The overarching scientific objective of this project is to advance diagnostics for a wide range of critical medical conditions using advanced microfluidics and nanobiotechnology integration. To achieve this goal, the project partners have recruited 15 young researchers (11 doctoral students and 4 post-docs). Below, the fellows are presenting their individual projects:

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Kushagr Punyani Kushagr is involved in adaptation of Deterministic Lateral Displacement (DLD) in the group of Prof. Jonas Tegenfeldt for assisting diagnosis of malaria and leishmaniasis. The primary aim of the project is to exploit alterations in deformability of erythrocytes upon infection with P. falciparum to enable fractionation of whole blood using DLD to diagnose malaria; and to enrich extracellular L. mexicana promastigotes from blood based on morphology. Possibility of DLD-based enrichment of other parasitic stages from whole blood is also being investigated. Besides, Kushagr is also involved in density based microfluidic separation of parasites from blood, and development of on-chip temporal-controlled cell lysis.
Bao Dang Ho has been a doctoral student in the group of Prof. Jonas Tegenfeldt since October 2014. In his project, his focus is on combining electrodeless dielectrophoresis (DEP) with deterministic lateral displacement (DLD) for separating biological particles, e.g. blood cells, parasites, bacteria, etc. Dielectrophoresis can be utilized to change either the distance between a particle and a post or the orientation of the particle itself in a bumper array (DLD device). In essence, the effective size of the particles changes, leading to the change in their position at the outlet of the DLD device and enabling separation. DEP has the advantage of flexibility, tunability, and versatility and can be a promising complement to DLD technique. Recently, Bao has investigated DEP effect on polystyrene beads with different surface charge coating and on the orientation of red blood cells in DLD devices. Experiments showed that DEP force is dependent on the amount of surface charge on polystyrene particles and in effect, particles having higher surface charge density would be displaced more in DLD devices. In a normal DLD device, RBCs appear as small as their thickness due to their vertical orientation near posts of a bumper array. Experiments on RBCs in DEP-DLD devices revealed that dielectrophoresis can help control the orientation of the cells – in particular, suppress the mentioned vertical alignment and make them appear as big as their diameter. In short, dielectrophoresis affects the RBCs’ orientation and control their effective size in a DLD device. Investigation on separation of bacteria in DEP-DLD device is in progress.
Figure: Orientation of red blood cells in a DLD device. (a) In the absence of an AC field (E = 0), RBCs align vertically near posts. (b) When electric field was turned on (V = 1000 Vpp, |E| ≈ 177 VRMS/cm), RBCs always orient horizontally like Frisbees flying in mid-air.
Dr. Zunmin Zhang Since January 2015, Zunmin has been working on dynamics of non-spherical deformable particles in obstacle arrays. His supervisors are Prof. Gerhard Gompper and Dr. Dmitry Fedosov at Forschungszentrum Juelich GmbH. The separation of biological particles and cells in microfluidic devices is very promising, but is also a difficult problem due to the complex properties of such particles including shape, deformability, and dynamics in fluid flow. On the other hand, various particle properties can be exploited for particle separation, since different particles show distinct behaviors under flow. Here, mesoscopic simulations provide a valuable tool for the understanding of the behavior of soft particles in microfluidic devices. The main goal of this research task is to extend our modeling approach to the study of lateral displacement (DLD) devices, and to suggest promising designs for the separation of soft particles in DLDs. Simulation approaches have been extended to be able to simulate the flow of various soft particles in different DLD geometries. Furthermore, the deformation and dynamics of spherical particles and red blood cells have been studied in DLDs with circular, triangular, diamond-, and L-shaped obstacles. This has led to a better understanding of the advantages of certain structures, and has triggered the testing of our hypotheses in the experiments of Kushagr Punyani and Bao Dang Ho. The next step will be to identify how various particle properties can be probed in DLDs and to test the sensitivity of device configurations to such properties. We hope that our results will help in the design of novel efficient DLDs.
Ewan Henry has been working on simulations for separating mixtures of deformable particles by use of obstacle arrays and external fields since April 2014. His supervisors are Prof. Gerhard Gompper and Dr. Dmitry Fedosov at Forschungszentrum Juelich GmbH. The separation of a mixture of different particles in microfluidic devices is possible if it is known how different particle properties can be contrasted in microfluidic flow. This can be often achieved through differences in particle deformation and dynamics in fluid flow. The main goal of this research task is to investigate how different particle in mixtures can be sorted in DLDs. As the first step, our mesoscopic simulation approach has been validated against the experimental measurements with red blood cells of Kushagr Punyani and Bao Dang Ho. In addition to a good agreement between the simulation and experimental results, the comparison has led to the discovery that red blood cells with different cytosol viscosity properties can be differentiated in DLDs. Thus, the cell viscosity can be employed as a parameter for sorting. The next step will be to investigate the deformability of red blood cells as a parameter for sorting. These results will help to make suggestions for an efficient DLD device, which can be used in malaria disease. Finally, we plan to investigate the effect of external fields (e.g., gravity, dielectrophoresis) on the efficiency of separation of different mixtures in DLDs. More information about the group
Dr. Genevieve Garriss joined the group of Prof. Birgitta Henriques Normark at KI in August 2014. The aim of her research in the LAPASO project is to gain a fundamental understanding of pneumococcal pathogenesis. To this end, the pneumococci are divided in subpopulations based on physical properties (ex.: single cells, diplococci, chains, capsule, pili) using sorting technology developed by other fellows. More information about the group
Vitor Oliveira has been a member of LAPASO since October 2014, when he started his research in the group of Prof. Birgitta Henriques Normark. His PhD project is titled “Biofilm formation by pathogenic Streptococcus pneumoniae and its impact on DNA uptake and bacterial virulence” and we are interested in setting up different in vitro biofilms models to study mechanisms involved in biofilm formation. We aim also to study biofilm development in vivo by setting up a mice model. We have already established a pneumonia model that will be used to study biofilm formation in the lungs and in the nasopharynx after pneumococcal challenge. By using the microfluidic label-free sorting technologies, such as deterministic lateral displacement (DLD) developed by Prof. Jonas Tegenfeldt´s group in Lund we aim to be able to sort and distinguish between single cells, diplococci and chain forming pneumocococci based on morphologic parameters. Subsequently, we will study subpopulations of pneumococci and their impact on virulence and biofilm formation. It is also our interest to understand the underlying mechanisms of how DNA is taken up by competent bacteria and role of the competence pilus. The exact structure and composition of the competence pilus, however, remains to be elucidated. In order to characterize the composition of the pneumococcal transformation pilus we will employ a broad range of techniques including microscopy, protein co-expression and a bacterial two-hybrid screen. By using technique we will be able to study the interactions between the proteins involved in pilus assembly. Hence, this will allow us to get more insights into the underlying mechanisms of DNA uptake. To further analyze the mechanisms of how DNA is taken up by competent bacteria, we will study transformation in culture grown bacteria as well as in bacteria in biofilms. We will compare transformation efficiencies and analyze different mutants, lacking key components to build up their transformation machinery, in their ability to take up DNA and to form biofilms.
Elisabeth Reithuber started her PhD work in the Prof. Birgitta Henriques Normark / Dr. Peter Mellroth in 2014. In the course of her PhD studies she wants to work on the acceleration of sepsis diagnosis with the help of microfluidic devices. Sepsis is an acute disease with high mortality where fast and precise diagnosis is crucial for a successful treatment. Therefore we aim for a diagnostic protocol avoiding the conventional time-consuming blood culture method by using microfluidic devices for specific enrichment of bacteria from blood or for investigation of white blood cell deformability change in response to bacterial infection. Late identification of the infection causing species and untargeted use of antibiotics also results in emerging antimicrobial resistance. Answering the need for new antimicrobials Elisabeth is involved in the identification and characterization of new chemicals against the frequently sepsis causing pathogen Streptococcus pneumoniae. We aim to find chemicals that activate the major pneumococcal autolysin LytA and further characterize the function of this peculiar enzyme.
Clément Regnault has been a member of Prof. Michael Barret's group since October 2014. Clément's research focus is on separation of leishmania and trypanosome parasites from blood for rapid diagnosis. Deterministic lateral displacement is a robust micro-total analysis system that exploits label-free fractionation of heterogeneous cell populations. Therefore, he is interested in the development of DLD devices enabling to carryout the separation of Leishmania promastigotes from blood. Moreover, separation of macrophages infected with parasites from healthy macrophages is also of interest from a diagnosis point of view. The differences in the physico-chemical properties of these two cell populations will be investigated; in particular, their differences in size, deformability and dielectric properties will be quantified. Following these experiments, DLD devices sensitive for these differences and aiming at enriching for parasite-infected cells will be designed and developed in collaboration with Lund University. They will then be tested on in vitro samples.
Figure: Leishmania infected cells.
Laura Ciuffreda has been a member of Dr. Lisa Ranford-Cartwright's group since September 2014. The main aim of her project is to develop a new diagnostic device malaria parasite-infected erythrocytes. This device will be based on microfluidics, which enables a label-free enrichment of P. falciparum ring-infected red blood cells (the only stage present in the peripheral circulation) from the blood, allowing an improved diagnosis of malaria, especially at low parasitaemia. This project will encompass an in-depth study of changes in mechanical and dielectric properties during the parasite development (focusing mainly on ring stage) in order to define at what point after invasion the ring-infected cells start to differ from the uninfected ones and therefore can be separated.
Figure: Giemsa stained smear of P. falciparum parasites in culture - A ring stage parasite (right) and a schizont stage (left) are shown.
Jorge Miguel Xavier In September 2014, Miguel Xavier moved to the University of Southampton to start his PhD under the framework of the LAPASO project in the group of Prof. Hywel Morgan on the use of microfluidic systems for skeletal stem cell sorting. Skeletal stem cells (SSC) are a sub-population of mesenchymal stem cells, found in the bone marrow and have shown osteogenic, chondrogenic and adipogenic differentiation potential. However, efficient isolation of these cells remains a challenge because they are scarce and lack a specific biochemical marker. Microfluidics devices could provide novel solutions for cell separation based on bio-physical features of single cells, for example stiffness and dielectric properties. We have shown that surrogate cells (MG-63) differ sufficiently in size and mechanical stiffness or deformability to indicate that these cells can be separated from leukocytes using techniques such as Deterministic Lateral Displacement (DLD) arrays.
Figure: the aim of this project is to isolate and purify Skeletal Stem Cells.
Walter Minella joined the research team of Elvesys in May 2014. Elevesys research task concerns the dynamic control of cell environment. Indeed, the sorting of particles by the means of techniques such as DLD or DEP, which are the core of LAPASO, strongly depends on the electrical and dialectical properties of the surrounding media. Being able to perform in flow tagging and washing of cells not only reduces the steps to accomplish before further analysis of the sample can be operated, but also allows integrated LOC applications. The main aim of our research is thus to develop devices capable of dynamically modifying particles environment during their transport by the means of combined diffusive and convective transport though permeable membranes. In this regard, extensive studies and modeling of both convective and diffusive transports need to be carried out in order to optimize the device and its throughput. These will be carried out with the support of Juelich and Ewan Henry. Eventually, these devices will be employed by other partners within LAPASO, such as Kushagr Punyani, Bao Dang Ho and Anke Urbansky, in order to enhance the sorting contrast of their setups, and hence, their performances.
Anke Urbansky Has been working in the group of Prof. Thomas Laurell at the Department of Biomedical Engineering, Lund University (Sweden) since June 2014. Her research focus is on acoustophoresis for sorting mesenchymal stem cells.
Carlos Honrado Has been working in the group of Prof. Hywel Morgan at the University of Southampton since September 2014. His research is to further develop microfluidic impedance cytometry for the label-free characterization and sorting of human pathogens.
Dr. Simone Tanzi Simone Tanzi is in LAPASO with the objective to develop microfluidic devices that can be fabricated on an industrial scale. His main tasks rely on understanding the needs for every sorting technique, designing and manufacturing of prototype devices based on sorting techniques developed in the consortium and identifying customised solutions for each application through feedback from laboratory and field tests. Different fabrication technologies such as hot embossing of polymers devices and wet etching of glass devices will be employed.
Dr. Ahmad Ahsan Nawaz has joined the group of Prof. Jochen Guck at TU Dresden in Oct 2015. Gucks’ group has recently published (Otto, O. et al., Nat. Methods 12, 2015), real-time deformation cytometry (RT-DC) for on-the-fly continuous analysis of cell deformation, within LAPASO project. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. These capabilities render RT-DC a unique and novel approach with diverse applications in basic biological research and medicine. Dr. Nawaz aims to utilize acoustic waves to sort the cells analysed via RT-DC. Surface acoustic waves (SAW) are sound waves produced on the surface of a lithium niobate substrate (piezo-electric material) when excited by a pair of inter-digital-transducer (IDTs) electrodes deposited onto such substrates. We intend to utilize standing surface acoustic waves (SSAW) for sorting the cells downstream of the RT-DC measurement channel. In this approach two IDT pairs will be deposited such that the RT-DC channel is centered. The two IDT pairs, when excited, result in constructive interference of the sound waves originating from each pair of IDT. Upon encountering the liquid flowing in the RT-DC microfluidic channel, the acoustic waves will leak into the liquid such that pressure nodes and anti-pressure nodes are formed within the channel. The acoustic radiation force will push the cell (in its wake) towards the pressure nodes. It is anticipated that use of SSAW could allow sorting of cells at a rate of up to 1000 cells/sec without adversely affecting the integrity or functions of the living cells. The marriage of acoustic separation with RT-DC will open new avenues of research in stem cell biology, pathology, microbiology, parasitology, medical diagnostics (e.g. early stage cancer detection), circulating tumor cell separation as well as drug discovery research.

 

 

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