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This month we interview Victor U. Weiss, Assistant Professor at TU Wien, in Vienna, Austria, about his focus on the analysis and electrophoretic separation of nanoparticles from organic, inorganic, and biological sources, and the concept behind nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA).
Q. When did you first encounter electrophoresis and what attracted you to the subject?
A: I first encountered the concept of electrophoresis during my studies at the University of Vienna, but only during my diploma thesis did I really start to get involved with the subject. At this time, my supervisor, Ernst Kenndler, had worked in the field of electrophoresis for some time and had published several papers on the separation of virus particles in a capillary electrophoresis setup. When I contacted him to see if there would be a chance to do my diploma thesis in his research group, he was affirmative and told me that there would be a commercially available chipbased electrophoresis setup intended for gel electrophoresis arriving in his research group in a few weeks; if I was interested I could start to transfer his already developed capillary electrophoresis (CE) separation setup of virus particles without a sieving matrix to the chip format—and that also meant adjusting the instrumental setup itself. As I am also very interested in biochemistry, I was very glad for this opportunity. The possibility of combining analytical techniques and biochemical questions in one thesis was very appealing to me. Also, at this time, analytical work with viruses was not very common and soundedexotic—who could know that 13 years later a viral pandemic would strike?
Q. Can you tell us more about your Ph.D. thesis?
A: My Ph.D. was a continuation of the work I did during my diploma thesis. During the latter, we finally succeeded in transferring the electrophoretic separation of fluorescently labelled rhinovirus particles (a virus responsible for common cold infections) to the format of commercially available, single-use chips without the use of an originally supplied sieving matrix. In order to do so, we modified the software of the instrument to allow for electrophoresis in the presence of electroosmosis. In contrast to conventional capillary electrophoresis setups, chip electrophoresis of fluorescently labelled rhinovirus particles was even possible in the absence of detergents. Therefore, we had the idea to combine virions and cell surface analogues in one sample to set up an in vitro model for early viral cell infection. This was actually the starting point of my Ph.D. thesis. We opted for liposomes as vesicles to mimic cell membranes. Liposomes are bionanoparticles themselves, and are formed of a lipid bilayer encapsulating an aqueous lumen. These nanoparticles are usually applied as carrier molecules in pharmaceutical or other applications. In nature, extracellular vesicles exhibit a corresponding role and can be found in cell/cell communication or for cargo transport, for example.
During my Ph.D. studies, I worked in the research groups of Ernst Kenndler and Dieter Blaas at the University of Vienna and the Medical University of Vienna. We decorated liposomes with recombinant receptor fragments and were able to show, not only with capillary electrophoresis but also for example, with electron microscopy techniques, that it is possible to target individual, discrete steps of viral uncoating (that is, the process of viral conformational changes during genome transfer through a lipid membrane in the course of cell infection) with our developed in vitro setup. We followed the attachment of virus particles to recombinant receptor molecules and receptor-decorated liposomes, as well as membrane permeabilization by virusderived peptides, viral proteins, and entire virions of human rhinovirus in a series of papers. Even some years after my Ph.D., during my postdoc years at TU Wien, I went back to a corresponding chip electrophoretic setup and demonstrated that it is possible to follow the release of the rhinoviral RNA genome with chip electrophoresis based on molecular beacons as fluorescent probes; only in the presence of a complementary genomic sequence (that is, the viral RNA) would these probes lighten up as the spatial proximity between a fluorophore and quencher is lost and corresponding species are detected.
Q. What electrophoretic techniques have you worked with?
A: I started to work with capillary electrophoresis on a conventional instrument with UV absorption detection either in the absence or the presence of detergents. Later on, I also worked on a conventional instrument with laserinduced fluorescence detection before transfering corresponding separations to the chip format. And there is, of course, nano electrospray gas-phase electrophoretic mobility molecular (nES GEMMA) analysis, which has played a big role in my research. I came into contact with the latter method when I started my postdoc career at TU Wien in the research group of Günter Allmaier, almost 12 years ago. After the retirement of Günter Allmaier, Martina Marchetti-Deschmann is now head of our research group “Mass spectrometric Bio and Polymer Analytics” at the Institute of Chemical Technologies and Analytics at TU Wien. Together with Ernst Pittenauer and Günter Allmaier, who is still active as a scientific advisor, we form a principal investigator (PI) team, covering a broad range of analytes and analytical techniques with our expertise, ranging from mass spectrometry (MS), mass spectrometric imaging, metabolomics, and lipidanalyses to nanoparticle characterization. And I am happy to still do (not exclusively but also) electrophoresis in the liquid- and the gas‑phase after all these years.
Q. What is the concept behind nES GEMMA? What applications could this technique potentially be useful for?
A: Nano electrospray gas-phase electrophoretic mobility molecular analysisin its current form was first described in 1996 by Stan Kaufman and colleagues in Analytical Chemistry, and a first prototype was run in the laboratory of Günter Allmaier and Wladyslaw Szymanski at the University of Vienna. The same setup is today also found under the name nES differential mobility analyzer (nES DMA), ion mobility spectrometer (macro-IMS), LiquiScan electrospray (ES), and scanning mobility particle sizer (SMPS). Analytes are electrosprayed in their native form from a volatile electrolyte solution. Subsequently, droplets are dried and at the same time charge equilibration of analytes occurs in a bipolar atmosphere induced, for example, by a 210Po α-particle source, a bipolar corona discharge process, a soft X-ray charger, or similar. After passage of this atmosphere, particles are in large parts either neutral and are not considered further or single-charged. The latter are then separated in a high laminar sheath flow of particle-free air and a tunable electric field. By variation of the field strength, only particles of a certain size can pass through the size separator (a differential mobility analyzer [DMA]) based on electrophoretic principles. Finally, particles are counted and particle numbers are related to the applied field strength for separation and, thus, ultimately to the surface-dry particle size itself, leading to a corresponding spectrum. We then combine several spectra via their median to correct for any spikes detected during individual runs. This separation method is in accordance with a recommendation of the EC for nanoparticle characterization (2011/696/EU from 18 October 2011) (1) as nanoparticle number-concentrations are reported.
nES GEMMA analysis was successfully applied by others as well as in our laboratory for the characterization of different nanoparticle materials. Protein aggregates were analyzed for instance, or viruses, virus-like particles, gold or silver nanoparticles, polymers, polysaccharides, DNA, just to name a few different analyte classes. Besides information on surface‑dry particle size, information on analyte heterogeneity and particle‑number concentration, as well as additional material present in a sample, is obtained. There is also the possibility to calculate the molecular weight of an analyte based on its surfacedry particle size in the gas‑phase when a corresponding correlation is applied. This is even possible for analytes for which classical native electrospray ionization (ESI)-MS is sometimes reaching its limits. On the other hand, for setting up such a correlation, MS data are needed, so both methods are actually complementary.
Q. Are there any challenges involved with using this technique?
A: For each novel analyte type you need to have an idea of how to transfer your nanoparticles in their native form to the gas-phase. For biomaterials, often non‑volatile buffer solutions are applied as solvents, or the sample includes high amounts of detergents. These solvents need to be exchanged to something volatile—ammonium acetate, for instance, because everything that is non-volatile will be detected later on. When non‑volatile buffer components are present in high concentrations, particularly in comparison to the actual analyte, this causes all kinds of undesired aggregates to be formed (between buffer components and buffer components and analytes), either already in solution or during the electrospray process. However, sometimes, and for some analytes, the original solvent system and its exchange to something volatile is challenging. One possibility is to apply offline dialysis or spin filtration steps or—and we did some work already in that respect—to combine gas-phase electrophoresis online with additional separation techniques such as size‑exclusion chromatography (SEC) or CE in the capillary of the nES part of the instrument used. Hence, non-volatile sample buffer components are removed from actual analytes prior to gas-phase electrophoresis.
Likewise, we are working on further instrumental developments in our research group; for example, the exchange of the radioactive 210Po α-particle source by a bipolar corona discharger. Such a replacement works perfectly and makes life in the laboratory much more easy because there are no longer any radioactive instrumental parts to be considered.
An interesting feature of gas-phase electrophoresis on the instrument used is the collection of nanoparticle material of a defined size on supports to allow for subsequent in-depth analyte characterization applying orthogonal methods. Thus, I am also working on the offline hyphenation of gas-phase electrophoresis with spectroscopic, spectrometric, and microscopic techniques for improved nanoparticle characterization. In addition, the comparison of nES GEMMA results to data obtained from orthogonal nanoparticle size determination techniques is of interest to me because the combination of methods often results in better interpretable data.
Q. Your primary focus is the analysis and electrophoretic separation of nanoparticles from organic, inorganic, and biological sources—what specifically attracted you to this area of research?
A: I think nanoparticle research is a really interesting topic—on the one hand, you have quite a number of nanomaterials available in nature, viruses, for example, or corresponding particles that have already been used for centuries, such as colloidal gold for fabrication of colourful glass windows. On the other hand, there is still much to learn about such materials, and nanomaterial characterization is something that can help tremendously in that respect. There is even the possibility to further develop instrumentation for nanoparticle characterization, like the work we did and are currently doing modifying the nES GEMMA instrument we used (2). To me, all these aspects make nanoparticle research a very challenging and interesting field.
Q. You have recently published a paper on characterizing virus-like particles using gas-phase electrophoresis (3). Why is this research important and what advantages could it offer over existing techniques?
A: Virus-like particles, or VLPs, are non‑infectious analogues of viruses. If you take a virus particle, it consists of a proteinaceous shell, sometimes also with an additional lipid envelope protecting the viral genome from the exterior until cell infection occurs. In the case of VLPs, this genomic material is missing, but otherwise VLPs resemble their parent viruses. This makes VLPs ideally suited for vaccination purposes, for example. There is also the approach of applying VLPs as carrier molecules, for example, in gene therapy because there is the possibility to encapsulate genomic material other than of virus origin in these particles. However, no matter what medical or pharmaceutical application, the thorough characterization of the resulting bionanoparticles is a necessary prerequisite. It is here that gas‑phase electrophoresis comes into play. In our experience, this method is exceptionally well suited for the characterization of bionanoparticles in terms of size, analyte homogeneity, number-concentration, or batch purity. Other methods might yield this information as well but at the cost of long analysis times with microscopic techniques, for example, because you have to consider a statistical valid dataset, or extremely customized, expensive instrumentations, such as native ESI-mass spectrometry. For the latter, you need highly pure and concentrated samples, which is usually a smaller requirement for gas-phase electrophoresis.
Q. Are there any challenges involved with using this technique?
A: Yes, definitely. As with every analytical technique you need to have your analyte in a high enough quantity and quality, but given that this is the case, you will probably gain valuable information on your analyte. One example would be human rhinovirus. For a long time it was applied in experimental setups after a standard preparation and purification protocol, which included sucrose density centrifugation and digestion steps. However, subsequent analyses—for instance when applying capillary electrophoresis—always showed a contaminating material present—only to a small extent in preparation batches but nevertheless present. Only upon application of gas-phase electrophoresis did we learn that this contaminating material is present in a greater amount than anticipated, and also in approximately the same surface-dry particle size range as our actual virions, although much more heterogeneous in size distribution. It took us some time to deduce that this additional material was related to lipid species. In the end we succeeded in improving batch polishing either via a lipase digestion step or by application of a chromatographic separation. Finally, thanks to gas-phase electrophoresis, we ended up with highly pure rhinovirus preparations.
Another example are liposomes. We can analyze these as well with
gas-phase electrophoresis and, besides the expected material in the size range to which vesicles were extruded, we also detected lower sized aggregates to different extents in different batches. First experiments hinted that these aggregates influence cell viability, and to my knowledge gas‑phase electrophoresis is one of very few methods capable of targeting that specific size range and keeping analytes in their native state.
Q. Another of your recent papers focuses on the offline hyphenation of nES GEMMA with matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS) for the analysis of liposomes (4). What led you to develop this approach and what advantages does it offer
A: Liposome analysis via gas-phase electrophoresis is another of my fields of research. Over the years it has been revealed that nES GEMMA is capable of yielding some very interesting results for these vesicles, for instance, identifying sample components that influence cell viability in subsequent experiments, as mentioned previously. Plus, as it is possible with gas-phase electrophoresis to size-collect nanoparticle material on sample supports for its subsequent characterization applying orthogonal analysis techniques, we also succeeded in collecting intact vesicles for further visualization experiments applying atomic force microscopy. Interestingly, only after passage of the nES GEMMA system and vesicle collection were we able to image intact vesicles. As soon as we tried to deposit vesicles from a liquid solution, liposomes burst open while drying on the surface.
Once we realized that a liposome size‑collection step is possible with a nES GEMMA setup, we also focused on the characterization of individual liposomes. In doing so, we did spectroscopic analyses of single liposomes. A logical additional approach was then based on mass spectrometry. Not only it is possible with this technique to unambiguously identify lipid species applied in the formation of vesicles but we envision that it will also be possible to target cargo molecules encapsulated within liposomes in the future, maybe even some contaminating material
Q. Where do you think this research could go in the future?
A: One very interesting question is do vesicles of different sizes—remember that liposomes are rather heterogeneous despite extrusion—differ in their lipid composition, despite being of the same batch? Or, do they encapsulate cargo molecules to a different degree independent of the actual nanoparticle size? Also, a similar technology to liposomes is currently applied for vaccination purposes. It is of importance if nanoparticles include cargo molecules, and we think that nES GEMMA in combination with mass spectrometry might help to deliver that answer.
Q. What projects are you working on next?
A: I am currently trying to get a project going analyzing SARS-CoV-2 viral particles. But obviously, with this project the infectivity of the analytes is a big issue that can perhaps be solved in the form of VLPs in the near future.
Victor U. Weiss is Assistant Professor at the Institute of Chemical Technologies and Analytics, TU Wien, in Vienna, Austria. His main areas of interest include the analysis and electrophoretic separation in the liquid- and the gas-phase of nanoparticle material from various sources (organic, inorganic, biological material) and the hyphenation of electrophoretic separations with orthogonal analysis methods. His work includes: an improved protocol for preparation of RV-A2; setup of an in-vitro model system for early viral cell infection; gas-phase electrophoretic-based molecular weight determination of viruses and virus-like particles; gas-phase electrophoresis of liposomes; offline hyphenation of gas-phase electrophoresis with spectroscopic, spectrometric, and microscopic techniques for liposomes; and comparison of nanoparticle size determination techniques.
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