enable one to cover interaction energies ranging from 10(-5) to 1 mN/m. The experimental data are well described by the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory revealing that the effective surface charge density changes slightly with concentration.

Dynamic force spectroscopy on the binding of monoclonal antibodies and tau peptides

Optical tweezers-assisted dynamic force spectroscopy (DFS) is employed to investigate specific receptor/ligand interactions on the level of single binding events. Here, the specific binding of two anti-human tau monoclonal antibodies (mAbs), HPT-110 and HPT-104, to synthetic tau-peptides with different phosphorylation patterns is analyzed. The specificity of HPT-110 to the tau-peptide containing a phosphorylation at Ser235 and of HPT-104 to the tau-peptide containing a phosphorylation at Thr231 is confirmed. Additionally, our approach allows for a detailed characterization of the unspecific interactions that are observed between HPT-104 and the peptide phosphorylated only at Ser235 and between HPT-110 and the peptide phosphorylated only at Thr231. By analyzing the measured rupture-force distributions it is possible to separate unspecific from specific interactions. Thereby for the latter characteristic parameters like the lifetime of the bond without force τ0, the characteristic length xts and the free energy of activation ΔG are determined. The results are in accordance with conventional ELISA tests but offer a much more refined insight.

Determining the Specificity of Monoclonal Antibody HPT-101 to Tau-Peptides with Optical Tweezers

Optical tweezers-assisted dynamic force spectroscopy is employed to investigate specific receptor-ligand interactions on the level of single binding events. In particular, we analyze binding of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to synthetic tau-peptides with two potential phosphorylation sites (Thr231 and Ser235), being the most probable markers for Alzheimers disease. Whereas the typical interpretation of enzyme-linked immunosorbent assay (ELISA) suggests that this monoclonal antibody binds exclusively to the double-phosphorylated tau-peptide, we show here by DFS that the specificity of only mAb HPT-101 is apparent. In fact, binding occurs also to each sort of monophosphorylated peptide. Therefore, we characterize the unbinding process by analyzing the measured rupture force distributions, from which the lifetime of the bond without force τ0, its characteristic length xts, and the free energy of activation ΔG are extracted for the three mAb/peptide combinations. This information is used to build a simple theoretical model to predict features of the unbinding process for the double-phosphorylated peptide purely based on data on the monophosphorylated ones. Finally, we introduce a method to combine binding and unbinding measurements to estimate the relative affinity of the bonds. The values obtained for this quantity are in accordance with ELISA, showing how DFS can offer important insights about the dynamic binding process that are not accessible with this common and widespread assay.

Optical tweezers setup with optical height detection and active height regulation under white light illumination

An optical tweezers setup with optical detection in three dimensions and active height regulation has been developed. The presented novel method to determine the relative height of a microparticle from its microscopic image is based on the analysis of the integrated light intensity of the main maximum of the diffraction pattern. After the determination of a master curve as reference, the height can be detected with an accuracy of up to 2?nm. The method is applicable under microscopic white light illumination and is simple to implement. As an example of measurements where active height regulation is indispensable, force?distance curves are discussed. Furthermore, the colloid height is calculated geometrically. In the range where the geometrical estimation provides reliable results, the values are found to be in quantitative agreement with the suggested algorithm.

FACS-sorted particles reduce the data variance in optical tweezers-assisted dynamic force spectroscopy measurements.

By combining optical tweezers-assisted dynamic force spectroscopy experiments with fluorescence activated cell sorting (FACS), we demonstrate a new approach to reducing the data variance in measuring receptor-ligand interactions on a single molecule level by ensuring similar coating densities. Therefore, the carboxyfluorescein-labelled monophosphorylated peptide tau226-240[pThr231] is anchored on melamine resin beads and these beads are sorted by FACS to achieve a homogeneous surface coverage. To quantify the impact of the fluorescence dye on the bond parameters between the phosphorylated peptide and the corresponding phosphorylation specific anti-human tau monoclonal antibody HPT-104, we perform dynamic force spectroscopy and compare the results to data using unsorted beads covered with the non-fluorescence peptide analogue. Finally, we demonstrate that the data variance of the relative binding frequency is significantly decreased by a factor of 3.4 using pre-sorted colloids with a homogeneous ligand coating compared to using unsorted colloids.