Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere1. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed2 technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies3,4. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

Although separation of polymers based on the combination of dielectrophoretic trapping and electrophoretic forces was proposed 15 years ago, experimental proof has not yet been reported. Here, we address this problem for long DNA fragments in a simple and easy-to-fabricate microfluidic device, in which the DNA is manipulated by electrophoresis and by electrodeless dielectrophoresis. By slowly increasing the strength of the dielectrophoretic traps in the course of the separation experiments, we are able to perform efficient and fast DNA separation according to length for two different DNA conformations: linear DNA (lambda (48.5-kbp) and T2 (164-kbp) DNA) and supercoiled covalently closed circular plasmid DNA (7 and 14 kbp). The underlying migration mechanism-thermally induced escape processes out of the dielectrophoretic traps in the direction of the electrophoretic force-is sensitive to different DNA fragments because of length-dependent DNA polarizabilities. This is analyzed in a second series of experiments, where the migration mechanism is exploited for the quantitative measurement of the DNA polarizabilities. This new and simple technique allows for the systematic characterization of the polarizability not only for DNA but also for other biomolecules like proteins. Furthermore, our results have direct implications to future biotechnological applications such as gene therapy and DNA vaccination.

Protein dielectrophoresis (DEP) has the potential to play an important role as a manipulation, fractionation, pre-concentration and separation method in bioanalysis and as manipulation tool for nanotechnological applications. The first demonstrations of protein DEP have been reported almost twenty years ago. Since then various experimental realizations to manipulate proteins by DEP as well as more targeted applications employing protein DEP have been demonstrated. This review summarizes the experimental studies in the field of protein DEP trapping and focusing as well as specific applications in separation, molecular patterning, on bioprobes and biosensors. While a comprehensive theoretical model describing protein DEP is still lacking we also attempt to provide an overview of the factors influencing protein DEP and relate to currently available theoretical models. We further point out the variations in experimental conditions used in the past to study the somewhat 20 proteins as well as the implications of protein molecular structure to the DEP response.

Dielectrophoresis (DEP) has demonstrated to be a versatile tool to manipulate micro- and nanoparticles with applications for positioning, separation and fractionation. Recent developments of DEP have also shown that DEP can be used for the manipulation of biomolecules, such as DNA. Here, we focus on the manipulation of proteins using insulator-based dielectrophoresis (iDEP). We designed suitable post arrays in a microfluidic channel and use numerical simulations to calculate the electric field distribution as well as concentration of proteins according to a convection-diffusion model for both negative and positive DEP. Experimentally, we find DEP trapping of mainly protein aggregates in phosphate buffer. However, when adding a charged zwitterionic detergent, we observed DEP streamlining of immunoglobulin G (IgG) and bovine serum albumin (BSA). Our experimental observations are in excellent agreement with numerical simulations and indicate positive DEP behavior of IgG and BSA under the employed experimental conditions. Our results demonstrate DEP streaming of proteins in an iDEP device for the first time and indicate the potential of protein DEP for separation and fractionation.

Control of surface properties in microfluidic systems is an indispensable prerequisite for successful bioanalytical applications. Poly(dimethylsiloxane) (PDMS) microfluidic devices are hampered from unwanted adsorption of biomolecules and lack of methods to control electroosmotic flow (EOF). In this paper, we propose different strategies to coat PDMS surfaces with poly(oxyethylene) (POE) molecules of varying chain lengths. The native PDMS surface is pretreated by exposure to UV irradiation or to an oxygen plasma, and the covalent linkage of POE-silanes as well as physical adsorption of a triblock-copolymer (F108) are studied. Contact angle measurements and atomic force microscopy (AFM) imaging revealed homogeneous attachment of POE-silanes and F108 to the PDMS surfaces. In the case of F108, different adsorption mechanisms to hydrophilic and hydrophobic PDMS are discussed. Determination of the electroosmotic mobilities of these coatings in PDMS microchannels prove their use for electrokinetic applications in which EOF reduction is inevitable and protein adsorption has to be suppressed.