A microfabricated dielectrophoretic trapping array for cell-based biological assays

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2001.Includes bibliographical references (p. 143-152).This thesis presents the development of a small planar array of microfabricated traps for holding single cells and performing assays on them. The traps use the phenomenon of dielectrophoresis-the force on polarizable bodies in a non-uniform electric field-to make potential energy wells. These potential energy wells are electrically switchable, arrayable, and amenable to batch fabrication. The trapping arrays have potential use as a cytometer for monitoring the dynamics of populations of single cells and then sorting those cells based upon those dynamics. To design such traps, I have developed a modeling environment that can absolutely predict the ability of DEP-based traps to hold particles against liquid flows, which are the dominant destabilizing force in these systems. I have used the common easy-to-fabricate planar quadrupole trap to verify the accuracy of these modeling tools, and in the process determined why planar quadrupole traps behave as they do. I next used the modeling tools to design an improved quadrupole trap-the extruded quadrupole-that has the potential to hold particles lOx-100x stronger. The extruded quadrupole trap consists of a set of microfabricated gold posts arranged in a trapezoidal fashion, to ease trap loading, and includes metal substrate shunts to improve performance. The fabrication process for small arrays of these traps uses electroplating of gold into an SU-8 mold to achieve the required geometries. The final section of the thesis details experiments using small arrays of these extruded quadrupole traps. Experiments were performed with beads to verify the strong nature of the trap and then with cells to demonstrate qualitative operation of the arrays and the ability to perform dynamic fluorescent assays on multiple single cells followed by sorting. The technology is now well poised to enable the development of biological assays that are currently unavailable.by Joel Voldman.Ph.D

Single-Cell Chemical Lysis on Microfluidic Chips with Arrays of Microwells

Many conventional biochemical assays are performed using populations of cells to determine their quantitative biomolecular profiles. However, population averages do not reflect actual physiological processes in individual cells, which occur either on short time scales or nonsynchronously. Therefore, accurate analysis at the single-cell level has become a highly attractive tool for investigating cellular content. Microfluidic chips with arrays of microwells were developed for single-cell chemical lysis in the present study. The cellular occupancy in 30-μm-diameter microwells (91.45%) was higher than that in 20-μm-diameter microwells (83.19%) at an injection flow rate of 2.8 μL/min. However, most of the occupied 20-μm-diameter microwells contained individual cells. The results of chemical lysis experiments at the single-cell level indicate that cell membranes were gradually lysed as the lysis buffer was injected; they were fully lysed after 12 s. Single-cell chemical lysis was demonstrated in the proposed microfluidic chip, which is suitable for high-throughput cell lysis

Manipulation and trapping of sub-micron bioparticles using dielectrophoresis

A non-uniform alternating electric field induces motion in polarisable particles called dielectrophoresis. The effect is governed by the relative magnitudes of the dielectric properties of the medium and the particles. The technology has been used to manipulate particles for biotechnological applications, including purification, fractionation and concentration of cells and micro-organisms. However, the lower size limit for the dielectrophoretic manipulation of particles was believed to be about 1 ?m, but recent work has proved otherwise. The dielectrophoretic movement and properties of latex beads and a simple rod-shaped virus, tobacco mosaic virus (TMV), have been measured using microfabricated electrode structures. Measurements have been made over a range of suspending medium conductivities, applied frequencies and electric field strengths. It is shown that under appropriate conditions both latex beads and tobacco mosaic virus particles can be selectively attracted to regions of high electric field strength located at the tips of microfabricated electrode structures. The ability to selectively trap and separate bio-particles has many potential applications in the area of biotechnology

Effects of Dielectrophoresis on Growth, Viability and Immuno-reactivity of Listeria monocytogenes

Dielectrophoresis (DEP) has been regarded as a useful tool for manipulating biological cells prior to the detection of cells. Since DEP uses high AC electrical fields, it is important to examine whether these electrical fields in any way damage cells or affect their characteristics in subsequent analytical procedures. In this study, we investigated the effects of DEP manipulation on the characteristics of Listeria monocytogenes cells, including the immuno-reactivity to several Listeria-specific antibodies, the cell growth profile in liquid medium, and the cell viability on selective agar plates. It was found that a 1-h DEP treatment increased the cell immuno-reactivity to the commercial Listeria species-specific polyclonal antibodies (from KPL) by ~31.8% and to the C11E9 monoclonal antibodies by ~82.9%, whereas no significant changes were observed with either anti-InlB or anti-ActA antibodies. A 1-h DEP treatment did not cause any change in the growth profile of Listeria in the low conductive growth medium (LCGM); however, prolonged treatments (4 h or greater) caused significant delays in cell growth. The results of plating methods showed that a 4-h DEP treatment (5 MHz, 20 Vpp) reduced the viable cell numbers by 56.8–89.7 %. These results indicated that DEP manipulation may or may not affect the final detection signal in immuno-based detection depending on the type of antigen-antibody reaction involved. However, prolonged DEP treatment for manipulating bacterial cells could produce negative effects on the cell detection by growth-based methods. Careful selection of DEP operation conditions could avoid or minimize negative effects on subsequent cell detection performance

Integrated Circuit / Microfluidic Chips for Dielectric Manipulation

Engineering and Applied Science

Applications of Microfabrication in Biosensor Technology

This thesis investigates the application of microfabrication techniques in biotechnology, embracing two major methodologies: biosensing and dielectrophoresis. In the first instance, the miniaturisation of biosensors for use in aqueous solutions was explored, focusing on the issues of insulator deposition and metal multilayer adhesion. Two different immobilisation strategies were used. The first involved the binding of the electroactive protein, cytochrome c, to a self-assembled monolayer in order to measure the cellular production of superoxide. The second was the entrapment of an enzyme, glucose oxidase (EC 1.1.3.4), within a polypyrrole film as the core of an amperometric sensor for glucose. In the latter case, a novel analytical technique was developed for characterising the sensor interface involving the use of XPS and FTIR. The technique was used to demonstrate the efficiency with the which enzyme could be bound within the film, and indicated a non-linear relationship between the concentration of entrapped enzyme and the its concentration in the polymerisation solution. To complement these studies, work in dielectrophoresis centred on methods for trapping and measuring single cell function within microfabricated electrode arrays. Although it was possible to hold a cell in close proximity to a biosensor array, difficulties concerning the sensitising of the electrochemical devices precluded measurements on single cells. However, by using a related technique it was possible, for the first time, to observe the dynamic activation of a single human neutrophil. The electrorotation technique was used to monitor changes in the physical character of the cell in order to identify the effects of chemotactic stimulation. Studies involving electrochemical and chemiluminescent measurements were performed to corroborate these findings

Synergism between particle-based multiplexing and microfluidics technologies may bring diagnostics closer to the patient

In the field of medical diagnostics there is a growing need for inexpensive, accurate, and quick high-throughput assays. On the one hand, recent progress in microfluidics technologies is expected to strongly support the development of miniaturized analytical devices, which will speed up (bio)analytical assays. On the other hand, a higher throughput can be obtained by the simultaneous screening of one sample for multiple targets (multiplexing) by means of encoded particle-based assays. Multiplexing at the macro level is now common in research labs and is expected to become part of clinical diagnostics. This review aims to debate on the “added value” we can expect from (bio)analysis with particles in microfluidic devices. Technologies to (a) decode, (b) analyze, and (c) manipulate the particles are described. Special emphasis is placed on the challenges of integrating currently existing detection platforms for encoded microparticles into microdevices and on promising microtechnologies that could be used to down-scale the detection units in order to obtain compact miniaturized particle-based multiplexing platforms