Rapid rotation of micron and submicron dielectric particles measured using optical tweezers

We demonstrate the use of a laser trap (‘optical tweezers’) and back-focal-plane position detector to measure rapid rotation in aqueous solution of single particles with sizes in the vicinity of 1 μm. Two types of rotation were measured: electrorotation of polystyrene microspheres and rotation of the flagellar motor of the bacterium Vibrio alginolyticus. In both cases, speeds in excess of 1000 Hz (rev s−1) were measured. Polystyrene beads of diameter about 1 μm labelled with smaller beads were held at the centre of a microelectrode array by the optical tweezers. Electrorotation of the labelled beads was induced by applying a rotating electric field to the solution using microelectrodes. Electrorotation spectra were obtained by varying the frequency of the applied field and analysed to obtain the surface conductance of the beads. Single cells of V. alginolyticus were trapped and rotation of the polar sodium-driven flagellar motor was measured. Cells rotated more rapidly in media containing higher concentrations of Na+, and photodamage caused by the trap was considerably less when the suspending medium did not contain oxygen. The technique allows single-speed measurements to be made in less than a second and separate particles can be measured at a rate of several per minute

AC electrokinetic studies of virus host interactions

Abstract available p. ii

Numerical modeling of dielectrophoretic effect for manipulation of bio-particles

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.This text describes different aspects of the design of a Doctor-on-a-Chip device. Doctor-on-a-Chip is a DNA analysis system integrated on a single chip, which should provide all of the advantages that stem from the system integration, such as small sample volume, fast and accurate analysis, and low cost. The text describes all of the steps of the on-chip sample analysis, including DNA extraction from the sample, purification, PCR amplification, novel dielectrophoretic sorting of the DNA molecules, and finally detection. The overview is given of the technologies which are available to make the integration on a single chip possible. The microfluidic technologies that are used to manipulate the sample and other chemical reagents are already known and in this text they are analyzed in terms of their feasibility in the on-chip system integration. These microfluidic technologies include, but are not limited to, microvalves, micromixers, micropumps, and chambers for PCR amplification. The novelty in the DNA analysis brought by Doctor-on-a-Chip is the way in which the different DNA molecules in the sample (for example, human and virus DNA) are sorted into different populations. This is done by means of dielectrophoresis – the force experienced by dielectric particles (such as DNA molecules) when subject to a non-uniform electric field. Different DNA molecules within a sample experience different dielectrophoretic forces within the same electric field, which makes their separation, and therefore detection, possible. In this text, the emphasis is put on numerical modelling of the dielectrophoretic effect on biological particles. The importance of numerical modelling lies in the fact that with the accurate model it is easier to design systems of microelectrodes for dielectrophoretic separation, and tune their sub-micrometre features to achieve the maximum separation efficacy. The numerical model described in this text is also experimentally verified with the novel microelectrodes design for dielectrophoretic separation, which is successfully used to separate the mixture of different particles in the micron and sub-micron range

Information Fusion for Improved Motion Estimation

studentship award number 98318229Motion Estimation is an important research field with many commercial applications including surveillance, navigation, robotics, and image compression. As a result, the field has received a great deal of attention and there exist a wide variety of Motion Estimation techniques which are often specialised for particular problems. The relative performance of these techniques, in terms of both accuracy and of computational requirements, is often found to be data dependent, and no single technique is known to outperform all others for all applications under all conditions. Information Fusion strategies seek to combine the results of different classifiers or sensors to give results of a better quality for a given problem than can be achieved by any single technique alone. Information Fusion has been shown to be of benefit to a number of applications including remote sensing, personal identity recognition, target detection, forecasting, and medical diagnosis. This thesis proposes and demonstrates that Information Fusion strategies may also be applied to combine the results of different Motion Estimation techniques in order to give more robust, more accurate and more timely motion estimates than are provided by any of the individual techniques alone. Information Fusion strategies for combining motion estimates are investigated and developed. Their usefulness is first demonstrated by combining scalar motion estimates of the frequency of rotation of spinning biological cells. Then the strategies are used to combine the results from three popular 2D Motion Estimation techniques, chosen to be representative of the main approaches in the field. Results are presented, from both real and synthetic test image sequences, which illustrate the potential benefits of Information Fusion to Motion Estimation applications. There is often a trade-off between accuracy of Motion Estimation techniques and their computational requirements. An architecture for Information Fusion that allows faster, less accurate techniques to be effectively combined with slower, more accurate techniques is described. This thesis describes a number of novel techniques for both Information Fusion and Motion Estimation which have potential scope beyond that examined here. The investigations presented in this thesis have also been reported in a number of workshop, conference and journal papers, which are listed at the end of the document

Applications to cancer research of "lab-on-a-chip" devices based on dielectrophoresis (DEP).

The recent development of advanced analytical and bioseparation methodologies based on microarrays and biosensors is one of the strategic objectives of the so-called post-genomic. In this field, the development of microfabricated devices could bring new opportunities in several application fields, such as predictive oncology, diagnostics and anti-tumor drug research. The so called "Laboratory-on-a-chip technology", involving miniaturisation of analytical procedures, is expected to enable highly complex laboratory testing to move from the central laboratory into non-laboratory settings. The main advantages of Lab-on-a-chip devices are integration of multiple steps of different analytical procedures, large variety of applications, sub-microliter consumption of reagents and samples, and portability. One of the requirement for new generation Lab-on-a-chip devices is the possibility to be independent from additional preparative/analytical instruments. Ideally, Lab-on-a-chip devices should be able to perform with high efficiency and reproducibility both actuating and sensing procedures. In this review, we discuss applications of dielectrophoretic(DEP)-based Lab-on-a-chip devices to cancer research. The theory of dielectrophoresis as well as the description of several devices, based on spiral-shaped, parallel and arrayed electrodes are here presented. In addition, in this review we describe manipulation of cancer cells using advanced DEP-based Lab-on-a-chip devices in the absence of fluid flow and with the integration of both actuating and sensing procedures

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

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

Electrical Characterization and Detection of Blood Cells and Stones in Urine

Urine contains an immense amount of information related to its physical, chemical, and biological components; hence, it is a promising tool in detecting various diseases. Available methods for detecting hematuria (blood in the urine) are not accurate. Results are influenced by many factors, such as, health and vitals of the patients, settings of the equipment and laboratories, which leads to false positive or false negative outputs. This necessitates the development of new, accurate, and easy-access methods that save time and effort. This study demonstrates a label-free and accurate method for detecting the presence of red and white blood cells (RBCs and WBCs) in urine by measuring the changes in the dielectric properties of urine upon increasing concentrations of both cell types. The current method could detect changes in the electrical properties of fresh urine over a short time interval, making this method suitable for detecting changes that cannot be recognized by conventional methods. Correcting these changes enabled the detection of a minimum cell concentration of 10² RBCs per ml which is not possible by conventional methods used in the labs except for the semi-quantitative method that can detect 50 RBCs per ml, but it is a lengthy and involved procedure, not suitable for high volume labs. This ability to detect a very small amount of both types of cells makes the proposed technique an attractive tool for detecting hematuria, the presence of which is indicative of problems in the excretory system. Furthermore, urolithiasis is also a very common problem worldwide, affecting adults, kids, and even animals. Calcium oxalate is the major constituent of urinary tract stones in individuals, primarily due to the consumption of high oxalate foods. The occurrence of urinary oxalate occurs by endogenous synthesis, especially in the upper urinary tract. In a normal, healthy individual, the excretion of oxalate ranges from 10 to 45 mg/day, depending on the age and gender, but the risk of stone formation starts at 25 mg/day depending on the health history of the individual. This study also addresses the detection of the presence of calcium oxalate in urine following the same label-free approach. This can be done by measuring the changes in the dielectric properties of urine with increasing concentrations of calcium oxalate hydrate (CaC₂O₄.H₂O). The current method could detect dynamic changes in the electrical properties of urine over a time interval in samples containing calcium oxalate hydrate even at a concentration as low as 10 μg/mL of urine, making this method suitable for detecting changes that cannot be recognized by conventional methods. The ability to detect a very small amount of stones makes it an attractive tool for detecting and quantifying stones in kidneys. Using a non-invasive method which also works as a precautionary measure for early detection of some severe ailments, holds a good scope. It forms the basis of the cytological examinations and molecular assays for the diagnosis of several diseases. This method can be considered a point-of-care test because the results can be instantaneously shared with the members of the medical team. Based on these results, it is anticipated that the present approach to be a starting point towards establishing the foundation for label-free electrical-based identification and quantification of an unlimited number of nano-sized particles

Microfluidic and Electrokinetic Manipulation of Single Cells

Traditional cell assays report on the average results of a cell population. However, a wide range of new tools are being developed for a fundamental understanding of single cell's functionality. Nonetheless, the current tools are either limited in their throughput or the accuracy of the analysis. One such technology is electrorotation. Although it is known to be unique in its capability for single-cell characterization, it is commonly a slow technique with a processing time of about 30 minutes per cell. For this reason, this thesis focuses on the development of a 3D electrode based electrorotation setup for fast and automatic extraction of a single cell's spectrum. For this purpose, new fabrication processes for 3D electrodes were developed to achieve high-resolution patterning of 3D metal electrodes. The first process we developed was a subtractive one based on passivated silicon structures and the second process was an additive one based on SU-8 photolithography. The additive nature of the second process enables high patterning resolution of electrodes and connection layers, while providing high conductivity thanks to the use of standard metal films. The electrodes have been characterized by different electrical measurements to ensure a proper connection and side-wall exposure. Furthermore, we characterized and compared the sheet resistance of planar and vertical layers. A further microfabrication process was developed for integrating the electrodes into microfluidic channels. The process was designed to enable the use of high numerical aperture lenses; for that purpose, a PDMS-mediated bonding process was engineered to seal the channels with a thin glass coverslip. Moreover, the development of a process to realize microfluidic access holes on the back of the wafer reduces the footprint of the chips and facilitates access for the microscope optics. Finally, a pressure-driven system was used together with the chips to achieve high control of liquid injections and to enable fast and precise flow stop. The combination of such a system, together with the dielectrophoretic forces that can be applied by the 3D electrodes, allows accurate positioning of single cells inside the 3D electrode quadrupole. The particles can then be analyzed by electrorotation. For this purpose, a custom Labview interface was built to coordinate the full setup and to acquire a full electrorotation spectrum in less than 3 minutes