Methods for performance characterization of artificial molecular motors

The overall aim of this thesis work is to characterize artificial biomolecular motors, specifically the Lawnmower, a motor based on the burn bridges mechanism, and the Tumbleweed, a protein motor that employs ligand specific DNA-proteins binding to step along DNA. An overview of their operational principles is given, and initial results of our studies are presented, along with a review of currently available methods to characterize artificial molecular motors. One way to characterize the expected 10-15 nm sized steps of Tumbleweed may be to harness the properties of waveguiding nanowires. An outline of such an approach is presented, and a detailed study of underlying physical phenomena is performed. Specifically, to understand what dimensions of nanowires should be used, we study the enhancement of fluorescence excitation, one of the phenomena contributing to guidance of light in gallium phosphide nanowires. We find experimentally, and confirm with modelling, that the nanowires with diameter around 110 nm and 10 nm thick Al2O3 coating maximize the enhancement for red fluorophores, and our modelling suggests that the optimum diameter shifts to smaller diameters for fluorophores with a shorter excitation wavelength. The nanowires are promising for many applications including biosensing.Plans for further studies are also presented. Specifically, molecular motor studies are proposed on the Tumbleweed or similar motors, and it is described how nanowires may be used to that end. As for the nanowire studies, these may be continued using fluorescence-lifetime imaging microscopy (FLIM) which would show how the nanowires affect fluorescence emission, and towards the goal to use nanowires in biosensing which might also involve fluorescent noble metal nanoclusters

Fluorescence excitation enhancement by waveguiding nanowires

The optical properties of vertical semiconductor nanowires can allow an enhancement of fluorescence from surface-bound fluorophores, a feature proven useful in biosensing. One of the contributing factors to the fluorescence enhancement is thought to be the local increase of the incident excitation light intensity in the vicinity of the nanowire surface, where fluorophores are located. However, this effect has not been experimentally studied in detail to date. Here, we quantify the excitation enhancement of fluorophores bound to a semiconductor nanowire surface by combining modelling with measurements of fluorescence photobleaching rate, indicative of the excitation light intensity, using epitaxially grown GaP nanowires. We study the excitation enhancement for nanowires with a diameter of 50-250 nm and show that excitation enhancement reaches a maximum for certain diameters, depending on the excitation wavelength. Furthermore, we find that the excitation enhancement decreases rapidly within tens of nanometers from the nanowire sidewall. The results can be used to design nanowire-based optical systems with exceptional sensitivities for bioanalytical applications

DNA Interaction with Head-to-Tail Associates of Cationic Surfactants Prevents Formation of Compact Particles

Cationic azobenzene-containing surfactants are capable of condensing DNA in solution with formation of nanosized particles that can be employed in gene delivery. The ratio of surfactant/DNA concentration and solution ionic strength determines the result of DNA-surfactant interaction: Complexes with a micelle-like surfactant associates on DNA, which induces DNA shrinkage, DNA precipitation or DNA condensation with the emergence of nanosized particles. UV and fluorescence spectroscopy, low gradient viscometry and flow birefringence methods were employed to investigate DNA-surfactant and surfactant-surfactant interaction at different NaCl concentrations, [NaCl]. It was observed that [NaCl] (or the Debye screening radius) determines the surfactant-surfactant interaction in solutions without DNA. Monomers, micelles and non-micellar associates of azobenzene-containing surfactants with head-to-tail orientation of molecules were distinguished due to the features of their absorption spectra. The novel data enabled us to conclude that exactly the type of associates (together with the concentration of components) determines the result of DNA-surfactant interaction. Predomination of head-to-tail associates at 0.01 M < [NaCl] < 0.5 M induces DNA aggregation and in some cases DNA precipitation. High NaCl concentration (higher than 0.8 M) prevents electrostatic attraction of surfactants to DNA phosphates for complex formation. DAPI dye luminescence in solutions with DNA-surfactant complexes shows that surfactant tails overlap the DNA minor groove. The addition of di- and trivalent metal ions before and after the surfactant binding to DNA indicate that the bound surfactant molecules are located on DNA in islets

Semiconductor nanowires for biosening

Semiconductor nanowires are known to enhance the signal of fluorophores in their proximity. In our recent work, we optimized GaP nanowires to maximize the enhancement of fluorescence excitation. For that, we used optical microscopy to measure the fluorescence photobleaching rate on the nanowires, proportional to local excitation. From the measurements and modelling, we show for red fluorophores (excitation wavelength 640 nm) that nanowires with diameters of 90–130 nm enable enhancement of over a factor of 5 compared to bulk solution. We also demonstrate that a 10 nm oxide layer on the nanowires enables functionalisation using biotin-streptavidin chemistry without hindering the enhancement. Such nanowires were used in our laboratory to detect fluorescently-labelled proteins on a single molecule level. Now we employ these results for detection of down to nanomolar concentrations of DNA in solution, and in our poster, we discuss these biosensors

Through the Eyes of Creators: Observing Artificial Molecular Motors : ACS Nanoscience Au

Inspired by molecular motors in biology, there hasbeen significant progress in building artificial molecular motors, usinga number of quite distinct approaches. As the constructs become moresophisticated, there is also an increasing need to directly observe themotion of artificial motors at the nanoscale and to characterize theirperformance. Here, we review the most used methods that tacklethose tasks. We aim to help experimentalists with an overview of theavailable tools used for different types of synthetic motors and tochoose the method most suited for the size of a motor and the desiredmeasurements, such as the generated force or distances in the movingsystem. Furthermore, for many envisioned applications of syntheticmotors, it will be a requirement to guide and control directed motions.We therefore also provide a perspective on how motors can be observed on structures that allow for directional guidance, such asnanowires and microchannels. Thus, this Review facilitates the future research on synthetic molecular motors, where observations ata single-motor level and a detailed characterization of motion will promote applications

Motility of an autonomous protein-based artificial motor that operates via a burnt-bridge principle

Abstract Inspired by biology, great progress has been made in creating artificial molecular motors. However, the dream of harnessing proteins – the building blocks selected by nature – to design autonomous motors has so far remained elusive. Here we report the synthesis and characterization of the Lawnmower, an autonomous, protein-based artificial molecular motor comprised of a spherical hub decorated with proteases. Its “burnt-bridge” motion is directed by cleavage of a peptide lawn, promoting motion towards unvisited substrate. We find that Lawnmowers exhibit directional motion with average speeds of up to 80 nm/s, comparable to biological motors. By selectively patterning the peptide lawn on microfabricated tracks, we furthermore show that the Lawnmower is capable of track-guided motion. Our work opens an avenue towards nanotechnology applications of artificial protein motors