Combined dielectrophoresis and impedance systems for bacteria analysis in microfluidic on-chip platforms

Bacteria concentration and detection is time-consuming in regular microbiology procedures aimed to facilitate the detection and analysis of these cells at very low concentrations. Traditional methods are effective but often require several days to complete. This scenario results in low bioanalytical and diagnostic methodologies with associated increased costs and complexity. In recent years, the exploitation of the intrinsic electrical properties of cells has emerged as an appealing alternative approach for concentrating and detecting bacteria. The combination of dielectrophoresis (DEP) and impedance analysis (IA) in microfluidic on-chip platforms could be key to develop rapid, accurate, portable, simple-to-use and cost-effective microfluidic devices with a promising impact in medicine, public health, agricultural, food control and environmental areas. The present document reviews recent DEP and IA combined approaches and the latest relevant improvements focusing on bacteria concentration and detection, including selectivity, sensitivity, detection time, and conductivity variation enhancements. Furthermore, this review analyses future trends and challenges which need to be addressed in order to successfully commercialize these platforms resulting in an adequate social return of public-funded investments

Single-cell microfluidic impedance cytometry: From raw signals to cell phenotypes using data analytics

The biophysical analysis of single-cells by microfluidic impedance cytometry is emerging as a label-free and high-throughput means to stratify the heterogeneity of cellular systems based on their electrophysiology. Emerging applications range from fundamental life-science and drug assessment research to point-of-care diagnostics and precision medicine. Recently, novel chip designs and data analytic strategies are laying the foundation for multiparametric cell characterization and subpopulation distinction, which are essential to understand biological function, follow disease progression and monitor cell behaviour in microsystems. In this tutorial review, we present a comparative survey of the approaches to elucidate cellular and subcellular features from impedance cytometry data, covering the related subjects of device design, data analytics (i.e., signal processing, dielectric modelling, population clustering), and phenotyping applications. We give special emphasis to the exciting recent developments of the technique (timeframe 2017-2020) and provide our perspective on future challenges and directions. Its synergistic application with microfluidic separation, sensor science and machine learning can form an essential tool-kit for label-free quantification and isolation of subpopulations to stratify heterogeneous biosystems

Developing integrated optical structures, with special respect to applications in medical diagnostics

In my dissertation, I described two label-free optical biosensors based on integrated optical (IO) structures for the sensitive, rapid detection of pathogens - bacterial cells, viral proteins - from fluid samples, which can serve as a basis for rapid clinical tests. These types of devices provide a specific, cost-effective, user-friendly and portable way of detection with sufficient sensitivity by changing the optical signal. Thus, in practice, they could potentially be used as point-of-care (POC) or home rapid diagnostic tests, offering a promising alternative to traditional laboratory assays. Their realization is supported by their integration with microfluidic channels in a lab-on-a-chip (LOC) device, for handling small volumes of fluid. Based on these aspects, biosensors were designed as waveguides, integrated in a microfluidic channel on a glass substrate, performing evanescent-field sensing. The detection method is based on the fact that the light, propagating in the waveguide with total internal reflections, penetrates into the surrounding media at a limited extent, which is called the evanescent field. Material can enter this space and become bound to the surface, which can change the phase of the light, propagating in the structure, or even scatter it into the surrounding medium. These phenomena offer the possibility of specific detection of pathogens, adhering to the surface, pre-coated with a biological recognition element, such as an antibody. As a first application, an electro-optical biosensor was developed with an evanescent field-based detection concept, aiming at label-free, rapid, selective and sensitive detection of bacteria from body fluids. The usability of the measurement principle, based on the processing of light-scattering patterns, caused by evanescent waves, scattered on target cells, was demonstrated by quantitative detection of Escherichia coli bacterial cells from their suspensions. One of the keys to the applicability of biosensors is their sensitivity. To increase it in case of this device, I applied the phenomenon of dielectrophoresis using the polarizability of the target cells. It provides the possibility to selectively collect cells on the surface of electrodes placed close to the waveguide and then detect them based on the evanescent field. To test this, I wanted to sense bacteria in an artificial urine sample containing somatic cells, in this case endothelial cells, mimicking urine in an inflammatory state. By optimizing the parameters of the measurements, a rapid, sensitive bacterial detection of about 10 minutes was achieved. The detection limit of the biosensor was comparable to the characteristic pathogen concentration in body fluids. Furthermore, selective bacterial detection was also achieved from a fluid sample containing somatic cells, mimicking inflammatory urine. In my dissertation, a second application is also presented, in this case a miniature IO Mach-Zehnder interferometer-based biosensor was developed for the specific quantitative detection of viral proteins. Thanks to the interferometric measurement principle, a fast and accurate detection of target proteins can be achieved. With this device, the aim was to investigate the potential neuroinvasion of coronavirus (SARS-CoV-2) infection, from which point of view the pathological effects of viral surface spike proteins on the blood-brain barrier are of great importance in the case of severe symptoms. Furthermore, infection may also cause adverse effects in the intestinal tract. Thus, the specific aim of this application was to evaluate the ability of the S1 subunit of the coronavirus surface spike protein to cross the human in vitro blood-brain barrier and intestinal epithelial biological barrier system models using the biosensor. Experiments were designed to use the sensor for specific, quantitative detection of spike proteins, that may have been passed through permeability assays on biological barrier models prepared by our collaborators. To reach the specific sensing of the target protein, the waveguide surface of the interferometer’s measuring arm was functionalized with specific S1 protein antibody. To achieve optimal, stable measurement conditions, the operating point of the interferometer was adjusted thermo-optically. The results of the experiments with the biosensor were in agreement with the ones of the conventional immunological tests (ELISA) carried out in parallel. It was possible to determine that S1 protein could pass through the two types of barriers in different amounts. The findings of the experiments with the integrated optical Mach-Zehnder interferometer biosensor demonstrate that this detection approach can be used for similar medical diagnostic purposes, and thus can contribute to the investigation of the adverse effects of SARS-CoV-2 on the human body

UTILIZING DIELECTROPHORESIS TO DETERMINE THE PHYSIOLOGICAL DIFFERENCES OF EUKARYOTIC CELLS

Type 1 diabetes affects over 108,000 children, and this number is steadily increasing. Current insulin therapies help manage the disease but are not a cure. Over a child’s lifetime they can develop kidney disease, blindness, cardiovascular disease and many other issues due to the complications of type 1 diabetes. This autoimmune disease destroys beta cells located in the pancreas, which are used to regulate glucose levels in the body. Because there is no cure and many children are affected by the disease there is a need for alternative therapeutic options that can lead to a cure. Human mesenchymal stem cells (hMSCs) are an important cell source for stem cell therapeutics due to their differentiation capacity, self-renewal, and trophic activity. hMSCs are readily available in the bone marrow, and act as an internal repair system within the body, and they have been shown to differentiate into insulin producing cells. However, after isolation hMSCs are a heterogeneous cell population, which requires secondary processing. To resolve the heterogeneity issue hMSCs are separated using fluorescent- and magnetic-activate cell sorting with antigen labeling. These techniques are efficient but reduce cell viability after separation due to the cell labeling. Therefore, to make hMSCs more readily available for type 1 diabetes therapeutics, they should be separated without diminishing there functional capabilities. Dielectrophoresis is an alternative separation technique that has the capability to separated hMSCs. This dissertation uses dielectrophoresis to characterize the dielectric properties of hMSCs. The goal is to use hMSCs dielectric signature as a separation criteria rather than the antigen labeling implemented with FACS and MACS. DEP has been used to characterize other cell systems, and is a viable separation technique for hMSCs

Improving the Design and Application of Insulator-Based Dielectrophoretic Devices for the Assessment of Complex Mixtures

Dielectrophoresis (DEP) is an electrokinetic (EK) transport mechanism that exploits polarization effects when particles are exposed to a non-uniform electric field. This dissertation focused on the development of high-performance insulator-based DEP (iDEP) devices. A detailed analysis of the spatial forces that contribute to particle movement in an iDEP device is provided. In particular, this analysis shows how particle size and shape affects the regions where particles are likely to be retained due to dielectrophoretic trapping. The performance of these trapping regions was optimized using a systematic approach that integrates the geometrical parameters of the array of insulating structures. Devices that decrease the required electrical potential by ~80% where found. The optimization strategy enabled the detection of structures that promote and discourage particle trapping. By combining the best and worst structures in a single asymmetric structure, a novel iDEP device was designed. This device selectively enriches the larger particles in a sample and drives the smaller particles away from the enrichment region. A quick enrichment and elution of large cells was achieved. This is important when dealing with samples containing eukaryotic cells, which can be harmed by the electrical treatment. Yeast cells were successfully separated from polystyrene particles in under 40 seconds using this device and a high cell viability of 85% was achieved. Finally, an enhancement of traditional iDEP devices is proposed, where some insulating posts are replaced by conducting structures. That is, insulating and conductive posts are intimately combined within the same array. The performance of this hybrid device is presented to show the advantage of using insulating structures with microelectrodes in the same array to dominate particle movement

EXPLORING THE ROLE AND IMPACT OF MICROSCALE PHENOMENA ON ELECTRODE, MICRODEVICE, AND CELLULAR FUNCTION

Microfluidic technologies enable the development of portable devices to perform multiple high-resolution unit operations with small sample and reagent volumes, low fabrication cost, facile operation, and quick response times. Microfluidic platforms are expected to effectively interpret both wanted and unwanted phenomena; however, a comprehensive evaluation of the unwanted phenomena has not been appropriately investigated in the literature. This work explored an attenuative evaluation of unwanted phenomena, also called here as secondary phenomena, in a unique approach. Upon electric field utilization within microfluidic devices, electrode miniaturization improves device sensitivity. However, electrodes in contact with medium solution can yield byproducts that can change medium properties such as pH as well as bulk ion concentration and eventually target cell viability. While electrode byproducts are sometimes beneficial; but, this is not always the case. Two strategies were employed to protect cells from the electrode byproducts: (i) coating the electrodes with hafnium oxide (HfO2), and (ii) stabilization of the cell membrane using a low concentration of Triton X-100 surfactant. Our results showed that both strategies are a plausible way to selectively isolate cell and reduce the risk of contamination from electrode byproducts. The design of a medium solution is also critical to minimize unwanted cell-medium interaction. Surfactants are frequently added to cell-medium solutions to improve sensitivity and reproducibility without disrupting protein composition of cell membranes or cell viability. In non-electrokinetic systems, surfactants have been shown to reduce interfacial tensions and prevent analyte sticking. However, the impacts of surfactant interactions with cell membranes have not previously been explored in electrokinetic systems. This work indicated the dynamic surfactant interactions with cell membranes which altered the cell membrane integrity. It is important that the effects of the chemical interactions between cells to be fully explored and to be separately attributed to reported cellular responses to accurate catalog properties and engineer reliable microfluidic electrokinetic devices. Finally, a comprehensive level of understanding led us to utilize dielectrophoresis in its full capacity as a tool to monitor the state and progression of virus infection as well as anti-viral activities of regenerative compound. Glycine was utilized as potential antiviral compounds to reduce porcine parvovirus (PPV) infection in porcine kidney (PK-13) cells. Our results demonstrate that the glycine altered the virus-host interactions during virus assembly. Thus, elucidating the mechanisms of these novel antiviral compounds is crucial to their development as potential therapeutic drugs

Isomotive dielectrophoresis for enhanced analyses of cell subpopulations.

As the relentless dream of creating a true lab-on-a-chip device is closer to realization than ever before, which will be enabled through efficient and reliable sample characterization systems. Dielectrophoresis (DEP) is a term used to describe the motion of dielectric particles/ cells, by means of a non-uniform electric field (AC or DC). Cells of different dielectric properties (i.e., size, interior properties, and membrane properties) will act differently under the influence of dielectrophoretic force. Therefore, DEP can be used as a powerful, robust, and flexible tool for cellular manipulation, separation, characterization, and patterning. However, most recent DEP applications focus on trapping, separation, or sorting particles. The true value of DEP lies in its analytical capabilities which can be achieved by utilizing isomotive dielectrophoresis (isoDEP). In isoDEP, the gradient of the electric field-squared is constant, hence, upon the application of electric field, all particles/cells that share the same dielectric properties will feel the same constant dielectrophoretic force i.e., translate through the micro-channel at the same velocity. However, DEP is not the only acting force upon particles inside an isoDEP device, other electrokinetics, including but not limited to electrothermal hydrodynamics, might act on particles simultaneously. Within this dissertation, electrothermal-based experiments have been conducted to assess the effect of such undesired forces. Also, to maximize the relative DEP force over other forces for a given cell/particle size, design parameters such as microchannel width, height, fabrication materials, lid thickness, and applied electric field must be properly tuned. In this work, scaling law analyses were developed to derive design rules that relate those tunable parameters to achieve the desired dielectrophoretic force for cell analysis. Initial results indicated that for a particle suspended in 10 mS/m media, if the channel width and height are below 10 particle diameters, the electrothermal-driven flow is reduced by ∼ 500 times compared to the 500 µm thick conventional isoDEP device. Also, Replacing glass with silicon as the device’s base for an insulative-based isoDEP, reduces the electrothermal induced flow by ∼ 20 times. Within this dissertation, different device designs and fabrication methods were attempted in order to achieve an isoDEP platform that can characterize and differentiate between live and dead phytoplankton cells suspended in the same solution. Unfortunately, unwanted electrokinetics (predicted by the previously mentioned scaling law analysis) prevented comprehensive isoDEP analysis of phytoplankton cells. Due to isoDEP device limitations and other complications, other techniques were pursued to electrically characterize phytoplankton cells in suspension. An electrochemical-based platform utilizing impedance spectroscopy measurements was used to extract the electrical properties of phytoplankton cells in suspension. Impedance spectroscopy spectra were acquired, and the single-shell model was applied to extract the specific membrane capacitance, cytoplasm permittivity, and conductivity of assumingly spherical cells in suspension utilizing Maxwell’s mixture theory of a controlled volume fraction of cells. The impedance of suspensions of algae were measured at different frequencies ranging from 3 kHz to 10 MHz and impedance values were compared to investigate differences between two types of cells by characterizing their change in cytoplasm permittivity and membrane capacitance. Differentiation between healthy control and nitrogen-depleted cultured algae was attempted. The extracted specific membrane capacitances of Chlamydomonas and Selenastrum were 15:57 ± 3:62 and 40:64 ± 12:6 mF/m2 respectively. Successful differentiation based on the specific membrane capacitance of different algae species was achieved. However, no significant difference was noticed between nitrogen abundant and nitrogen depleted cultures. To investigate the potential of isoDEP for cell analysis, a comparison to existing dielectrophoresis-based electrokinetic techniques was encouraged, including electrorotation (ROT) microfluidic platforms. The ROT microfluidic chip was used to characterize M17, HEK293, T-lymphocytes, and Hela single cells. Through hands-on experience with ROT, the advantages and disadvantages of this approach and isoDEP are apparent. IsoDEP proves to be a good characterization tool for subpopulation cell analysis with potential higher throughput compared to ROT while maintaining simple fabrication and operation processes. To emphasize the role of dielectrophoresis in biology, further studies utilizing the 3DEP analytical system were used to determine the electrical properties of Drosophila melanogaster (Kc167) cells ectopically expressing Late embryogenesis abundant (LEA) proteins from the anhydrobiotic brine shrimp, Artemia franciscana. Dielectrophoretic-based characterization data demonstrates that single expression of two different LEA proteins, AfrLEA3m and AfrLEA6, both increase cytoplasmic conductivity of Kc167 cells to a similar extend above control values. The extracted DEP data supported previously reported data suggesting that AfrLEA3m can interact directly with membranes during water stress. This hypothesis was strengthened using scanning electron microscopy, where cells expressing AfrLEA3m were found to retain their spherical morphology during desiccation, while control cells exhibited a larger variety of shapes in the desiccated state

Ultrafine Dielectrophoresis-based Technique for Virus and Biofluid Manipulation

abstract: Microfluidics has shown great potential in rapid isolation, sorting, and concentration of bioparticles upon its discovery. Over the past decades, significant improvements have been made in device fabrication techniques and microfluidic methodologies. As a result, considerable microfluidic-based isolation and concentration techniques have been developed, particularly for rapid pathogen detection. Among all microfluidic techniques, dielectrophoresis (DEP) is one of the most effective and efficient techniques to quickly isolate and separate polarizable particles under inhomogeneous electric field. To date, extensive studies have demonstrated that DEP devices are able to precisely manipulate cells ranging from over 10 μm (mammalian cells) down to about 1 μm (small bacteria). However, very limited DEP studies on manipulating submicron bioparticles, such as viruses, have been reported. In this dissertation, rapid capture and concentration of two different and representative types of virus particles (Sindbis virus and bacteriophage M13) with gradient insulator-based DEP (g-iDEP) has been demonstrated. Sindbis virus has a near-spherical shape with a diameter ~68 nm, while bacteriophage M13 has a filamentous shape with a length ~900 nm and a diameter ~6 nm. Under specific g-iDEP experimental conditions, the concentration of Sindbis virus can be increased two to six times within only a few seconds, using easily accessible voltages as low as 70 V. A similar phenomenon is also observed with bacteriophage M13. Meanwhile, their different DEP behavior predicts the potential of separating viruses with carefully designed microchannels and choices of experimental condition. DEP-based microfluidics also shows great potential in manipulating blood samples, specifically rapid separations of blood cells and proteins. To investigate the ability of g-iDEP device in blood sample manipulation, some proofs of principle work was accomplished including separating two cardiac disease-related proteins (myoglobin and heart-type fatty acid binding protein) and red blood cells (RBCs). Consistent separation was observed, showing retention of RBCs and passage of the two spiked protein biomarkers. The numerical concentration of RBCs was reduced (~70 percent after one minute) with the purified proteins available for detection or further processing. This study explores and extends the use of the device from differentiating similar particles to acting as a sample pretreatment step.Dissertation/ThesisDoctoral Dissertation Chemistry 201

Diamond MEMS Biosensors: Development and applications

This research focuses on the development a dielectrophoresis-enhanced microfluidic impedance biosensor (DEP-e-MIB) to enable fast response, real-time, label-free, and highly sensitive sensor for bacterial detection in clinical sample. The proposed design consists of application of dielectrophoresis (DEP) across a microfluidic channel to one of the impedance spectroscopy electrodes in order to improve the existent bacterial detection limits with impedance spectroscopy. In order to realize such a design, choice of electrode material with a wide electrochemical potential window for water is very important. Conventional electrode material, such as gold, are typically insulated for the application of DEP, and they fail when used open because the DEP voltages avoiding electrolysis do not provide enough force to move the bacteria. First, the use of nanodiamonds (ND) seeding gold surface to widen the electrochemical potential window is examined, since diamond has a wider potential window. ND seed coverage is a function of sonication time, ND concentration, and solvent of ND dispersion. Examining these parameters allowed us to increase the ND surface coverage to ~35%. With the highest ND coverage achievable, such electrodes are still susceptible to damage from electrolysis, however yield a unique leverage for impedance biosensing. When NDs is seeded at a 3x3 interdigitated electrode array, which act as electrically conductive islands between the electrodes and reduce the effective gap between the electrodes, thus allowing to perform impedance spectroscopy in solutions with low electrical conductivity such as ITS. The changes obtained in resistance to charge transfer with bacterial capture is nearly twice than that obtained with plain electrodes. Secondly, the feasibility of using boron-doped ultra nanocrystalline diamond (BD-UNCD) to apply DEP is tested without constructing a 3x3 IDE array. BD-UNCD electrodes can be used for DEP through tagging of the bacteria with immunolatex beads. This allows applying a larger DEP force on the bacteria. Since historically bead based assays are plagued with problems with non-specific binding, the role of different parameters including bead bioconjugation chemistry, bead PEGylation, BD-UNCD surface PEGylation, and DEP on specific and non-specific binding are tested. Most importantly DEP increases the specific binding and PEGylation of beads decreases the specific binding. Finally, a 3x3 IDE array with BD-UNCD was fabricated, and used impedance spectroscopy to test the suitability of BD-UNCD IDEs for impedance biosensing. The huge electrode resistance and the charge transfer resistance at BD-UNCD IDEs poses a problem for impedance biosensing as it will lead to lower sensitivity. BD-UNCD is the material of choice for applying DEP at open electrodes however gold is the choice of material for designing the chip interconnects. So the BD-UNCD layer should be as thin as possible and the interface between gold IDEs and the solution phase during DEP. The findings in this dissertation put us closer to realizing a DEP-eMIB