New methods to study single proteins
Rediscovery of dielectrophoresis
The phenomenon of dielectrophoresis holds great potential as a means of manipulating different particles and cells, yet there are gaps in understanding when it comes to applying it on very small scales. We spoke to Dr Sergii Pud about his work in developing a label-free tool to study proteins and molecules, and how it could lead to deeper insights into protein dielectrophoresis. A dielectric material develops a dipole when placed in an electric field, causing electrons to shift within it. When the dipole is placed in a sufficiently non-uniform electric field, different parts of it will experience different forces, and as a result the dipole will start moving in the direction of the gradient of the electric field. “This effect is called dielectrophoresis,” explains Dr Sergii Pud, an Assistant Professor in the lab-ona-chip group at the University of Twente. In his research, Dr Pud is now developing new tools and methods to study this effect, which could open up new possibilities in separating and manipulating different types of particles. “Dielectrophoresis has been used extensively in manipulating ensembles of nanoparticles, including sub-micron sized particles as well as proteins,” outlines Dr Pud. “There has however not been a lot of research into applying dielectrophoresis with single entities.”
Rediscovering dielectrophoresis
3D render of the electrodes
This topic is at the core of Dr Pud’s research, inspired to a large degree by the earlier development of a label-free optical methodology called iSCAT (Interferometric Scattering Microscopy), which allows scientists
Chip with electrodes made in MESA+ cleanroom.
to effectively see single protein molecules free of labels and assess their size. The iSCAT methodology doesn’t provide much time to study the dynamics of these molecules, so Dr Pud aims to extend the observation time by arresting the motion of these molecules using dielectrophoresis. “We use cleanroom nanotechnology to make pointy tips that are only 20 nanometres apart. The nanogap between two gold electrodes creates an electric field to trap proteins and particles,” he says. A field somewhere between 106 and 107 volts per meter is thought to be sufficient to trap a single globular protein. Moreover,
using these electrodes, researchers also aim to develop the ability to hold it in the same position and release it. “Once a particle arrives, our setup is able to detect that something is in the trap, and act upon it,” continues Dr Pud. Dielectrophoretic force is very specific to the electric properties of the trapped object, therefore measuring this force can yield a lot of valuable new information about the particles. Measuring the DEP force is challenging, as this force is very local to the gap between the electrodes. “That will tell us something about the size of the particle and its polarisability or embedded dipole moment,” outlines Dr Pud. “We gradually attenuate the trapping electric field parameters to the point when the particle leaves the trap. This includes amplitude and the frequency of the field. Particles and proteins interact with an electric field differently at different frequencies. We can make a spectra, which will be a signature of a protein.” This goal was part of the initial vision of the project, but as research has progressed Dr Pud says it has become apparent that the methods can also be used in other ways as well. “The methodology we develop is promising to study how proteins bind to small molecules detecting proteins binding small molecules,
Actuation of polystyrene nanoparticles using dielectropherisis
Rediscovery of dielectrophoresis for labelfree manipulation and interrogation of single protein molecules SEM image of the electrodes and the COMSOL simulation of the electric field in the electrodes.
something that is very hard to measure with labelled techniques,” he continues. “A lot of protein-small molecule interactions don’t significantly change either the volume of the protein or its configuration, yet this binding can change the dipole moment.” The project team are working to develop the methods to a level where these types of changes of the dipole moment can be seen by researchers, and to further improve their sensitivity. Currently Dr Pud is preparing a paper on trapping polystyrene particles 30 nanometres in size, then the plan is to study proteins, which are an order of magnitude
Dielectrophoretic force is very specific to the electric properties of the trapped object, therefore measuring this force can yield a lot of valuable new information about the proteins and their dynamics. This is the focus of our lab’s research. smaller. “Most proteins are in the range of 3-10 nanometres in size,” he says. The expectation is that the gap between electrodes will need to be close to the size of the protein being studied if it is to be immobilised effectively. “We’re confident we can reach a gap of 10 nanometres, and that could be sufficient to trap proteins,” continues Dr Pud. “A PhD student in the project will focus solely on proteins, and another is dedicating a lot of time to improving the trapping strategies.”
Protein measurements This research is currently ongoing, with Dr Pud and his colleagues in the project hoping to gain meaningful measurements from
Schematic layout of the single-particle dielectrophoretic trapping experiment. Fluorescent polystyrene beads of ~45 nm diameter are attracted, trapped and actuated in the gap. The time traces on the bottom right represent examples of fluorescence signals from particles trapped with actuation at 1000Hz and at 250Hz and the upper part of the panel shows corresponding Fourier spectra of the optical signal.
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proteins, which is a major challenge. The problem here is that dielectrophoresis was initially theoretically described for rather large objects, and the standard equation (the Clausius-Mossotti relation) used to calculate the dielectric constant does not fully reflect the situation with respect to smaller objects. “The situation when you place a submicronsized particle in solution is highly complex. The particle is surrounded by a cloud of ions and is slightly charged, so it’s forming an extra dipole, on top of its polarisability,” explains Dr Pud. The existing equation still works to a degree on micron-sized particles, but Dr
Pud says the effect of this cloud of ions is increasingly apparent as you move towards smaller objects. “This cloud plays a huge role. We can’t really think of polarisability for proteins, because they already have their own dipole moment on top of that,” he continues. As researchers apply more sophisticated techniques to study protein dielectrophoresis, Dr Pud believes it will be possible to learn more about how it works, opening up wider possibilities in sorting and manipulating proteins. The project’s research represents an important contribution in this respect. “We’re building a system that will be able to capture how a single protein interacts with an electric field,” says Dr Pud.
Project Objectives
This project is aimed at creating a new tool for label-free studying of protein physical parameters in real time. In a nutshell we plan studying protein molecules one by one through engaging them in a game of molecular pingpong and observing their behaviour in it.
Project Funding
The project has been funded by NWO Veni initiative and through internal funding of EEMCS Faculty (University of Twente).
Project Partners
This project is a collaboration between BIOS Lab-on-a-chip group and group of Michel Orrit (Leiden University).
Contact Details
Project Coordinator, Dr. Sergii Pud University of Twente Faculty of Electrical Engineering, Mathematics and Computer Science Carré C2409 P.O. Box 217 7500 AE Enschede The Netherlands T: +31 5 348 93590 E: s.pud@utwente.nl W: https://www.utwente.nl/en/eemcs/ bios/research/Lab-on-a-chip%20 tools%20for%20nanobiology/Protein%20 actuation%20spectroscopy/ Dr. Sergii Pud
Dr. Sergii Pud is an Assistant Professor at the BIOS (Lab-on-a-Chip) group, University of Twente, concentrating on the intersection of biological research with nanotechnology. His expertise lies in developing nanodevices for meticulous single-cell and single-molecule studies. With a primary focus on understanding of protein interactions with electric fields, his work is directed towards refining our knowledge of biological systems and propelling biomedicine forward with power of nanotechnology.
Project team.
EU Research
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