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

Advanced dielectrophoretic cell separation systems

This thesis describes experimental and theoretical investigations into new particle handling and separation methods and techniques. It makes a major contribution to the rapidly expanding field of cell separation technology. A novel dielectrophoretic cell separation system has been developed, which is capable of processing large sample volumes (~50mL) in a flow through system. Previously reported dielectrophoretic cell separator systems typically process sample volumes in the 100mL range. The electrode configuration developed for this work allows the isolation and concentration of single particle types from large sample volumes; a method which could be further developed into a new rare-cell separation technology. In addition, a new technique of particle fractionation was developed termed ‘Dielectrophoretic Chromatography’. A cell separation chip was designed and built using standard micro-fabrication techniques. Experimental work was undertaken to demonstrate the function and limitations of the device. Numerical modelling of the particle motion in the device is presented and compared with experimental work for a number of different particle types, applied voltages and fluid flow rates. The dielectrophoretic separation system comprises a microfluidic channel, of cross-section 100mm x 10mm and length 50mm, with two sets of interdigitated microelectrode arrays. The first set of arrays, with characteristic electrode size 40mm, called a focussing device, has electrodes patterned onto the top and bottom surfaces of the flow channel. The second electrode array, which is part of the same device, has an electrode array patterned only on the bottom of the channel. Two sizes of secondary electrode array were used 20mm and 40mm. AC voltages (from 1V to 10V peak) are applied to the microelectrode, with a frequency between 10kHz to 180MHz. A dielectrophoretic force is exerted on the particles as they flow along the channel. The first electrode array uses negative dielectrophoresis to focus the stream of particles entering the device into a narrow sheet (one particle diameter thick) midway between the upper and lower channel surfaces. The second electrode array, down stream from the first is separately controllable

Higher Order Electrokinetic Effects for Applied Biological Analytics

abstract: Microfluidic systems have gained popularity in the last two decades for their potential applications in manipulating micro- and nano- particulates of interest. Several different microfluidics devices have been built capable of rapidly probing, sorting, and trapping analytes of interest. Microfluidics can be combined with separation science to address challenges of obtaining a concentrated and pure distinct analyte from mixtures of increasingly similar entities. Many of these techniques have been developed to assess biological analytes of interest; one of which is dielectrophoresis (DEP), a force which acts on polarizable analytes in the presence of a non-uniform electric fields. This method can achieve high resolution separations with the unique attribute of concentrating, rather than diluting, analytes upon separation. Studies utilizing DEP have manipulated a wide range of analytes including various cell types, proteins, DNA, and viruses. These analytes range from approximately 50 nm to 1 µm in size. Many of the currently-utilized techniques for assessing these analytes are time intensive, cost prohibitive, and require specialized equipment and technical skills. The work presented in this dissertation focuses on developing and utilizing insulator-based dielectrophoresis (iDEP) to probe a wide range of analytes; where the intrinsic properties of an analyte will determine its behavior in a microchannel. This is based on the analyte’s interactions with the electrokinetic and dielectrophoretic forces present. Novel applications of this technique to probe the biophysical difference(s) between serovars of the foodborne pathogen, Listeria monocytogenes, and surface modified Escherichia coli, are investigated. Both of these applications demonstrate the capabilities of iDEP to achieve high resolution separations and probe slight changes in the biophysical properties of an analyte of interest. To improve upon existing iDEP strategies a novel insulator design which streamlines analytes in an iDEP device while still achieving the desirable forces for separation is developed, fabricated, and tested. Finally, pioneering work to develop an iDEP device capable of manipulating larger analytes, which range in size 10-250 µm, is presented.Dissertation/ThesisDoctoral Dissertation Chemistry 201

ELECTROKINETICS-ASSISTED ELECTRICAL SENSORS FOR RAPID DETECTION OF BACTERIA

Department of Mechanical Enginering (Mechanical Engineering)An array of microfabricated interdigitated electrodes (IDEs) is the most commonly used form of electrode geometry for dielectrophoretic manipulation of biological particles in microfluidic biochips owing to simplicity of fabrication and ease of analysis. However, the dielectrophoretic force dramatically reduces as the distance from the electrode surface increasestherefore, the effective region is usually close to the electrode surface for a given electric potential difference. Here, I present a novel two-dimensional computational method for generating planar electrode patterns with enhanced volumetric electric fields, which I call the ???microelectrode discretization (MED)??? method. It involves discretization and reconstruction of planar electrodes followed by selection of the electrode pattern that maximizes a newly defined objective function, factor S, which is determined by the electric potentials on the electrode surface alone. In this study, IDEs were used as test planar electrodes. Two arrays of IDEs and respective MED-optimized electrodes were implemented in microfluidic devices for the selective capture of Escherichia coli against 1-??m-diameter polystyrene beads, and I experimentally observed that 1.4 to 35.8 times more bacteria were captured using the MED-optimized electrodes than the IDEs (p < 0.0016), with a bacterial purity against the beads of more than 99.8%. This simple design method offered simplicity of fabrication, highly enhanced electric field, and uniformity of particle capture, and can be used for many dielectrophoresis-based sensors and microfluidic systems. Dielectrophoresis (DEP) is usually effective close to the electrode surface. Several techniques have been developed to overcome its drawbacks and to enhance dielectrophoretic particle capture. Here a simple technique was presented of superimposing alternating current DEP (high-frequency signals) and electroosmosis (EOlow-frequency signals) between two coplanar electrodes (gap: 25 ??m) using a lab-made voltage adder for rapid and selective concentration of bacteria, viruses, and proteins, where the voltages and frequencies of DEP and EO were controlled separately. This signal superimposition technique enhanced bacterial capture (Escherichia coli K-12 against 1-??m-diameter polystyrene beads) more selectively (>99 %) and rapidly (~30 s) at lower DEP (5 Vpp) and EO (1.2 Vpp) potentials than those used in the conventional DEP capture studies. Nanometer-sized MS2 viruses and troponin I antibody proteins were also concentrated using the superimposed signals, and significantly more MS2 and cTnI-Ab were captured using the superimposed signals than the DEP (10 Vpp) or EO (2 Vpp) signals alone (p < 0.035) between the two coplanar electrodes and at a short exposure time (1 min). This technique has several advantages, such as simplicity and low cost of electrode fabrication, rapid and large collection without electrolysis. Electrokinetic technologies such as AC electro-osmosis (EO) and dielectrophoresis (DEP) have been used for effective manipulation of bacteria to enhance the sensitivity of an assay, and many previously reported electrokinetics-enhanced biosensors are based on stagnant fluids. An effective region for positive DEP for particle capture is usually too close to the electrode for the flowing particles to move toward the detection zone of a biosensor against the flow directionthis poses a technical challenge for electrokinetics-assisted biosensors implemented within pressure-driven flows, especially if the particles flow with high speed and if the detection zone is small. Here, a microfluidic single-walled carbon nanotubes (SWCNTs)-based field-effect transistor immunosensor was presented with electrohydrodynamic (EHD) focusing and DEP concentration for continuous and label-free detection of flowing Staphylococcus aureus in a 0.01?? phosphate buffered saline (PBS) solution. The EHD focusing involved AC EO and negative DEP to align the flowing particles along lines close to the bottom surface of a microfluidic channel for facilitating particle capture downstream in the detection zone. For feasibility, 380-nm-diameter fluorescence beads suspended in 0.001?? PBS were tested, and 14.6 times more beads were observed to be concentrated on the detection area with EHD focusing. Moreover, label-free, continuous, and selective measurement of S. aureus in 0.01?? PBS was demonstrated, showing good linearity between the relative changes in electrical conductance of the SWCNTs and logarithmic S. aureus concentrations, a capture/detection time of 35 min, and limit of detection of 150 CFU/mL, as well as high specificity through electrical manipulation and biological interaction.ope

Electrokinetic Transport, Trapping, and Sensing in Integrated Micro- and Nanofluidic Devices

Thesis (Ph.D.) - Indiana University, Chemistry, 2009Microfluidics is rapidly becoming a mature field, and improved fabrication methods now routinely produce sub-micrometer features. As device dimensions shrink, physical phenomena that are negligible at larger length scales become more important, and by integrating nanofluidic elements with microchannels, new analytical techniques can be developed based on the unique behavior of matter at the nanoscale. This work addresses the fabrication, operation, and application of in-plane nanochannels and out-of-plane nanopores in lab-on-a-chip devices. In planar nanofluidic devices, we demonstrate a method to produce micro- and nanoscale features simultaneously with a single UV exposure step and evaluate flow control and sample dispensing with nanofluidic cross structures. Modification of the pinched injection method makes it applicable to variable-volume, attoliter-scale injections, including the smallest volume electrokinetically-controlled injections to date. As an alternative approach, track-etch nanopore membranes are explored as out-of-plane nanofluidic components. The random distribution of pores in these membranes is overcome by lithographic and microchannel-based methods to isolate and address specific pores. Microfluidic isolation improves mass transport to the pore(s), provides easy coupling of electrical potentials, and facilitates additional sample processing steps up- and downstream. These integrated microchannel-nanopore devices are used for diffusion-based dispensing, electrokinetic trapping, and resistive pulse sensing. In a high pore density device, diffusion-based dispensing establishes a stable chemical gradient for bacterial chemotaxis assays. For lower pore density devices, the nanopores are the most resistive components in the fluidic circuit, and application of an electric potential produces localized regions of high electric field strength and field gradient. These high field regions are applied to electrokinetic trapping of particles and cells in multiple-pore devices and to single particle detection by resistive pulse sensing in devices with a single isolated pore. To better understand factors influencing ion current in single nanoscale conduits, we systematically examine ion current rectification as a function of pore diameter, ionic strength, and pH to improve understanding of ion current through nanopores and to characterize preferred operating parameters for sensing applications. These results are applied to detection of virus capsids, and future work is proposed to investigate capsid assembly

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

Optical manipulation and advanced analysis of cells using an innovative optofluidic platform

This doctoral research project aims to analyse complex processes of living cells using Digital Holographic Microscopy (DHM) as a three-dimensional (3D) imaging tool. DHM is a real-time, high-throughput, label-free and quantitative phase imaging technique which permits advanced cell analysis in microfluidic environment. In particular, an innovative optofluidic platform is implemented, composed of a DHM modulus and aided by holographic optical tweezers (HOT) for optical manipulation and a fluorescence modulus. This platform has been used for blood disease screening, cell manipulation studies and tracking of migrating cells. In this thesis, three main topics have been investigated. The first topic focuses on diagnostics, which plays several critical roles in healthcare. Here a novel and cost-effective approach for detecting real blood disorders such as iron-deficiency anaemia and thalassemia at lab-on-chip scale is shown. In addition, cell dynamics studies were performed by DHM. In particular, a study regarding the temporal evolution of cell morphology and volume during blue light exposure is reported. The second topic aims to investigate cell mechanics. To this end, the capabilities of HOT were used to enable the generation and the independent high-precision control of an arbitrary number of 3D optical traps. The combination of HOT and DHM provides the possibility to manipulate cells, detect nano-mechanical cell response in the pN range, and reveal cytoskeleton formation. To confirm the formation of the cytoskeleton structures after the stimulation, a fluorescence imaging system was used as control. Finally, the third topic focuses on cell manipulation using an innovative electrode-free dielectrophoretic approach (DEP) for investigating smart but simple strategies for orientation and immobilization of biological samples such as bacteria and fibroblast. In particular, the light-induced DEP is achieved using ferroelectric iron- doped lithium niobate crystal as substrate. In this way, a dynamic platform that can dynamically regulate the cell response has been developed. In this case, DHM is going to be used as a time-lapse imaging tool for the characterization of dynamic cell processes. In conclusion, the results show that DHM is a highly relevant method that allows novel insights into dynamic cell biology, with applications in cancer research and toxicity testing. In addition, this study could pave the way for detecting and quantifying circulating tumor cells and for providing multidimensional information on tumour metastasis. In this framework, the optofluidic platform is a promising tool for both identification and characterization of “foreign” cancer cells in the blood stream in order to achieve an early diagnosis

Particles Separation in Microfluidic Devices, Volume II

Microfluidic platforms are increasingly being used for separating a wide variety of particles based on their physical and chemical properties. In the past two decades, many practical applications have been found in chemical and biological sciences, including single cell analysis, clinical diagnostics, regenerative medicine, nanomaterials synthesis, environmental monitoring, etc. In this Special Issue, we invited contributions to report state-of-the-art developments in the fields of micro- and nanofluidic separation, fractionation, sorting, and purification of all classes of particles, including, but not limited to, active devices using electric, magnetic, optical, and acoustic forces; passive devices using geometries and hydrodynamic effects at the micro/nanoscale; confined and open platforms; label-based and label-free technology; and separation of bioparticles (including blood cells), circulating tumor cells, live/dead cells, exosomes, DNA, and non-bioparticles, including polymeric or inorganic micro- and nanoparticles, droplets, bubbles, etc. Practical devices that demonstrate capabilities to solve real-world problems were of particular interest

Microfluidics for waterborne pathogen separation and detection

There are millions of Cryptosporidium-attributable cases annually in children aged <24 months in the sub-Saharan Africa and India, Pakistan, Bangladesh, Nepal, Afghanistan regions, respectively, and ~202,000 Cryptosporidium-attributable deaths”. Improved monitoring is one solution to this challenge; however, detection of this pathogen is particularly challenging, particularly in regard to determining viability information. This thesis explores the development of novel protocols and devices for Cryptosporidium parvum (C. parvum), through usage of nanoparticles (NPs) and microfluidic methods. NP lysis approaches were developed as a low-cost, one-step rapid method with the possibility to then integrate lysis and molecular detection into one microfluidic device. Different materials, exposure times and concentrations were explored and ZnO NPs were found to be as effective as the traditional freeze-thaw protocol. Dielectrophoretic microfluidic devices were designed, prototyped and optimised for viability-based separations. Fabrication was attempted via laser ablation for the purpose of generating microchannels on PMMA sheets and Physical Vapour Deposition via an E-Beam system to investigate the deposition of electrodes; however, lift-off solvents were incompatible with PMMA. Electrode design modifications were implemented to optimise performance and efficacy of oocyst separation, based on viability, was assessed via an excystation assay where at the outlet collecting non-viable oocysts viability was found to be 5.9% and the other outlets showed a viability of 81.8% and 88.4%. The original sample provided had a viability of 89.7%. The work here adds to the growing number of studies investigating new ways of lysis and detection for C. parvum. The lysis and detection of the oocyst is currently highly intensive and a number of new methods of miniaturisation such as µPCR when integrated with a lysing and filtration device to create a more precise method of finding the presence of C. parvum in water samples. The DEP based separation device builds on the work of previous studies such as Su et al. and others in order to separate C. parvum based on its viability status in two different devices. This is a significant step showing that label-free separation of oocysts of the same species can be separated based on their viability