ACTAUNIVERSITATISUPSALIENSISUPPSALA2006

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 166

Microfluidic Devices forManipulation and Detection ofBeads and Biomolecules

MATS JÖNSSON

ISSN 1651-6214ISBN 91-554-6523-4urn:nbn:se:uu:diva-6746

Tillägnad

Morfar o Mormor

“ The times they are A-changin’ “

Bob Dylan

List of papers

I Peter Nilsson, Mats Jönsson and Lars Stenmark. “Chip Mounting and Interconnection in Multi Chip Modules for Space Applications”. Jour-nal of Micromechanics and Microengineering, 11, No 4 2001, pp 339-343.

II Jenny Samskog, Sara K. Bergström, Mats Jönsson, Oliver Klett, Mag-nus Wetterhall, Karin E. Markides. ”On-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface”. Electrophoresis, Volume 24, Issue 11, No. 11 2003, pp 1723-1729.

III M. Jönsson, F. Aldaeus, L. E. Johansson, U. Lindberg, S. Hamp, G. Jonsson, J. Roeraade, Y. Bäcklund. ”A Study of Biological Particles in Bio-MEMS Devices using Dielectrophoresis”. In proceedings of the fifth Micro Structure Workshop, pp 65-69, 30-31 Mars 2004, Ys-tad.

IV Mats Jönsson, Ken Welch, Sven Hamp, Maria Strømme. ”Bacteria counting with impedance spectroscopy in a micro probe station”. Ac-cepted for publication in Journal of Physical Chemistry B.

V Mats Jönsson, Ulf Lindberg. “A planar polymer microfluidic electro-capture device for beads immobilisation”. Submitted to Journal of Mi-cromechanics and Microengineering.

VI Henrik Anderson, Mats Jönsson, Lars Vestling, Ulf Lindberg, Teodor Aastrup. ”Quartz crystal microbalance biosensor design: I. Sensor re-sponse and performance”. Submitted to Sensors and Actuators B, Chemical.

VII Mats Jönsson, Henrik Anderson, Teodor Aastrup, Ulf Lindberg. ”Quartz crystal microbalance biosensor design: II. Sample transport”. Submitted to Sensors and Actuators B, Chemical

VIII Mats Jönsson, Leif Nyholm, Ulf Lindberg. “Design and fabrication of a miniaturised Electrical Field-Flow Fractionation instrument with ITO electrodes”. Manuscript

Reprints were made with permission from the publishers

Comments on the author’s contribution to appended papers

I. Part of fabrication, evaluation and writing.

II. Part of design, microfabrication and small part of writing.

III. Part of planning. All microfabrication. Part of evaluation and writ-ing.

IV. Significant part of planning, evaluation and writing. All microfabri-cation and major part of experimental work.

V. Major part of planning and writing. All design, fabrication and evaluation.

VI. Part of planning, and writing. Major part of resonator characterisa-tion.

VII. Major part of planning, modelling, analysis and writing.

VIII. Major part of planning, evaluation and writing. All design and fabri-cation.

Related work not included in this thesis

“Method and device for capturing charged molecules travelling in a flow stream”. Patent no: EP1572574. The author is one of eight in-ventors.

Rik ter Veen, Mats Jönsson, Jesper Gantelius, Ulf Lindberg, Karin Caldwell. ”Development of a micronized Electrical Field-Flow Fractionation instrument with an improved electrode system”. Pre-sented at the 225th ACS National Meeting, Elopto 2003 March 24-27, 2003, New Orleans, Louisiana, USA.

Quan Du, Benoit De Pradier, Gerald Jesson, Mats Jönsson, Harold Swerdlow , and Zicai Liang. “A silicon-based DNA micro array substrate with enhanced signal intensity”. Submitted to BMC Jour-nal of Biotechnology.

Contents

Preface ............................................................................................................9

Introduction...................................................................................................11

Objectives .....................................................................................................12

Background and Methods .............................................................................13Micro Structure Technology ....................................................................13Electrocapture Technology.......................................................................15Dielectrophoresis......................................................................................16Electric Impedance Spectroscopy ............................................................17Quartz Crystal Microbalance....................................................................17Electric Field-Flow Fractionation ............................................................18

The Lab-on-Chip concept .............................................................................19Sample transport.......................................................................................19Manipulation and Detection .....................................................................20A Lab-on-Chip demonstrator ...................................................................21Fabrication issues .....................................................................................22

Summary and Outlook ..................................................................................24

Summary of appended papers .......................................................................26

Sammanfattning på svenska..........................................................................34

Acknowledgements.......................................................................................36

References.....................................................................................................38

Abbreviations

BCB Bisbenzocyclobuthane BVD Butterworth-van Dyke CD Capture device CE Capillary electrophoresis DC Direct current DEP Dielectrophoresis DEPIM Dielectrophoretic impedance measurement DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid ECD Electrocapture device ECI Electrocapture instrument ECT Electrocapture technology EFFF Electric field-flow fractionation EIS Electric impedance spectroscopy EOF Electroosmotic flow ESI Electrospray ionization FFF Field-flow fractionation FIA Flow injection analysis iDEP Insulator-based dielectrophoresis ITO Indium-tin-oxide KISS Keep it simple, stupid! LC Liquid chromatography LCCE Liquid chromatography capillary electrophoresis LOC Lab-on-Chip MS Mass spectrometry MST Microstructure technology OM Optical microscopy PDMS Poly(dimethylsiloxane) PS Polystyrene QCM Quartz crystal microbalance SPR Surface plasmon resonance TOF Time of flight UV Ultra violet µTAS Micro-total analysis system

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Preface

My mind was set on building micro devices and systems enabling continu-ous flow manipulation and analysis of cells, DNA, enzymes, etc, etc. In my imagination the design would take some careful thinking but the fabrication would be a “piece of cake”. The manufactured reliable lab-on-chip (LOC) should be delivered with joyful delight to my project collaborators. Some redesign would be necessary after the proof of principle measurements. This might have been naive but the words in the approved grant applications are still clearly pictured before me.

The true story of the presented research is however not as fancy as the grant application promised. As the ideas matured, the main efforts was put in investigating and fabricating subpart devices of the planned systems and to validate the desired functions. In the end, however, some cells were charac-terised and some fluorescent beads were manipulated in a number of novel microfluidic devices, proudly presented in this thesis, and the future still looks bright (shades are recommended).

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Introduction

The concept of Lab-on-chip (LOC) is already well established and the num-ber of review articles written on how to benefit from miniaturization and integration increase every month. Analysis would be faster, and less sample and reagents would be consumed. Integrated sample preparation can result in a substantial time, cost (instrument, labour and sample), and space savings. The detection limit is forced down which make single-particle detection possible.The interest in using microdevices for bio-applications started to grow in the 1980’s and in 1990 Manz et al. [1] introduced the concept of micro-total-analysis-system (µTAS). The first µTAS conference was held in 1994 in Enschede, The Netherlands, and has until today grown to an internationally renowned annual event. To manufacture a functional microfluidic device for bioanalysis applications, knowledge of smart design, micro fabrication, and several disciplines of biotechnology and chemistry (e.g. electro chemistry, surface chemistry, cell biology, etc), are all needed simultaneously.

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Objectives

The urge for miniaturised analysis instrument for faster and more cost effec-tive analysis in the chemistry lab, at the hospital or at the large pharmaceuti-cal company, is the main underlying reason the work performed. Before this objective can be realised and come into use for regular people, there are many areas where considerable research and development must be done. Miniaturised devices performing sample transport, bead immobilisation, cell manipulation and counting, and bio-particle detection could be some steps towards the true Lab-on-chip. The more “down-to-earth” aim of this thesis is to present a few projects where design, fabrication, and characterisation of such microdevices have been performed.

If this thesis should present an answer to one question it would be… How do you build low-cost micro devices to study small particles? The wide-ranging answer presented in this thesis is one of many in today’s large community of Lab-on-chip researchers but it has hopefully contributed by taking one step in the right direction.

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Background and Methods

In this thesis subfunctions of analysis systems have been miniaturised in order to increase the overall efficiency. Sample delivery, manipulation, sepa-ration, and detection are done using many different methods [2-9]. The func-tions studied in this work are:

Sample transport Manipulation Detection

Biological samples, e.g. E. coli cells have been manipulated and detected. To evaluate microdevice performance, polymer (polystyrene or latex) beads are preferably used due to uncomplicated sample preparation procedures compared to case of biological sample and less sample composition varia-tion. The beads and cells studied in this have diameter ranging from 50 nm to 10 µm. In order to understand the presented work the methods and tech-nologies used will first be presented briefly. The methods include:

Micro Structure Technology Electrocapture Technology (ECT) Dielectrophoresis (DEP) Electric Impedance spectroscopy (EIS) Quartz Crystal Microbalance (QCM) Electric Field-Flow Fractionation (EFFF)

Micro Structure Technology The manufacture of the presented micro devices includes a wide range of fabrication techniques from traditional silicon micro structuring to all-polymer device manufacture, for example:

Thin film deposition Photo lithography Bonding and lamination Poly(dimethylsiloxane) (PDMS) moulding

Metallic thin films e.g. aluminium, titanium or gold are often used as elec-trode material in microfluidic devices. Evaporation and sputtering methods are the most common methods to deposit the film onto solid bulk substrates.

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A photo lithography process is used to pattern the electrode film on e.g. glass substrates. A liquid photo-sensitive polymer film is spun onto the sub-strate and selectively exposed to light through a shadow mask. Afterwards the polymer film is developed analogously to a traditional photo film. The polymer film left is now covering the electrode film in the desired pattern. The uncovered electrode film is etched away, realising the desired electrode pattern. A reversed process, called “lift-off”, is also common where the polymer is deposited and patterned first, and then the electrode film is depos-ited onto the polymer. The polymer is then dissolved and hence selectively lifting off undesired electrode film layer. A patterned titanium electrode layer on a glass slide is seen in Figure 1.

Figure 1. Glass slide, 35 x 70 mm, with sputtered Ti-electrodes patterned by photo-lithography. The inset microscope photo shows a close-up of the electrode pattern with distance 30 µm between opposite electrode tips (a hair has a diameter of ap-proximately 100 µm).

Traditionally, geometrical structures are etched from e.g. silicon sub-strates as in paper I. The substrate is laminated to a lid substrate to create an enclosed channel by means of bonding or gluing. In paper V and VIII, the channel geometry presented is constructed from two substrates and a poly-mer spacer film. The channel geometry is cut out from the spacer, and the spacer thickness defines the channel height. The spacer is then laminated between the substrates and sealed by thermal curing (paper VIII) or by using an adhesive film (paper V).

Micro moulding of PDMS from moulds prepared by photo lithography and etching is a popular method for rapid prototyping of microfluidic de-vices [10]. In paper II, replication of capillaries for forming a four-way channel crossing has been utilised. For further details on microfabrication the reader is referred to textbooks in MST, e.g. [11-13].

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Electrocapture Technology The ECT was first presented by Park and Swerdlow in a capillary device [14]. By ECT, charged bio molecules and particles can be immobilised in a flow channel by applying a local electric field [14-19]. As the flow passes, charged bio molecules are locally captured and concentrated. By switching the flowing fluid to other liquids, the sample can be cleaned, desalted, or chemically modified e. g. enzymaticly digested [17, 19]. A stacking princi-ple explaining the electrocapture phenomena has been suggested [14] and is also discussed in paper V.

For high throughput electrocapture separation a planar microfluidic de-vice is more suitable as parallel channels could be utilised. In paper V a pla-nar electrocapture device is presented. A commercial ECT-instrument for sample preparation has recently been presented, see figure Figure 2 [20].

Figure 2. The first commercial Electrocapture Instrument (ECI).

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Dielectrophoresis

Dielectrophoresis (DEP) was discovered by H. Pohl [21] in 1951. Dielectro-phoresis refers to the motion of polarisable particles suspended in an electro-lyte and subjected to a non-uniform electric field [22-24]. The field creates

dipoles in the material and therefore the particles starts to move towards increasing or decreasing field gradients as illustrated in

Figure 3. The direction and strength of the DEP-force exerted on the parti-cles due to this field depends on the size of the particles, the permittivity of particles and medium, respectively, and the frequency of the applied electri-cal field according to eq. 1 and 2.

Figure 3. Schematic drawing of a polarisable particle in an inhomogeneous electric field.

The DEP-force on a particle is given by

23 Re2 EKrF mDEP [1]

where r is the radius of the particle, E is the electric field vector and K, ac-cording to the Clausius-Mossotti equation, is

**

**

2 mpmpK [2]

where *p and*m is the frequency-dependent complex permittivity for the

particle and the medium, respectively. During the last decade this method has been used to separate different

particles and biological samples [25-28]. Mostly, DEP-trapping is used to

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sort out material of interest either by trapping the material or by trapping “the rest” letting the sample material pass.

Electric Impedance Spectroscopy Studies of living cells and tissue by dielectric characterisation e.g. Electric Impedance Spectroscopy (EIS) have been going on for 50 years [29]. Con-ductivity and permittivity are often measured by EIS. The ability of the par-ticles to follow the alternating electric field is measured, i.e. determination of speed of redistribution of the mobile charges on the surface and around the particle. The smaller the particle the faster is the response. The frequency limit where the particle polarisation cannot follow the speed of alternation, called the relaxation frequency, is a measure used to characterise particles [24, 30].

Models have been developed to account for the measured impedance re-sponse [23, 29, 31-35]. EIS has recently been utilized to monitor biomass concentration of Bacillus Thuringensis [36], to identify culture phases by transient capacitance measurements of permittivity [37], and to detect bacte-rial metabolism [38, 39]. The bacteria counting presented in paper IV is based on impedance spectrometry measurements and a method for extracting conductivity data from raw data disturbed by polarization effects at lower frequencies [40, 41].

Quartz Crystal Microbalance QCMs have been applied as biosensors and surface science studies for the last two decades [42-52]. The technology is based on findings by Sauerbrey in the 1950’s [53]. The relationship between the mass adsorbed to a QCM device and the resonance frequency can be described with the Sauerbrey equation:

Am

vff

22 [3]

where f is the resonance frequency, the density of quartz, v the shear wave velocity in quartz, A the electrode area, and m the sample mass. The equa-tion is valid if the added mass is small in relation to the mass of the crystal and if the added mass forms an evenly distributed rigid layer on the active sensor area. For studies of biological systems in liquid environments this will not be the case, since biomolecules bound to a sensor surface cannot be considered completely rigid and since the biomolecules will coordinate wa-

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ter which will enhance the sensor signal. However, Muratsugu and co-workers have shown with radio-isotope labelling methods that the linearity between adsorbed mass and frequency response will persist under these con-ditions, but with a coefficient of about 4 governing the frequency-to-mass relationship [54]. Mecea have addressed the created megagravity field in the crystal to explain the excellent sensitivity compared to other microbalances [55]. The behaviour of a quartz crystal resonator can be described by an electric equivalent model e.g the Butterworth-van Dyke (BVD) model which describes the mechanical and electric characteristics of the resonator [56].

Electric Field-Flow Fractionation In EFFF an electric field perpendicular to a hydrodynamic flow-field results in a separation effect of particles with respect to size and susceptibility to the field, i.e. due to mass density ( and electrophoretic mobility (µ) [57, 58] as illustrated in Figure 4. Subtypes of FFF dependent on a force gradient i.e. electrical [59, 60], thermal [61] and dielectrophoretic FFF [62, 63], could benefit from miniaturisation. The scaling effects in EFFF have been studied in detail by Gale et al. [64, 65]. Theoretically, advantages of higher electric field with decreased flow channel heights are expected and experimental studies of successful fractionation of nanoparticles have been reported [66-69]. Fouling of electrodes is a problem in DC electrochemical methods e.g. EFFF and DEP due to adhesion of particles, degradation of the electrode surface, or that the thin film electrodes are detached from the substrate sur-face as a result of interference in the film-substrate interface [70]. A minia-turised low cost EFFF-device with indium-tin-oxide (ITO) electrodes is pre-sented in paper VIII.

Figure 4. Schematic of the field-flow fractionation principle.

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The Lab-on-Chip concept

In this chapter, sample transport, manipulation and detection will be dis-cussed and some examples from research and commercial instruments are highlighted. In the last section, aspects of design, fabrication, and integration of the functions into a LOC are discussed.

Sample transport Transport of sample is important and necessary in microfluidic devices. However the main effort is often put on the separation or detection function as it is considered the more important and challenging task to accomplish, leaving the sample delivery to be done in a conventional way by available equipment. Flow injection analysis (FIA) [71] is a method where the profile of an injected sample plug is analysed in detail. The dispersion of the plug gives information mainly on diffusion of the sample particles.

Electroosmotic flow (EOF) is preferred when a non-dispersed sample plug is required but the applied high voltage, typically 500 V/cm, could dis-qualify the method from being integrated together with sensitive low voltage or high-frequency detection methods.

In the Biacore instrument, hydrodynamic sample and buffer delivery is used and the sample delivery is implemented in the sensor chamber [72]. The chamber has two inlets and one outlet. By controlling the buffer and sample flow, the sample propagates rapidly over the detection spots in the middle of the chamber in an ingenious fashion. This kind of “virtual valve” is a result of smart design, not by new microfabrication techniques or inte-gration of sub-parts, but by integration of functions in one simple chamber with three ports for inlet and outlet. Another example of a smart transport method is the pillar technology applied in the 4-cast chip consisting of an open channel structure with arrays of micro pillars [73]. The propagation of fluid along the channel is controlled by the distance between the pillars and the length of the channel.

The batch manufacturing possibilities, feasible through miniaturisation and lithography processes have been utilised to design and demonstrate mul-ti-channel networks. Simultaneous tests of several fluorescent markers and multiple tests for statistics have been demonstrated by Thorsen et al. in a PDMS-microsystem [74]. However, the use of a peripheral syringe pump

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system to deliver fluid into the micro device is still the most common sample delivery technique.

The flow behaviour of sample transport to a QCM-sensor surface is dis-cussed in paper VII, indicating the importance of sample transport effi-ciency. The design of the sensor and sensor chamber becomes a trade-off between a sharp sample plug distribution and potential sensor performance.

Manipulation and Detection In the research towards µTAS or LOC, a vast variety of methods for manipu-lation and detection are applied. The different methods used in this work will be compared with each other and optional methods and a number of related successful works with respect to function, method and applications are summarised. The different samples, i.e. beads and biological samples, e.g. cells, are often studied using the same methods.

The manipulation methods ECT, DEP, and EFFF are all used for sample preparation as described in the background section, while EIS, QCM, optical fluorescence microscopy, and UV-measurements are used for detection. Mass spectrometry (MS) is an excellent, though both expensive and time consuming detection method but is chosen not o be described in this sum-mary.

As capturing methods ECT and DEP could be comparable to acoustic [75, 76] and mechanical [77, 78] methods.

Dielectrophoretic impedance measurements (DEPIM) [79] and insulator-based dielectrophoresis (iDEP) [80] are related methods representing com-bined or further developed methods from the DEP-concept. Sample purifica-tion and separation by other FFF-methods [57, 81, 82], e.g. thermal and cy-clical EFFF stand for the competition against the EFFF-technology illus-trated in paper VIII. Liquid Chromatography (LC) [83] and the combination of LC and capillary electrophoresis (CE) combined into LCCE for MS-detection, presented in paper II, are examples of other alternative preparation methods.

Detection by QCM or Surface Plasmon Resonance (SPR) is used for simi-lar applications and have earlier (1995) been found to be comparable [84]. Methods for biomass monitoring, as alternative to EIS, have been suggested by Schügerl [85], e.g. conductivity measurements and optical density meas-urements.

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A Lab-on-Chip demonstrator Versatile open DEP-trap chips are described in paper III. The open system is uncomplicated so continuous measurements could be performed without interruption due to lengthy cleaning procedures or chip failures. However the performance and robustness of multi-functional fluidic chips will be en-hanced through technological and methodological development and we will see the commersial LOC to come true in the future. In agreement with this conviction, a multifunctional DEP-chip have been designed and fabricated where sample transport, separation/trapping and detection could be per-formed in the same chip as illustrated in Figure 5. Sample transport is to be performed by peripheral pumps in this case. The sample could focused in the centre of the channel by negative DEP before reaching the series of five manipulation electrode arrays, individually controllable via separate contact pads. In the end of the channel, detection electrodes are positioned, se figure 5d. The detection electrodes are identical to the ones used in paper IV, which enables for EIS on-chip detection. Optical on-chip detection is also possible through the transparent glass substrate by means of, e.g. UV-light or fluores-cence measurements.

Figure 5. Multifunctional DEP-chip: a) CAD-drawing including flow channel ge-ometry, bottom substrate with sample manipulation electrode pattern and lid sub-strate with focusing and detection electrodes. b) microscope picture of DEP-manipulated beads, diameter 0.5 and 2.0 µm respectively. c) picture of Aspergilluscells trapped between electrode pairs, electrode distance 100 µm. d) picture of elec-trodes for detection by impedance spectroscopy or conductivity measurements.

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The fluorescent beads and the Aspergillus cells seen in figure 5b and c, re-spectively, are trapped by positive DEP on identical electrode arrays as in the DEP-chip in Figure 6 but on an open device sealed by a cover glass. The fabricated device, described above, consisting of two glass substrates with electrode pattern is hold together by a spacer with a cut channel slit, as seen in Figure 6. The spacer, in this case, is a double coated adhesive tape of thickness 100 µm.

Figure 6. Photo of the assembled multifunctional DEP-chip (90x35x4mm) with fluidic interface for sample delivery via tubing and wiring for electrical connection to manipulation and detection instruments.

Fabrication issues From experience of MST-design over the years, my way of designing micro devices have progressed from building as complicated devices as possible, to design simple devices, and if possible, to avoid bonding processes. A col-league recently began her dissertation speech by saying

"…I have been accused to have chosen to study the bonding. That's not true! There are some in the audience that may disagree, but I would say that nobody choose to study bonding. At least not I! I would much rather have fabricated a lot of different fancy microchips for many analysis purposes with a lot of fabrication methods, chip materials, and coatings. But I was forced to study the bonding, because it was difficult. Either there was a leakage prob-lem or too low strength in the bond. The main conclusion from my work is: do not underestimate the problems with bonding!" [86]

The abbreviation KISS (Keep it simple, stupid) is a good guideline when designing micro devices. The word simple could mean many things. You can design a simple single, straight channel, e.g. a Flow Injection Analysis (FIA) device, and have a peripheral macro-detector and advanced data analysis

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methods. The use of open microdevices have the advantage of not needing to seal the channel and thus the cleaning process in between runs becomes faster although the disadvantages of sample evaporation, sample volume definition, and positioning is more problematic.

There is a need for a clean-room environment to produce micro electrode patterns but the rest of fabrication and assembly is suitable to be carried out in regular laboratory facilities. This advantage reduces cost and production time substantially.

A few selected examples of state-of-the-art microdevices for analysis is present below. Morgan et al. perform single nano particle impedance and fluorescence analysis in an excellent multi functional chip [28] even though the peripheral measurement system supporting the chip becomes complex. Baldwin et al. [87] have integrated a three-electrode electrochemical detec-tion, including a reference electrode, on-chip together with a CE separation function to enhance the measurement precision. By using insulating pillar arrays in a flow-channel to achieve a non uniform electric field in the fluid , the need of metal or other conducting electrodes inside the sample chamber is eliminated in the iDEP-device.[80]

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Summary and Outlook

The already mentioned benefits from miniaturisation, i.e. that it enables for faster analysis at a lower cost (labour, reagents/chemicals and instrument cost) is well known. In the mid 1990’s articles on new tools for miniturisa-tion were reported as latest news. Still, new tools are developed, mainly in polymers, but the applications have become the dominant development driver. As the miniaturisation could enable smarter integration and enhanced automation, the numbers of subparts included and needed to demonstrate an integrated microfluidic device have increased. The device design needs some careful thinking and the fabrication has to deal with process yield and ro-bustness aspects. The measurements set-up must meet integration require-ments with adequate software control of the different subparts to perform matched measurement sequences. The system hinted on here is no short-term one-person project.

The work behind this thesis takes no quantum leap towards the true LOC, but shows the ongoing continuous development. The work on LCCE, paper II, has made integration possible in a simple and low-cost PDMS-device. The planar electrocapture device, paper V, is not yet competitive to the ear-lier developed capillary device but the planar design opens for integration, e.g. multi-channel devices and branched channel designs not feasible with the earlier device. In the devices for ECT and EFFF in paper V and VIII the optical transparency of the used substrates enables for optical detection not possible before. The presented devices could be divided into open and flu-idic systems.

The systems studied are hybrid systems, meaning that some functions are miniaturised and some are carried out by conventional peripheral equipment. In the future the degree of integration will increase but the realistic devel-opment is that sub-functions need to be explored first, in order to identify further integration possibilities and benefits.

The status of the LOC-demonstrator project described above reflects the present state-of-the-art of LOC. Separate functions are demonstrated and the potential for integration is enabled by miniaturised devices or microfabrica-tion. The next step is to put the effort into integration of the sub-functions. There is a threshold to overcome in performing measurements to achieve quantitative results that are as accurate and reliable as those obtained by conventional instruments. This challenge is both positive and negative. The standard is set, which eliminates efforts in areas where miniaturisation is not

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advantageous or beneficial while in other situations ideas are not given enough time to grow in order to show their potential. However the share of articles including miniaturised analysis systems in chemical, medical, and biological publications will continue to increase.

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Summary of appended papers

In this section, the most interesting results from each paper are summarised.

Paper IChip Mounting and Interconnection in Multi Chip Modules for Space Applications.

In this paper, a process for fabricating a three dimensional multi-chip mod-ule for space applications is presented. The aim is to test the reliability as well and to discuss the process steps involved. Bisbenzocyclobuthane (BCB) fulfils the requirements for space qualification and has therefore been used as interdielectric material. Integrated circuit chips have been mounted and recessed in silicon using BCB as glue. Metal interconnects of thin film alu-minium are isolated with spin-on deposited BCB as an interlayer dielectric. Two layers of BCB, thickness 30 µm, are needed to achieve chip edge step coverage. Via interconnections, minimum dimensions 100 x 100 µm2 have successfully been patterned in a 30 µm thick BCB layer. Current-voltage characteristics were measured to validate the connections.

Paper IIOn-column polymer-imbedded graphite inlet electrode for capillary electrophoresis coupled on-line with flow injection analysis in a poly(dimethylsiloxane) interface.

A method for coupling an electrophoretic driven separation to a liquid flow, using conventional fused-silica capillaries and a soft polymeric interface is presented. A novel design of the electrode providing high voltage to the elec-trophoretic separation was also developed. The electrode consisted of a con-ductive polyimide/graphite embedded coating immobilized onto the capillary electrophoresis (CE) column inlet. This integrated electrode gave the same separation performance as a commonly used platinum electrode. The on-column electrode also showed good electrochemical stability in chronoam-perometric experiments. In addition, with this electrode design, the electrode position relative to the inlet end of the CE column will always be constant and well defined. The on-line flow injection analysis (FIA)-CE system was used with electrospray ionization (ESI)-time of flight (TOF)-mass spec-trometry detection. The preparation of the PDMS (poly(dimethylsiloxane)) interface for FIA-CE is described in detail and used for initial tests of the on-column polymer-imbedded graphite inlet electrode, see Figure 7. In this interface, a pressure-driven liquid flow, a make up CE electrolyte and a CE

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column inlet meet in a two-level cross (95 µm ID) in the PDMS structure, enabling independent flow characterization.

Figure 7. Schematic of the FIA-CE connection with the small contact area in the middle. The photos illustrates the PDMS cross section (upper left), and a sharpened fused-silica capillary (lower right).

Paper IIIA Study of Biological Particles in Bio-MEMS Devices using Dielectrophoresis.

Escherichia coli bacteria have been studied using different dielectrophoretic microsystems. In the range 10 kHz to 20 MHz, alignment, movement, pearl chains formation and rotation of bacteria have been observed, see Figure 8.

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a) b) Figure 8. Different behaviour of E. coli cells under influence of a DEP-field at dif-ferent frequencies, a) strong orientation at 2 MHz. b) Horizontal orientations at 20 MHz. The distance between opposite electrodes is 30 µm.

Paper IVBacteria counting with impedance spectroscopy in a micro probe station.

A new method to quantify the density of viable biological cells in suspen-sions is presented. The method is implemented by low-frequency impedance spectroscopy and based on the finding that immobilized ions are released to move freely in the surrounding suspension when viable Escherichia coli cells are killed by a heat-shock. The presented results show that an amount of ions corresponding to ~ 2 108 unit charges are released per viable bacte-rium killed.

A micro probe station with co-planar Ti electrodes was electrically char-acterized and used as measuring unit for the impedance spectroscopy re-cordings. This unit is compatible with common microfabrication techniques, and should enable the presented method to be employed using a flow-cell device for viable bacteria counting in miniaturized on-line monitoring sys-tems.

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Figure 9. Conductivity curves for glucose buffer and for three different concentra-tions of viable and dead E. coli solutions. The inset shows a magnification of the high-frequency part of the E. coli solution conductivities.

Paper VA planar polymer microfluidic electrocapture device for beads immobilisation.

A planar microfluidic electrocapture (ECD) device that captures and concen-trates beads by a local electrical field has been assembled and tested. The presented device also enables on-line observations of electrically manipu-lated beads in a micro channel. The design is based on a sandwich construc-tion from a glass substrate and a PMMA-lid, using a patterned polyimide spacer to form the channel, as illustrated in Figure 10.

Capture and release of 2 µm polystyrene beads, as seen in Figure 11, have been performed successfully at an applied potential of 300 V in 25 min. The captured sample plug and its parabolic shape is studied and discussed. The planar capture device design opens for using the electrocapture principle in future microfluidic tools, e.g. a fluidic switch.

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Figure 10. Cross section view of the planar capture device. The channel is seen between the glass slide and the PMMA lid part. Inlet and outlet connections and the ion selective membranes are integrated in the lid part.

Figure 11. Capture of beads and start of release of 2µm beads in the planar capture device. The channel height is 0.8 mm.

Paper VI Quartz crystal microbalance biosensor design: I. Sensor response and performance

This paper investigates a novel QCM biosensor with a small and rectangular flow cell along with a correspondingly shaped crystal electrode. The sensor was evaluated with impedance analysis and compared to standard circular sensor crystals and sensor crystals with small circular electrodes, see Figure12. Comparative QCM measurements on an antibody-antigen interaction system were carried out on the rectangular and standard circular sensor sys-tems. Impedance analysis and subsequent data extraction of the three differ-ent sensor crystals showed that the smaller sensors had significantly higher Q-values in air, but that liquid load on the electrodes lowered the Q-values radically for all crystals. Under liquid load, Q-values for the standard circu-lar and the rectangular sensors were similar whereas the Q-value for the small circular sensor was 50% higher. QCM experiments showed that the

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QCM system with rectangular crystal electrodes was fully functional in a liquid environment. The rectangular system showed higher and more rapid responses for series of antibody injections, albeit at a higher noise level than the standard system. The study elucidate a significant potential for improve-ment of sensor performance by optimising the sensor electrode size and shape together with the flow-cell geometry.

Figure 12. Photograph of the examined quartz crystals. The crystal diameter is 8 mm.

Paper VII Quartz crystal microbalance biosensor design II: Sample transport.

The influence of flow cell geometry on sample dispersion in a quartz crystal microbalance (QCM) biosensor system was investigated. A circular and a rectangular flow chamber and corresponding sensor electrodes were studied experimentally and modelled using a coupled Navier-Stokes and convection-diffusion model. Finite element simulations showed that dispersion phenom-ena in a flow cell can be significantly reduced with the rectangular flow cell compared to a circular system, as illustrated in Figure 13.

Experimental results from measurement of the time-dependent viscosity change of Dimethyl sulfoxide (DMSO) indicate that the sample delivery system has a predominant effect on the dispersion of the whole sensor sys-tem. Consequently, improvement of the sensor flow cell should be accompa-nied with improvement of the sample delivery system. With reference to kinetic studies of biological interactions, the current dispersion should have little effect on the results for studies of interaction pairs with relatively slow to normal binding rates such as antibody antigen interactions. Incentive for further development of the flow cell and sample delivery system exists pri-marily for applications with high reaction rates such as for certain receptor ligand interactions.

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Figure 13. Simulated sample plug profile showing normalized concentration for circular and rectangular geometries. The sample volume is 50 µL.

Paper VIII Design and fabrication of a miniaturised electrical field-flow fractionation system with ITO electrodes.

The design and fabrication of a micro EFFF-device with ITO electrodes is presented and retention of 50 nm PS-particles suspended in 100 µM NH4HCO3 is demonstrated. The choice of electrode material, microfabrica-tion issues and integration of sample injection and detection functions are addressed and discussed.

Figure 14. a) Schematic cross section view of the ITO µ-EFFF device with attached nanoport for inlet/outlet and electric connections. b) The channel geometry, width 5 mm, length 50 mm, height 50 µm, is cut out from the polymer spacer.

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Figure 15. The ITO µ-EFFF device assembled with nanoports, fused silica capillar-ies, and wires. The channel geometry, 50 mm long, is visible through the ITO-glass substrates. The device dimensions are 70x45x8 mm.

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Sammanfattning på svenska

Den engelska titeln på avhandlingen kan översattas till Mikrosystem för han-tering, behandling och detektion av partiklar och biomolekyler. Avhandling sammanfattar ett antal arbeten utförda i syfte att föra utvecklingen framåt mot förverkligandet av multifunktionella analysinstrument i mikroskala, så kallade Lab-on-Chip (LOC). Figur 1 visar ett mikrosystem med flera integre-rade funktioner. Strävan mot snabbare och bättre kemisk och biologisk ana-lys är drivkraften bakom utvecklingen av dessa mikrochip. Sjukvården och läkemedelsindustrin är exempel på användare som i slutändan kan dra fördel av effektiviseringen.

Komplexiteten hos ett mikrosystem ökar avsevärt när flera funktioner, t.ex. provtransport, provhantering och detektion, skall integreras på ett och samma chip. Detta förstärker behovet av klok och enkel design för att till-verka robusta och billiga mikrofluidala system.

I avhandlingen visas exempel på design, tillverkning och karaktärisering av mikrosystem som utför ovanstående funktioner. Genom enkel design och val av material som glas och plast är det möjligt att i stor utsträckning utföra tillverkningen utanför renrumsmiljö. Det mest kritiska steget vid tillverkning av fluidala mikrosystem är sammanfogningen av de olika delarna till ett slutet kanalsystem. Fogningsproblemet har lösts genom att använda sig av dubbehäftande polymerfilmer. I filmen definieras en öppning som bestäm-mer flödeskanalens geometri och tjockleken på filmen bestämmer kanalhöj-den. Genom användandet av öppna mikrosystem kan fogningsproblematiken undvikas.

Ett planart Electrocapture-instrument har konstruerats (artikel V) i vilket polymerpartiklar, beads, med diameter 2 µm har immobiliserats, fångats, med elektriska krafter och sedan släppts iväg. Den planara designen möjlig-gör vidare integration av funktioner.

Retention, fasthållande, av nanopartiklar av polymer med diameter 50 nm har utförts medels elektrisk fältflödesfraktionering i ett system med transpa-ranta tennoxidelektroder (ITO) (artikel VIII). UV-detektion tillämpades utanför chippet men de genomskinliga elektroderna möjliggör detektion direkt på chippet med t.ex. fluorescensmikroskopi.

PDMS är ett silikongummimaterial som används mycket för att gjuta ka-nalstrukturer i mikroskala. Ett fyrvägskors gjutet i PDMS presenteras i arti-kel II. Kanalkorset gör det möjligt att kombinera två separationsmetoder,

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vätskekromatografi och kapillärelektrofores, och på så sätt erhålla en förbätt-rad separation.

I ett kommersiellt analys-instrument (Attana 80 Bioanalyzer) vars sensor består av en kvartskristall-resonator (QCM) utvärderas en rektangulär sen-sorgeometri och jämförs med en traditionell rund geometri. Prestandan ut-värderats med hjälp av impedansanalys. I artikel VI utförs analyser av de båda geometrierna som påvisar högre känslighet för den rektangulära sen-sorn. Vid vätskebelastning uppmättes likvärdiga Q-värden för både rek-tangulär och cirkulär sensor. Provtransporten i sensorkammaren studeras i artikel VII genom modellering och experimentella mätningar. Simulerings-resultaten påvisar signifikant mindre dispersion av en injicerad fyrkantplugg för den rektangulära sensorn, medan experimentella resultat däremot uppvi-sar liten skillnad i dispersion. Diskrepansen i resultaten beror troligtvis på att dispersionen i hela systemet inte beaktas i modelleringen.

En metod att räkna bakterier eller celler baserad på konduktivitetsmät-ningar med impedansspektroskopi har framgångsrikt verifierats genom att detektera skillnader i konduktivitet mellan levande och döda E. coli-cellermed en mikroprob (artikel IV). Metoden kan tillämpas för snabb övervak-ning av cellodling vilket är eftertraktat av läkemedelsindustrin.

Sammanfattningsvis ger det presenterade arbetet flera exempel på an-vändbar design och lämpliga metoder för tillverkning av mikrosystem. Med användande av kunskap inom mikrosystemteknik har instrument i mikroska-la för hantering och detektion av biologiska prov demonstrerats och för-hoppningsvis bidragit till att ett steg till kunnat tas mot användbara Lab-on-Chip.

Figur 16. Multifunktionellt mikrosystem (90x35x3mm) för fokusering, separation och detektion av partiklar och biomolekyler.

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Acknowledgements

Professor Sören Berg is gratefully acknowledged for accepting me as Ph. D. student in the beginning and Professor Ilia Katardjiev for taking over at the finish-lineUlf Lindberg is gratefully acknowledged for being my supervisor, for having trust in me, introducing me to all interesting projects, all joyful discussions, and for letting me see the commercial side of research. Direktör Bertil Hök, thank you for invaluable help with strategy and coach-ing in the last hour

The Grand old Mik-Mek group, Kerstin, Johan B, Henrik, Örjan, Kjell, Carola, Ylva, Carolina, Johan K, Pelle, Mattias, Rostis, Bo, …..... it was you who attracted me to get stuck in academia. Thank you! I miss you all! En epok går nu i graven! Hej då Mik-Mekmacken. Nu finns du inte mer…… Special thanks to the great friends: Kerstin and Bejjan for always being around somewhere. The encouragement from Dr. Kratz is acknowledged, Johan A. for leading the way, and Hanna for being Hanna. A big hug to all current and former members of the Solid state electronics department and the other MST-groups at the Ångström laboratory who have contributed to the friendly atmosphere. “High five” to the “space-bunch” at ÅSTC for being so cool. Thanks to the MSL-staff for taking over the lab-responsibility, especially to Angelo for your dedication to keep the pattern generator alive.

Thank you Henrik Anderson, at Attana AB, for showing determination at late hours. Essential additional help from Lars, Ventsi, Johan K. and Örjan was very much appreciated Ett glatt tack till DEP-folket, Gunnar, Fredrik, Larsa, Sven, Ylva och Johan R., för ett mycket trevligt och givande projekt. Juan Astorga-Wells, Gerald Jesson, Tomas Bergman, and Hans Jörnvall for the Capture Device journey. It’s a success! Maria Strömme and Ken Welch, Thank you for adopting my initial idea and for letting me take part in the high quality and high speed research project we launched.

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Thank you Sara B, Jenny, Oliver for collaboration in the LCCE-project, and Karin Caldwell, Rik ter Veen and Adam Feiler for the effort to towards frac-tionation.Leif Nyholm is gratefully acknowledged for contributing with the electro-chemistry competence needed in most of my projects and appreciation to Jan Obertur for building the measurement set-up.

Tack till Pär Fernkvist för E. coli bakterier, praktisk cellkunskap o schyssta passar.Stefan Tiensuu is remembered for a very fruitful discussion. The enthusiasm of Gerald Jesson is appreciated. You deserve to become a Ph. D. in the future.

Tusen kramar o tack till Sara, utan dig hade jag inte orkat kämpa på och tro att det skulle gå vägen. Hej o hå, stå på tå!

Mor o Far, tack för att ni givit mig den där självklara tryggheten som alltid finns där.

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ACTAUNIVERSITATISUPSALIENSISUPPSALA2006

AbstractList of papersComments on the author’s contribution to appended papersRelated work not included in this thesis

ContentsAbbreviationsPrefaceIntroductionObjectivesBackground and MethodsMicro Structure TechnologyElectrocapture TechnologyDielectrophoresisElectric Impedance SpectroscopyQuartz Crystal MicrobalanceElectric Field-Flow Fractionation

The Lab-on-Chip conceptSample transportManipulation and DetectionA Lab-on-Chip demonstratorFabrication issues

Summary and OutlookSummary of appended papersSammanfattning på svenskaAcknowledgementsReferences