Design and production of polymer based miniaturised bio …€¦ · 3.4.1 Introduction to Surface Modification 43 3.4.2 Surface modification for bonding 44 3.4.2.1 Adhesion Promoting

Design and Production of Polymer Based

Miniaturised Bio-analytical Devices

Sebastiaan Garst

B. Eng (Mech)

Submitted for the degree of:

Master of Engineering

Industrial Research Institute Swinburne University of Technology

Melbourne Australia

2007

Abstract

The aim to provide preventive healthcare and high quality medical diagnostics and

treatment to an increasingly ageing population caused a rapidly increasing demand for

point-of-care diagnostic devices. Disposables have an advantage over re-usable units

as cross-contamination is avoided, no cleaning and sterilising of equipment is required

and devices can be used out of centralised laboratories. To remain cost-effective, costs

for disposables should be kept low. This makes polymer materials an obvious choice.

One method for the realisation of fluidic micro devices is the stacking of several

layers of microstructured polymer films. Reel-to-reel manufacturing is a promising

technique for high-volume manufacturing of disposable polymer bio-analytical

devices. Polyethylene terephthalate (PET) and cycloolefin copolymer (COC) were

selected as suitable polymer substrate materials and polydimethyl siloxane (PDMS) as

membrane layer.

Bonding of polymer films with the help of adhesives carries the risk of channel

blocking. Despite this drawback, no other method of bonding PDMS to a structural

layer could be identified. Bonding with solvents avoids channel blocking issues, but

adversely affects biocompatibility.

Thermal diffusion processes enable bonding of COC and PET without the use of any

auxiliary material. The extensive process times requires for thermal diffusion bonding

can be considerably shortened by pre-treating the material with plasma or UV

exposure. Welding with the use of a laser energy absorbing dye was demonstrated to

be particularly suitable for selective bonding around channels and reservoirs.

None of the assessed bonding methods provide a generic solution to all bonding

applications. Instead, the selection of an appropriate technique depends on the

intended application and the required level of biocompatibility. Since this selection

has implications on the feasibility and reliability of microfluidic structures on the

device, design rules which ensure design for production have to be established and

followed.

iii

Acknowledgements

This work would not have been written without the help and support of a number of

colleagues and friends. First and foremost I would like to thank Dr Matthias

Schuenemann for providing lots of support and guidance in his role as supervisor. His

excellent suggestions about the structure and contents in this work have been

invaluable. Additionally, I am very grateful for his assistance in preparing

publications and presentations.

A big thanks also goes out to Dr Matthew Solomon. Matt has been a great support

during most of the practical work that was carried out to complete the research in this

thesis. Furthermore his suggestions and feedback about the contents of the thesis have

been very helpful and well appreciated.

To Prof. Erol Harvey, for giving me the initial opportunity to work in his research

group as undergraduate student and afterwards for his support and encouragement to

start the work that is presented in this thesis.

A special thanks to all colleagues and friends at IRIS Scoresby and MiniFAB. Dr

Jason Hayes for being a critical but very helpful friend, Micah Atkin for his expertise

and many helpful discussions and Dave Wynne and Carl Bottcher at Artimech for

providing lots of complimentary engineering input and materials when needed.

This work would not have been carried out without the financial support of the

Cooperative Research Centre for Microtechnology for which I am grateful.

Sincere thanks to my parents for the great way they raised me, for their unconditional

support and encouragement and their understanding while completing this task on the

other side of the world.

I especially would like to thank my partner, Esther Engelage, for her patience, love,

encouragement and understanding during many weekends and late evening that were

spent putting this work together. Without her support this work would not have been

completed.

iv

Declaration

This thesis contains no material which has been accepted for the award of any other

degree or diploma, except where due reference is made in the text of the thesis. To the

best of my knowledge, this thesis contains no material previously published or written

by another person except where due reference is made in the text of the thesis.

Signature of Candidate:

Sebastiaan Garst

Dated:

Contents

ABSTRACT II ACKNOWLEDGEMENTS III DECLARATION IV LIST OF ABBREVIATIONS VII 1 INTRODUCTION 1 2 MINIATURISATION AND BIO-ANALYTICAL DEVICES 2

2.1 EMERGING POINT-OF-INTEREST APPLICATIONS IN BIOTECHNOLOGY 2 2.2 BIO-ANALYTICAL PROCESSES 3 2.3 MINIATURISATION TECHNOLOGIES & MICROMACHINING 5 2.4 MINIATURISED BIO-ANALYTICAL DEVICES 6 2.5 MATERIALS FOR BIO-ANALYTICAL DEVICES 8 2.6 CURRENT CHALLENGES FOR POLYMER BASED BIO-ANALYTICAL DEVICES 8 2.7 CURRENT CHALLENGES IN PACKAGING OF POLYMER BASED BIO-ANALYTICAL DEVICES 10

3 STATE OF ART 12 3.1 INDUSTRIAL MICROMANUFACTURING TECHNOLOGIES 12

3.1.1 Basic Cost Analysis 12 3.1.2 Requirements for Fabrication of Polymer Bio-analytical Devices 13 3.1.3 Fabrication Strategies for Polymer Microdevices 14

3.1.3.1 Laboratory Based Micro-Fabrication Techniques 14 3.1.3.2 Small Scale Fabrication and Prototyping of Polymer Microdevices 16 3.1.3.3 Medium Scale Manufacturing of Polymer Micro Devices 17 3.1.3.4 High-volume Manufacturing of Polymer Micro Devices 17 3.1.3.5 Overview of Scales in Manufacturing Capacity 23 3.1.3.6 Overview and Selection of High Volume Manufacturing Strategies 24

3.2 MATERIAL SELECTION FOR POLYMER BIO-ANALYTICAL DEVICES 25 3.2.1 Substrate Materials 25

3.2.1.1 Requirements for Polymeric Substrate Materials 25 3.2.1.2 Polymeric Substrate Materials in Microtechnology 27

3.2.2 Membrane Materials 29 3.2.2.1 Actuation 29 3.2.2.2 Requirements for Polymeric Membrane Materials 30 3.2.2.3 Polymeric Membrane Materials in Microtechnology 30

3.3 BONDING AND SEALING 32 3.3.1 General Bonding Theory 32 3.3.2 Mechanical Interlocking 32 3.3.3 Electronic Theory 33 3.3.4 Theory of Weak Boundary Layers and Interfaces 34 3.3.5 Adsorption Theory 36 3.3.6 Diffusion Theory 37 3.3.7 Chemical Bonding Theory 38 3.3.8 Current Bonding Methods in Miniaturised Polymer Assemblies 40

3.4 SURFACE MODIFICATION TECHNOLOGIES 43 3.4.1 Introduction to Surface Modification 43 3.4.2 Surface modification for bonding 44

3.4.2.1 Adhesion Promoting Surface Modification Techniques in Industry 44 3.4.2.2 Adhesion Promoting Surface Modification Techniques for Polymer Micro Analytical

Devices 46 3.4.2.3 Challenges in Surface Modification to Promote Adhesion in Microdevices 48

3.4.3 Surface Modification Techniques to Control Reactions in Microfluidic Devices 49 3.4.4 Surface Modification Techniques to Decrease Permeability 52 3.4.5 Challenges in Surface Treatment of Polymer Based Bio-analytical Devices 55

4 EXPERIMENTAL METHODOLOGY 56 4.1 MATERIAL SELECTION 56 4.2 SUBSTRATE CLEANING 59 4.3 SURFACE MODIFICATION 59

4.3.1 Introduction 59 4.3.2 UV-Irradiation 59 4.3.3 Wet Chemical Treatment 61 4.3.4 Characterisation of Modified Surfaces 62

4.4 BONDING TECHNIQUES 65 4.4.1 Bonding Systematics 65 4.4.2 Thermal Diffusion Bonding 67 4.4.3 UV Assisted Bonding 69 4.4.4 Chemical Etching 70 4.4.5 Bonding of composite substrate materials 70 4.4.6 Solvent bonding 71 4.4.7 Laser Welding 72

4.4.7.1 Laser Welding Techniques 72 4.4.7.3 Reverse Conductive Welding 73 4.4.7.4 Laser Welding with Use of an Absorbing Dye 74

4.4.8 Adhesive Bonding 76 4.5 BOND CHARACTERISATION 77

4.5.1 Test Methods 77 4.5.2 Bond Interface Imaging 78 4.5.3 Bond Strength Measurements 78

4.5.3.1 Tensile testing 78 4.5.3.2 Wedge Cleavage Testing 81 4.5.3.3 Peel Testing 82 4.5.3.4 Pressure Testing 82

4.5.4 Application specific testing 84 4.5.5 Design for Pressure Testing 84 4.5.6 Durability Testing 86

5 EXPERIMENTAL RESULTS AND DISCUSSION 88 5.1 MATERIAL SELECTION 88

5.1.1 Substrate materials 88 5.1.2 Membrane Materials 93

5.2 BONDING 96 5.2.1 Thermal Diffusion Bonding 96

5.2.1.1 Thermal Diffusion Bonding of PET 96 5.2.1.2 Crystallinity in PET 99 5.2.1.3 Thermal Diffusion Bonding of COC 100 5.2.1.4 Thermal Diffusion Bonding between Substrates and Elastomers 104

5.2.2 UV-Assisted Bonding 104 5.2.3 Chemical Etching 113 5.2.4 Bonding of Composite Substrate Materials 116 5.2.5 Solvent Welding 118 5.2.6 Laser Welding 122

5.2.6.1 Through Transmission Laser Welding 122 5.2.6.2 Reverse Conductive Welding 124 5.2.6.3 Laser Welding with the use of an Absorbing Dye 126

5.2.7 Adhesive Bonding 133 5.2.8 Summary Results Bonding Experiments 135

5.3 DESIGN RULES 137 6 CONCLUSIONS AND OUTLOOK 140

6.1 MANUFACTURING 140 6.2 MATERIALS 140 6.3 BONDING 141 6.4 DESIGN RULES 146 6.5 SUGGESTIONS FOR FUTURE WORK 146

7 REFERENCES 148 8 APPENDICES 156 APPENDIX 1: PRESSURE TESTING OF DIFFUSION BONDED PET 156 APPENDIX 2: HOT LAMINATION OF COC 157

vii

List of Abbreviations

General Abbreviations

DNA Deoxyribose Nucleic Acid

ELISA Enzyme Linked Immuno Sorbent Assay

IR Infrared

LOC Lab on a Chip

MEMS Micro Electromechanical System µµµµTAS micro Total Analysis System

Nd:YAG Neodymium Yttrium Aluminium Garnet

PCR Polymerase Chain Reaction

POC Point-of-Care

POI Point-of-Interest

RCW Reverse Conductive Welding

RNA Ribose Nucleic Acid

TTLW Through Transmission Laser Welding

UV Ultraviolet

viii

Abbreviations of Polymers

ABS Acrylonitrile-butadiene-styrene

ACM Acrylic Rubber

BR Butadiene Rubber

CA Cellulose Acetate

CAB Cellulose Acetate Butyrate

CIIR Chlorobutyl Rubber

COC Cyclo Olefin Copolymer

CPE Chlorinated Polyethylene

CR Polychloreprene

CSM Chlorosulphonated Polyethylene

ECO Epichlorohydrin

EPDM Ethylene Propylene-diene monomer

EPM Ethylene Propylene Rubber

FVMQ Fluorosilicone rubber

HDPE High Density Polyethylene

IIR Butyl Rubber

IR Isoprene Rubber

LDPE Low Density Polyethylene

NBR Nitrile Rubber

NR Natural Rubber

PA Polyamide

PBT Polybutylene Terephthalate

PC Polycarbonate

PDMS Polydimethylsiloxane Rubber

PEEK Polyetheretherketone

PEI Polyetherimide

PET Polyethylene Terephthalate

PETG Polyethylene Terephthalate -Glycol

PI Polyimide

PMMA Polymethylmethacrylate

PMP Polymethylpentene

POM Polyoxymethylene

PP Polypropylene

PPOX Polypropylene oxide

PS Polystyrene

PSU Polysulfone

PTFE Polytetrafluoroethylene

PVC Polyvinyl Chloride

PVDC Polyvinylidene Chloride

PVDF Polyvinylidene Fluoride

RTVS Room Temperature Vulcanizing Silicone

SBR Styrene Butadiene Rubber

SU-8 Photo-curable epoxy

TPE Thermo Plastic Elastomers

1 Introduction 1

1 Introduction

A growing interest in disposable point-of-care diagnostic devices has prompted

developments in the fabrication of polymer microfluidic devices. Currently, only very

limited products classifiable as polymer bio-analytical devices are commercially

available. Particularly devices with a high level of complexity have not yet made a

market entry. The limited availability of polymer microfluidic devices is primarily

due to significant fabrication challenges.

The scope of this thesis is to develop a fabrication method for miniaturised polymer

bio-analytical devices. Fabrication elements that need addressing are design for

production, manufacturing strategies, material selection and technology aspects like

bonding and biocompatibility. Challenges standing in the way of high-volume

manufacturing need identifying and solutions to these challenges are to be defined by

researching and developing appropriate techniques.

The work in this thesis is structured in a number of steps (see figure 1.1). Firstly,

miniaturisation is combined with bio-analytical processes and polymer materials in a

short overview in chapter 2. This chapter summarises challenges that arise from

miniaturising bio-analytical processes in polymer materials. Chapter 3 reviews the

current state of technology in respect to the formulated challenges. Outcomes derived

from this review function as input for the experimental methodology section in

chapter 4. Experimental results are analysed and outcomes are reviewed for use in

manufacturing techniques in chapter 5. The work is concluded with a summary of

outcomes as well as recommendations for future work in chapter 6.

Figure 1.1: Structure of work

Definition of scope and aim of work

Overview of miniaturisation and bio-analytical processes

Identification of challenges in the fabrication of miniaturised bio-analytical devices

Analysis of current state of technology in respect to the formulated challenges

Definition of experimental methodology

Experimental work

Analysis of experimental outcomes

Recommendation for fabrication

2 Miniaturisation and Bio-analytical Devices

2

2 Miniaturisation and Bio-Analytical Devices

2.1 Emerging Point-of-Interest Applications in Biotechnology

Currently, medical testing as well as food and water pathogen testing are carried out

in central laboratories at a low cost per analysis. The results of these test generally

become available within a few hours. However, shipping the sample, allocating

testing time, reviewing the outcomes and responding back to the point of interest add

to a significant delay in time and obstruct a rapid diagnosis [Tüdıs 2001]. In addition

to delaying medical interventions the wait for test results is an added expenditure in

other industries. For instance, the meat industry in the US is obliged by the US

Department for Agriculture to keep raw meat products in stock until test results are

confirmed [Alocilja 2003]. With tests being carried out in centralised laboratories,

stock is retained for 24 hours or longer, reducing cash flow and shelf life of the

product (see also figure 2.1 [Schuenemann 2004]).

In medical applications, the demand for rapid diagnosis and health monitoring to

assist in medical decision making is even more present. Additionally, the growing

awareness of personal health and healthy lifestyles introduces an increasing demand

for health monitoring at a low cost. Portable bio-analytical devices have the potential

to shift diagnostic testing from a centralised laboratory to the point of interest.

Figure 2.1: Process flow of pathogen testing at central laboratory level and point-of-interest level

Sample taken Test ordered by customer

Sample labelled and stored Sample transported to lab

Sample sorted to analyzer Sample prepared

Results reviewed by lab staff Sample analyzed

Action taken Results reported to customer

Sample taken Test initiated

Results reviewed Sample analysed

Action taken

Cen

tra

l L

ab

ora

tory

Po

int

of

Ap

pli

cati

on


3

2.2 Bio-analytical Processes

A basic understanding of bio-analytical processes is essential for a successful

development of manufacturing techniques for bio-analytical devices. For that purpose,

two generalised process families are reviewed: Immunosensing and genosensing.

Immunosensing is the analysis of antibodies, antigens and haptens. An antibody is a

protein used by the immune system to identify and neutralise unwanted objects such

as bacteria and viruses. Antigens stimulate an immune response and in particular the

production of antibodies. Antibodies have a high affinity towards their specific

antigens and haptens. This affinity can be used to detect the presence and

concentration of targeted antibodies, antigens or haptens in a sample. The affinity

binding can be detected in an analytical assay using a label. The label is a functional

molecule that is bound to one of the reactants and can be detected when a specific

binding took place. A large variety of labels is available for this purpose. Most

popular label assays are Enzyme-Linked ImmunoSorbent Assays (ELISA) where one

component of the assay is bonded to a substrate [Thijssen 1985].

Step 1: Coat well with capture antibody

Step 2:Wash off excess antibodies

Step 3: Add sample containing antigens

Step 4: Wash off excess sample

Step 5: Add second antibody (detection)

Step 10: Measure colour change

Step 9: Add colouring substrate

Step 8: Wash off excess antibodies

Step 7: Add enzyme labelled antibody

Step 6: Wash off excess antibody

Figure 2.2: Process flow for a sandwich type ELISA

An ELISA processes consists of a number of processing steps (see figure 2.2). The

process is started by washing sample material over a pre-treated well. Further washing

steps are aiming at removing excess sample material and attaching label enzymes to


4

the immobilised sample antibodies or antigens. The labelled components are detected

by the enzyme emitting a chromogenic or fluorescent signal which can be quantified

using a spectrophotometer or optical device.

Another bio-analytical process is genosensing, which is the analysis of deoxyribose

nucleic acid (DNA) or ribose nucleic acid (RNA). DNA is a nucleic acid that contains

the genetic instructions for the development of cellular organisms and most viruses.

DNA molecules generally appear in a double helix formation, whereas RNA is found

in a single strand, acting as template to form DNA.

DNA and RNA detection can be used for diagnostic detection of pathogens, bacterial

infections, genetic diseases and viral infections. For an analysis to be reliable, a

sufficient amount of target DNA needs to be present in the sample. Often samples

only contain very small amounts of target DNA. Therefore the DNA or RNA needs

amplifying before analysis can take place. One technique that enables amplification is

Polymerase Chain Reaction (PCR) [Mullis 1990]. PCR is a reaction method where

DNA molecules are multiplied exponentially using a DNA polymerase enzyme,

nucleotides, primers and buffer solutions. In addition, a pre-defined temperature

cycling protocol is required to control the reaction (see figure 2.3).

PICTURE REMOVED in ELECTRONIC VERSION

(Copyright)

Figure 2.3: PCR cycle. Step 1: Melting, denaturation breaks the double strands; Step 2: Annealing, primer binding; Step 3: Elongation during which DNA polymerase complements the strand. [From Van Gerwen 2000]


5

The thermal cycling protocol consists of three steps. The first step, melting (94 oC –

98 oC), denatures the DNA molecule, resulting in single stranded DNA. At a

temperature of 37 oC – 65 oC primers bind at the wanted location of the DNA strand,

called annealing. A further increase to 72 oC, (for Taq polymerase) allows nucleotides

to complete the DNA to a double strand (elongation). After this step, the cycle starts

again, theoretically doubling the number of DNA molecules per cycle. Typically, 20-

30 cycles are carried out to replicate enough DNA for detection.

After cycling, the amplified DNA molecules need detection to finish the analytical

process. Generally, PCR end-products are analysed using electrophoresis or by

hybridisation probes. Electrophoresis is a method of separating ionic species by their

charge and frictional forces. A typical electrophoresis technique is capillary

electrophoresis, allowing the separation of DNA species based on their size to charge

ratio in the interior of a small capillary filled with an electrolyte. Separated DNA

molecules can be detected in the capillary using UV or fluorescent techniques.

Hybridisation probes are short pieces of DNA that are labelled with a tag (optically or

radioactively detectable). A hybridisation step is carried out at denaturing

temperatures, forcing DNA molecules into single strands. The known sequence of the

probe can bind to complementary DNA strands during the hybridisation. A positive

binding results in the probe returning a positive signal (optically or radioactively

detectable). Often probes are part of a large array (1000x1000) enabling multiplexing

of DNA analysis [Fodor 1993].

2.3 Miniaturisation Technologies & Micromachining

Reducing physical dimensions of products is generally not the main driving force

behind miniaturisation. For instance, in the 1960s it was understood that by arraying

large numbers of micro-sized transistors on a single chip, microelectronic circuits

could be built that dramatically improved functionality, performance, and reliability,

all while reducing cost and decreasing volume. It is these advantages of

micromachining over conventional machining that fuelled the successful

miniaturisation of many products.


6

For years, microtechnology was primarily associated with the microelectronics

industry. Photolithography was used to define micro-sized features on silicon wafers.

It was only later that the field of micro electromechanical systems (MEMS) emerged.

MEMS products feature three-dimensional structures that also fulfil a mechanical

function, e.g. accelerometers, gyrometers, pressure sensors and inkjet nozzles. MEMS

products are typically manufactured using planar processing techniques. Processing

techniques include surface and bulk micromachining of glass or silicon substrates

[Madou 2002].

Although the microelectronics and MEMS industry contributed to significant cost

savings using miniaturisations techniques, materials and fabrication methods are still

complex and rather expensive. Emerging developments in polymer microfabrication

technology are aiming at a further cost reductions in MEMS devices [Becker 2000].

Polymer replication methods such as injection moulding and embossing are

particularly suited for microfluidic applications. A successful establishment of

microfabrication methods in polymers potentially opens up a huge market for

disposable miniaturised products.

2.4 Miniaturised Bio-analytical Devices

The drive to miniaturise bio-analytical devices has significantly developed during the

last decades. Despite the successful miniaturisation of a gas chromatograph in the 70’s

[Terry 1979], it was only in 1990 when the first Micro Total Analysis Systems

(µTAS) paper was published [Manz 1990]. Since then the field of µTAS, often

referred to as Lab on a Chip, has seen an exponential growth.

Miniaturisation forms a key aspect of point-of-care/point-of-interest bioanalytical

devices. In addition to allowing for portability of the equipment, the reduction in size

permits lower costs per diagnostic test as sample and reagent volumes drop. Another

benefit of miniaturising bio-analytical assays is the higher achievable analytical

performance that is facilitated by the different fluid dynamics present in microfluidic

devices [Kricka 1998]. As a consequence of changing fluid dynamics, however, fluid


7

handling in the microfluidic domain can not be performed by simple miniaturisation

of conventional analytical processes. In addition it is important to realise that that a

high degree of miniaturisation has a significant effect on the surface- to-volume ratio

that which is at least an order of magnitude higher than in conventional micro-tubes

used in processes like polymerase chain reaction. At a certain miniaturisation level,

the increased influence of surface effects may well upset the advantages gained by

miniaturisation.

To ensure a reliable analytical process, the size of the sample to test needs to match

the expected concentration of target molecules or colony-forming units in the sample

(see figure 2.4). The absence of sufficient target molecules in a sample reduces the

statistical probability of target detection, prohibiting the reliability of the test. This

requirement puts a limit to the level of miniaturisation and in particular of pathogen

DNA testing devices [Schuenemann 2004].

1 l10 l

-15 10

-12 l 10

-9 l 10

-6 l 10

-3 l

One m

olecule per sample

Sample Size

Con

cen

trati

on

of

Targ

et

Mole

cule

in

Sam

ple

Figure 2.4: Sample size vs. concentration of target molecules


8

2.5 Materials for Bio-analytical Devices

Initial developments of miniaturised bio-analytical systems were based on techniques

adapted from silicon micromachining technologies. Devices were realised in either

glass [e.g. Lagally 2000, Lagally 2001] or silicon [e.g. Northrup 1993, Northrup

1998]. Although well established for microtechnology, these materials and associated

fabrication techniques have certain disadvantages, including high material costs, large

number of fabrication steps, geometrical limitations and undesired surface chemistry

[Becker 2000].

The very high cost of silicon and glass based analytical devices dictates extensive

cleaning after testing in order to reuse the expensive device. Such a cleaning step

further adds cost to each diagnostic test. More importantly, re-use carries the risk of

cross-contamination, affecting the reliability of testing. To avoid this risk, the test

device must be disposed of after use. However, to allow for cost-effective disposables,

alternative materials and associated fabrication methods are required.

Polymer substrate materials have been accepted as a promising substitute to glass and

silicon. Their low cost and their wide range of properties open up a great potential for

the realisation of cost-effective disposable analytical devices. Although significant

developments in micro-fabricating polymer devices have been reported [Becker

2000], considerable research is required to unite materials, fabrication techniques and

biological processes.

2.6 Current Challenges for Polymer based Bio-analytical Devices

A wide range of substrate polymers have been used to fabricate prototypes of

microfluidic devices. For bio-analytical devices, the choice of polymer materials is

determined by the more strict requirements for these devices. For instance, the

majority of published sensor concepts in miniaturised analytical devices utilise optical

detection technologies [Auroux 2002]. Substrate polymers must be optically

transparent at the wavelengths utilised for optical detection techniques. Only a few

polymer materials can be identified that meet this requirement.


9

The interaction of substrate polymers with reagents and samples in microfluidic

devices is generally large due to high surface-to-volume ratios. The diagnostic

integrity of a device is therefore heavily influenced by material properties such as

water vapour permeability, water adsorption and bio-compatibility. Substrate

materials with suitable material properties need selecting to withstand reduced

analytical performance or even process failure. Alternatively, surface treatment

methods need identifying to counteract undesired materials properties.

The commercial success of a disposable bio-analytical device is dependant on the

establishment of a cost-effective manufacturing process. Important elements of

polymer bio-analytical device manufacturing are polymer structuring and bonding

techniques. Several polymer micro-structuring methods have been explored. Amongst

them are a number of replication processes including hot-embossing, (micro)-

injection moulding and thermoforming [Becker 2000, Schuenemann 2004]. In other

processes, laser cutting has been applied to form channels and structures in thin

polymer layers [Roberts 1997, Atkin 2003]. This approach has proven to be more

flexible and versatile and therefore very suitable for prototyping purposes and

medium scale production. Further investigations are required into alternative

structuring techniques, for instance die-cutting of thin films.

To enable the formation of three-dimensional microfluidic circuits and devices,

microstructures like channel and reservoirs need to be closed after structuring. This

sealing and bonding of channels by a capping layer has to be carried out without

clogging channels, changing geometries or changing surface chemistry. Lamination

has been demonstrated to cap one layer devices with the use of a thin polymer film

[Roberts 1997]. Other published methods include laser welding, adhesive bonding

and thermal diffusion bonding [Becker 2000]. All of these methods represent a big

challenge for use in high volume manufacturing. Additional bonding methods

therefore need exploration to ensure a commercially viable production method for

miniaturised bio-analytical devices.


10

2.7 Current Challenges in Packaging of Polymer Based Bio-

analytical Devices

Packaging is one of the most important aspects of micro-engineered parts and is often

the deciding factor between a commercially successful product and a laboratory

prototype. A commercially viable design for polymer point-of-care analytical systems

is dependant on a smart division between disposable and reusable system parts.

Expensive elements (e.g. thermal management, actuators and detection sensors)

should be part of the re-usable host device, while disposable parts are kept as simple

as possible to ensure a cost-effective test cartridge.

A major packaging challenge for the disposable cartridge is fluid handling and

control. Since micro-pumps and micro-valves are in direct contact with samples and

reagents, they cannot be separated from the disposable. To achieve cost-effective

pumping on board the analytical device, the pump chamber (to be integrated into the

disposable) needs spatial separation from the pump actuator (to be integrated into the

non-disposable host device). Actuation of the pump chamber can for example be

realised by pneumatics. In this approach the pneumatic circuitry forming part of the

disposable while the pneumatic source is kept external. This displacement method can

be used to form a peristaltic pump consisting of three pump chambers actuated in an

appropriate sequence [see also Schuenemann 2004]. Such pumping method relies on a

flexible membrane that deflects into the pump chamber upon actuation. Integrating a

polymeric membrane into the fluidic package requires a careful selection of suitable

materials and bonding techniques.

To cater for both pneumatics and microfluidics, several levels of microchannels are

required within the device. A promising method to realise multilayer packages is by

stacking and bonding several layers of microstructured films (see figure 2.5). In

addition to being a cost-effective method for fabricating disposables, this approach is

very well suited for high-volume manufacturing [Mehalso 2001].


11

fl_hole

fl_pipemembranepn_pipepn_linepn_hole

Microfluidic circuit

Pneumatic distribution

Figure 2.5: Multilayer package fabricated from vertically assembled polymer sheets

The architecture of multilayered microfluidic devices can become rather complex

when a large number of functions are integrated over a number of layers. The design

of the individual layers of the package has to meet a large number of constraints to

ensure functionality without compromising reliability or manufacturing costs. For

instance, non-disposable items such as heaters and sensors are not part of the

packaging but are placed at the interface between host and disposable. This separation

forces the package designer to ensure a good thermal conductive path while reducing

the influence of the thermal capacity of the polymer substrate. The observation of

strict design rules is required to assist the designer in developing a cost-effective and

reliable packaging.

3 State of the Art 12

3 State of Art

3.1 Industrial Micromanufacturing Technologies

3.1.1 Basic Cost Analysis

The objective of the work in this report is to develop fabrication methods for

disposable analysis units. This disposable is only one part of a bioanalytical system, as

it needs interfacing and a host device to interact with. The disposable unit, however, is

the consumable that needs replacing after every test and therefore the part that

requires a manufacturing technique capable of high volume production.

To be cost-effective and competitive, it is essential to keep the cost-of-ownership per

individual disposable as low as possible. The manufacturing costs of a product can be

split in a number of categories. Some cost elements depend on the manufacturing

method, while other costs remain unaffected, like material and overhead costs (table

3.1.1). Fabrication and assembly and to a smaller degree testing are major cost

determining factors. Design for fabrication is required to reduce these costs to a

minimum, allowing for cost-effective manufacturing [Kals 1996].

The costs associated with fabrication, assembly and testing heavily depend on the

number of products manufactured (figure 3.1.1). The costs attached to small series or

a prototyping run of a product are often several magnitudes higher than the final costs

of a device manufactured in large quantities. The different methods – from small scale

manufacturing to high volume manufacturing – are connected with a shift from labour

intensive to capital intensive production. The large volume of products manufactured

makes the high investment in production capacity sustainable and allows for lower

costs with large enough volumes.

Table 3.1.1: Effect of production method on product cost

Cost balance product

Cost component

Desi

gn

Ma

rke

ting

Ove

rhea

d

Ma

teri

al

Te

stin

g

Fa

brica

tion

Ass

em

bly

Relationship between production method and cost of product 0 0 1 1 3 4 4

Legend: Large effect 4 ► 3 ► 2 ► 1 ►0 No effect


Prototyping Small runs

High Volume Manufacturing

Volume of Product

Costs per product

Low Volume Series production

Effect of production volume on product costs

Figure 3.1.1: Costs per product vs. volume of product

3.1.2 Requirements for Fabrication of Polymer Bio-analytical

Devices

A careful consideration of requirements for polymer bio-analytical disposables is

required before designing for fabrication. First is there the need for selecting a

suitable polymer for the specific bioanalytical disposable. This polymer needs to be

compatible with the process on the microdevice, as described in the next chapter.

However, more important are the fabrication issues that arise with the specific

polymer. For example, fluorinated polymers are difficult to bond, while acrylic

polymers (PMMA) are unsuitable for die-cutting because of their brittleness.

Furthermore, the process on the device does often require a surface treatment on the

material, to improve biocompatibility and/or to promote adhesion. The most

prominent requirements are presented in table 3.1.2. A further detailed description of

requirements associated with materials, bonding and surface modification can be

found in chapters 3.2.1.1, 3.3.8 and 3.4.1.


Fabrication requirements for polymer bio-analytical devices

Requirement: Specification:

Ability to create multilayered 3D structures

1x3 substrate layers for fluid handling 1x3 substrate layers for pneumatic actuation 1 membrane layer for fluidic actuation

Accurate layer alignment +/- 50 µm lateral/longitudinal +/- 0o 2’ rotational (based on 75 x 25 mm platform)

Structuring of microchannels Minimum channel width of 200 µm

Reliable bonding method Handle internal pressures > 300 kPa Handle buckling (50 x 5 mm deflection at centre of chip)

Surface treatment to promote bonding Treatment to be carried out with a moving polymer web speed of >100 mm/s in web-based production

Surface treatment to improve biocompatibility

Treatment to be carried out with a moving polymer web speed of >100 mm/s in web-based production

High volume production >100000 products

Low cost < 2 A$ per product (not including auxiliary parts eg. arrays, processors etc.)

3.1.3 Fabrication Strategies for Polymer Microdevices

3.1.3.1 Laboratory Based Micro-Fabrication Techniques

The manufacturing of micro sized parts is far from being a new technology. In the

1920’s Swiss watch makers already applied fine-blanking techniques to fabricate

gears with high precision. A few decades later, fine blanking was used in different

industries to produce large numbers of small, micro-sized parts [Kren 2002]. Even

other conventional methods are capable of producing structures in the range of a few

10 µm (milling, cutting, sawing, and turning). An advantage of these techniques is

that they cover a wide variety of materials and no mask fabrication or lithography

steps are required. However, these techniques are not always adequate to produce

complex geometries and structures on a micro scale.

Table 3.1.2: Example of fabrication requirements for polymer bio-analytical devices


To allow for higher resolution and smaller structures, micro devices have been

fabricated with techniques adopted from the microelectronics industry. A historic

example of such a microdevice is a miniaturised gas chromatograph fabricated into a

silicon wafer [Terry 1979]. Recent reports describe bulk micromachining in both glass

and silicon for the fabrication of diagnostic microdevices albeit on a small scale,

because of the costs involved with the silicon/glass processing techniques [Belgrader

1998].

It is only quite recently that a shift can be observed in the use of polymers for

miniaturised diagnostic devices [Van den Berg 2000]. Unfortunately, this field is still

immature as evidenced by limited publications related to fabrication methods other

than in a laboratory environment.

To make a clear distinction between the different scales and therewith the magnitude,

the fabrication processes have been categorised into four scales. They are laboratory,

small (prototyping), medium and large scale as illustrated earlier in the cost analysis.

The relationship between these manufacturing scales and production numbers as well

as throughput and flexibility is represented in figure 3.1.2.

Figure 3.1.2: Manufacturing scales and related production quantities


3.1.3.2 Small Scale Fabrication and Prototyping of Polymer Microdevices

Most of the early micro fabrication techniques in polymers were derived from the

silicon microelectronics industry. To form a polymer micro analytical device, an

inverse silicon master was formed and used to hot emboss into Poly-(methyl

methacrylate) (PMMA) [Martynova 1997]. This method was very compatible with

existing infrastructure in most MEMS laboratories and could also be applied on many

other thermoplastic polymers. Similar to this method is the casting of a silicone-based

elastomer (PDMS) on the inverse silicon master mentioned before [Ekström 1990].

This method is frequently applied for prototyping purposes as it allows for an easy

fabrication of a microfluidic device. Although the need for a hot embossing tool is

eliminated, access to microstructuring equipment is still required to form the silicon

master.

In a more direct approach, a negative photoresist (SU-8) was deposited on a wafer and

structured with the use of a mask and photolithography technology [Guerin 1997].

Photolithographic techniques with the use of laser energy have been applied

frequently for the rapid fabrication of miniaturised polymer devices. Particularly

excimer laser ablation systems that operate in the ultraviolet (UV) region have proven

their capability for structuring a wide range of polymers [Roberts 1997]. Other laser

systems using visible and infrared (IR) light emission have been reported, however

the quality and resolution of the structures were inferior to the excimer laser process

[Brian 1999]. More recently other UV laser technologies emerged that are capable of

structuring polymers as well as metals and ceramics with an acceptable quality and

resolution. The shorter wavelengths of these high harmonic (3ω and 4ω) Nd:YAG

lasers cause reduced thermal damage and are therefore able to produce smaller and

more densely packed structures [Atkin 2002].

To form functional structures like reservoirs and microchannels, a substrate requires

capping/sealing after microstructuring its surface. Most methods for sealing rely on a

laminate or another planar substrate that adheres to the structured substrate [Becker

2000]. A number of microstructured layers can be stacked on top of each other to

form a device with an increased level of complexity by forming three dimensional

fluidic circuits [Webster 1997]. A variety of bonding techniques have been presented


that enable bonding in a laboratory environment. The vast majority of these methods

are time intensive and therefore only suitable for small scale manufacturing. More

details of the different bonding methods are available in section 3.3.

Characterised by a low throughput at high costs, small scale manufacturing is

unsuitable for the manufacturing of commercial quantities. However, it does offer

good flexibility to produce different structures and to work with different materials.

Therefore these small scale manufacturing abilities are well suitable for proof of

concept and prototyping purposes.

3.1.3.3 Medium Scale Manufacturing of Polymer Micro Devices

Medium scale manufacturing is characterised by the production of relatively small

commercial quantities. The production method in this scale is not as labour intensive

as small scale manufacturing. Additionally, production is carried out with tools that

enable faster replication, such as templates and product specific aids. Processing

steps, however, are still separated with very limited automation.

Small scale production relies on the replication of previous parts. Parts can be formed

by methods such as embossing and moulding. These techniques and the preparation of

an (inverted) master tool are discussed in paragraph 3.1.3.1. The advantage over small

scale production is the efficiency in which parts are made and in particular the way

the parts are assembled, e.g. with specific alignment tools. The more efficient

processing reduces manual labour. However, a minimal number of parts is required to

break-even with the small scale manufacturing method to justify the higher production

costs associated with additional tooling.

3.1.3.4 High-volume Manufacturing of Polymer Micro Devices

The fabrication of several hundreds of thousands or more identical products during a

longer period of time is considered high-volume or mass fabrication. Typically, this

fabrication method is characterised by a rather product oriented, functionally


organised manufacturing layout to create a high efficiency. Large production numbers

justify the high investment in manufacturing equipment that is associated with this

type of manufacturing. Several workstations are placed in parallel or in-line with

serial processing steps. Especially the connection and tuning between these stations

and the overall process is a critical requirement to ensure good efficiency and high

throughput [Kals 1996].

Different processing methods are employed in high volume manufacturing.

Depending on the complexity of the product and the level of integration, they can

consist of a single process method or a combination of two or more, for example in

the automotive industry. Different high volume manufacturing methods have been

reviewed for use in polymer miniaturised devices.

Batch processing

In a batch process a large quantity or a number of products are fabricated in a single

process. Typically, a product is formed by a sequence of single processes. In

microtechnology, batch processing is very common in the fabrication of integrated

circuits like microprocessors. A silicon wafer is used as substrate which is subjected

to a number of subsequent subtractive and additive techniques to form electronic parts

like logic gates and transistors. After these processing steps have finished, the wafer is

diced to form discreet electronic devices (figure 3.1.3).

In another batch process polymer bank notes are printed using the Intaglio printing

principle where sheets or coupons containing several tens of notes are processed.

Recent techniques combine this printing principle with embossing in polymer notes

where the embossed features are in the micro domain [Noteprinting 2004]. Equivalent

to wafer processing the sheets are cut into individual notes to finish the process.

Silicon wafer fabrication techniques have been applied in a similar fashion to create

miniaturised bio-analytical devices. However, the costs to set up a silicon wafer

processing factory plus the costs of each wafer processed significantly exceeds the

allowable cost for a disposable device [Madou 2002].



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Figure 3.1.3: 6-Inch silicon wafer ready for dicing into 16 discreet devices [Corman 2000]

Although not reported in microtechnology literature yet, polymer microdevice

manufacturing could well benefit from batch fabrication methods. A large number of

miniaturised devices can be formed at the same time in a sequence of processes, e.g.

die cutting, bonding, drilling and surface modification. In a final fabrication step, a

large sheet containing multiple devices can be cut into individual products.

The feasibility of batch processing for fabrication is primarily dependant on the

footprint of a device. Batch processing is cost-effective in the semiconductor industry

as a large number of devices are produced on one wafer. This processing method

becomes less effective, however, when space restrictions only allow for very limited

numbers of parts to be manufactured per wafer. It is therefore important to consider

the number of parts per batch and the effect of this number on production costs per

part.

A disadvantage of batch processing is the high tooling costs. An embossing step, for

example, would require an array of stamps, rather than one tool to form the multiple

devices in one batch. Furthermore the batch process provides a limited level of

flexibility. Each design variation in the product involves an expensive modification of

the full array of (identical) tools for each processing step. One advantage, however, is

the limited requirements for human labour. Labour is only required to transport parts


in between processing sequences. The total labour per part is therefore insignificant.

Additionally, this minimal human involvement reduces the risk of errors in the

process. However, if an error is made, the whole batch comprising large numbers of

parts is typically destroyed.

Serial Processing

Serial processing is manufacturing whereby the product follows a sequence of

workstations. Typically, these stations operate non- or semi-automated, using buffers

to maintain a good product flow. The throughput in such process is heavily dependant

on the slowest workstation. In contrast to batch processing, that also may be a serial

process, serial processing is typically handling one or very few parts per production

step. In high-volume manufacturing, serial processing is more suitable for products

with a limited complexity that do not require many careful production steps such as

assembly and alignment. These steps consume a large amount of production resources

per part, reducing its cost-effectiveness. A major advantage of serial processing on the

other hand its high flexibility as only one tool per production step requires

replacing/modifying.

A few typical serial processes such as embossing and casting have already been

discussed (see chapter 3.1.3.2). Injection moulding is another process that is often

found in the large scale manufacturing of plastic parts. This manufacturing technique

has found many applications in the fabrication of products with micro-sized features,

e.g. compact discs. The critical dimensions that can be replicated using this technique

are determined by the aspect ratio (the ratio of structural height to width). Micro-sized

products with a low aspect ratio, e.g. the submicron pits in compact discs can be

manufactured using conventional injection moulding. However, higher aspect ratios

cause a loss of replication quality due to the resin freezing before completion of the

mould filling. To overcome this problem a modified variant of injection moulding has

been developed, called micro-injection moulding [Heckele 2004, Piotter 2001].

When focusing on miniaturised bio-analytical applications, (micro)-injection

moulding has been the enabling fabrication method in many cases. An often cited

example of such a device is the disposable I-STAT® blood analyser cartridge (figure

3.1.4a) [Erickson 1993]. This device consists of a thin-film silicon sensor that is


incorporated in an injection moulded cartridge containing microchannels and

reservoirs. Other examples of injection moulded microfluidic parts are a 96-well

microtiterplate (figure 3.1.4b) and a diffusion detection system (figure 3.1.4c).

PICTURE REMOVED

in ELECTRONIC

VERSION

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PICTURE REMOVED in

ELECTRONIC VERSION

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PICTURE REMOVED in

ELECTRONIC VERSION

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Source: [Erickson 1993] Source: [Boehringer Ingelheim 2004] Source: [Weigl 1999]

Figure 3.1.4: Injection moulded microfluidic parts a) I-Stat disposable blood analyser, b) 96-well microtiterplate, c) Diffusion detection system

These and many other (micro)-injection moulded parts are typically characterised by

simplicity in the product; they only consist of one or a small number of parts without

the requirement for accurate alignment and complicated assembly steps. Additionally,

injection moulded parts have a limited minimum thickness; thin parts often require

ribs and other structural reinforcements to allow for an intact retrieval form the tool.

When stacking multiple planar injection moulded layers, the thickness of a device

easily exceeds 10 mm, as a result of the minimum thickness of each individual layer.

Injection moulding offers one major advantage. The costs associated with

infrastructure are limited as the moulding machines can be used for different products

and can therefore be spread over the different products. The injection moulding tool is

product specific and the costs for this have to be supported by this particular product.

However, standardisation of products can contribute to a further cost reduction by

using tool specific inserts. These inserts allow for the fabrication of a different

product for only a fraction of the price of a complete new injection moulding cavity.

This easy separation between product specific moulding tool and machine creates a

relatively flexible production capacity and allows for the production of a variety of

products without the need for an expensive product-dedicated production line.


Reel-to-Reel manufacturing

Reel-to-reel fabrication, often referred to as web-based manufacturing, is a common

production method when working with materials like films and thin sheets e.g. in food

packaging. This technique utilises films coming from a reel to form a product by

operations like cutting, folding, coating, bonding, etc. (see figure 3.1.5). After

processing the film is either fed back onto a reel (hence the term reel-to-reel

manufacturing) e.g. in the printing industry or cut to form separate products, e.g. in

food packaging to form bags etc. As the process is highly automated, only minimal

human interaction is required to maintain the process.

A web-based production line is mostly dedicated to one specific product or a very

similar product range. A high degree of standardisation within the product range

allows for the fabrication of different products by making simple tool changes, such

as a different die-cutter, embossing or thermoforming tool (see also figure 3.1.6).

Additionally, it is possible to change appearances, for instance by different labels or

prints, but the form and shape of the product typically remain unchanged as the

process is quite rigid and very sensitive to big changes.


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Figure 3.1.5: Reel to reel manufacturing setup (Source: RPS Automation)


1

1

1

1

1

1

1

2

2

3

5 B

B

B

B

B

4

7 BPP 1

i

i

i1. Course cutting2. Fine Blanking3. Thermoforming4. Deposition5. Electr. Print6. Graphical Print7. Pick & Place Robot8. PackagingB Bonding Uniti Optical InspectionP Printing Unit

8

Reel 1: Capping/blisters

Reel 2: Fine structuring

Reel 3: Fine structuring + Electr. Printing

Reel 4: Structural

Reel 5: Structural

Reel 6: Elastomeric Membrane

Reel 7: Graphical Layer

Figure 3.1.6: Schematic layout of a web-based manufacturing setup for the manufacturing of a seven-layer polymer bio-analytical device

A major advantage of web-based manufacturing is the very high throughput that can

be achieved. Furthermore the process is very efficient as the result of minimal labour

requirements. For the process to be cost-effective, large quantities of products need to

be manufactured, especially as initial costs for the production capacity are generally

high. Small repeating costs, however, allow for extremely low costs per product

provided that sufficient products are being manufactured [see also Mehalso 2002].

3.1.3.5 Overview of Scales in Manufacturing Capacity

Each magnitude of production volume in manufacturing has different key attributes.

Prototyping for instance is very flexible, while large scale manufacturing is often a

very rigid process that cannot easily undergo changes in the production process. The

most critical aspects of manufacturing have been summarised in table 3.1.3.

Table 3.1.3: Comparison of different scales of manufacturing

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Prototyping 4 1 4 4 3 1 1 1

Medium Scale Manufacturing 3 3 2 2 2 2 3 3

Large Scale Manufacturing 2 4 1 1 1 4 4 4

Legend: Very suitable 4 ► 3 ► 2 ► 1 ►0 Unsuitable


3.1.3.6 Overview and Selection of High Volume Manufacturing Strategies

The high-volume manufacturing methods discussed in paragraph 3.1.3.4 each provide

specific manufacturing characteristics (see table 3.1.4). None of these methods are

generic and suitable for all forms of manufacturing. The cost of manufacturing is

mainly influenced by factors including fabrication, assembly and testing (see

paragraph 3.1.1). To keep costs to a minimum, these fabrication steps require

automation to minimise human labour costs per part. Web-based manufacturing is

very suitable as fabrication, assembly and testing are cost-effectively integrated into

one manufacturing system. A drawback of this manufacturing method is the relatively

large production volume required to recover a high initial investment. However, the

focus on producing large quantities of relatively complex disposable parts supports

the choice for web-based manufacturing. Investigations into fabrication techniques for

disposable polymers devices in this work are therefore based on this manufacturing

strategy.

Table 3.1.4: Comparison of different high-volume manufacturing techniques

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Batch Processing 3 4 1 2 3 3 3 3

Serial Processing 2 3 3 3 2 3 2 3

Reel to reel 3 4 3 1 1 4 4 4

Legend: Very suitable 4 ► 3 ► 2 ► 1 ►0 Unsuitable


3.2 Material Selection for Polymer Bio-analytical Devices

3.2.1 Substrate Materials

3.2.1.1 Requirements for Polymeric Substrate Materials

Polymer substrate materials for miniaturised bioanalytical devices are limited to a

narrow selection of suitable materials; every bio analytical application will introduce

specific demands that affect the material choice. However, the vast majority of

bioanalytical applications have very related material requirements [Hupert 2003]. The

available polymer selection can therefore be narrowed to comply with these standard

requirements.

A first distinction is made between thermoplastic and thermoset polymers. The latter

is formed by strong cross-linking of adjacent polymer chains and cannot be reflowed

when heated up. Thermoplastics are characterised by polymer chains that are only

weakly bonded by Van der Waals forces. The chains can move around freely when

provided sufficient energy in the form of heat. This is an essential requirement for the

majority of fabrication processes in polymers as described in chapter 3.1. A

consequence of selecting thermoplastic substrate materials, however, is the need for a

polymer that will not collapse as a result of external or internal heating of the device.

A common micro biological process like polymerase chain reaction (PCR) [Woolley

1996] is subjected to temperatures as high as 95 0C and therefore requires a polymer

with a mechanical stability up to at least this temperature. In general, the thermal

stability can be determined by looking at the glass transition temperature (Tg) of the

material. However, when a material is highly crystalline, mechanical properties are

maintained above Tg and therefore the maximum structural use temperature is close to

the melting temperature (Tm) of the material [Strong 2000].

Biocompatibility of the substrate materials with the assay is a second important

requirement for a good analytical performance. Firstly, the polymer must not inhibit

the performed reaction on the device. Potential inhibitors are additives applied in

plastics such as plasticisers or UV stabilisers. Such additives might leech from the

plastic when subjected to the operational conditions of the device, eg. raised

temperatures or chemical solutions. However, the raw polymer material also needs

consideration as this might affect the chemistry on the device. Another important


point of consideration is the influence of surface effects as the surface-to-volume ratio

in microchannels is significantly larger than in traditional analytical processes.

[Daniel 1998, Schoffner 1996]. This can potentially lead to significant non-specific

attachment of proteins on the surface [Nicolau 2002]. Additionally, water vapour

permeability of the surface allows for the absorption and diffusion of reaction fluids.

Both protein attachment and absorption/diffusion inhibit processes such as PCR,

reducing its yield. As a last requirement the material must withstand the different

reagents and fluids that are in contact with the substrate. Aggressive reagents and

fluids may lead to collapsing microstructures when polymers show solubility, for

instance with the use of organic solvents [Hupert 2003].

Detection of reaction products forms a fundamental part of the bio-analytical device.

Most detection methods rely on optical techniques, for instance to detect fluorescent

dyes attached to proteins or to characterise spots on micro arrays [Burns 1998, Reyes

2002]. An optical transparent material in the visual and UV range is essential for

devices that depend on such detection techniques. In addition, the material must not

autofluoresce when exposed to the detection light source as this autofluorescence

hampers the optical detection process.

The commercial availability of a polymer material, especially in the form a film, is an

important factor. A limited number of suppliers can potentially put the commercial

success of a device in a dangerous position when the key material becomes

temporarily or even permanently unavailable. Therefore a widely supplied material is

more favourable and strategic than a special engineering type of polymer material that

can only be supplied by one or two suppliers. Examples of materials with limited

availability in film are COC and PMP (polymethyl pentene). However, these materials

offer highly favourable properties. A correct judgement between the advantages and

disadvantages of the different materials is therefore essential.

Substrate materials potentially pose a health and environmental risk during the

manufacture, use, and disposal of the product. In addition, as consumers become more

concerned about environmental characteristics of materials, the material choice may

ultimately impact how marketable a product is. Examples of potentially hazardous

materials are chlorinated polymers and polymers that contain particular plasticisers


for softening. These polymers contain ingredients that are carcinogenic and endocrine

disrupters, posing a potential future liability. Care has to be taken during the selection

of substrate materials to avoid the use of such environmentally unfriendly materials.

3.2.1.2 Polymeric Substrate Materials in Microtechnology

A wide variety of polymers have been applied in microtechnology. The emphasis,

particularly for use in microfluidics, has been on polycarbonate (PC) and

polymethylmethacrylate (PMMA) [Thomson 2003, Hupert 2003]. These polymers are

transparent and generally meet the basic requirements for miniaturised bioanalytical

devices.

Another material that has seen some applications in microfluidics is polyethylene

terephtalate (PET) [Atkin 2002, Rossier 2002]. This transparent polymer is

encouraging because of its widespread use in the packaging and printing industry. Its

application in these industries provides an extensive knowledge base in respect to

fabrication and processing. The availability of a wide range of different grades of the

material is another beneficial aspect [DuPont 2003]. This knowledge of existing

technologies in combination with PET can be used to the advantage of polymer

microdevice fabrication.

Polydimethylsiloxane (PDMS) is another transparent, elastomeric material that uses

soft lithography and moulding as enabling technologies. This material has often been

applied in a research environment for the fabrication of analytical microdevices for

instance to analyse and detect DNA fragments [Effenhauser 1997]. PDMS became a

very popular material as it allows for the quick and easy fabrication of multiple

devices by moulding it against a micromachined master. Although quite suitable for

the fabrication of prototypes, it also introduces a number of limitations. PDMS only

provides limited substrate strength due to its elastomeric character and is extremely

hard to bond to another material. Besides those mechanical issues PDMS possesses

extremely high water vapour permeability. This causes problems in an analytical

micro-assay like bubble formation, sample evaporation and protein adsorption [Shin

2003].


A newer group of materials, cycloolefin copolymers (COC), were recently introduced

into microtechnology [Choi 2003]. Although this material has only been on the

market for a short period of time, it has great potential in microanalytical processes

for its high chemical stability, low permeability rates and very good optical properties

[Topas 1998]. One drawback of a new material like COC is the limited availability as

only two manufacturers currently offer the resin (Ticona GmbH, Germany and Zeon

Corporation, Japan). Additionally, the availability of films is very limited as well as

knowledge about the material in terms of processing, e.g. bonding and surface

modification.

Recent research into organic light emitting diodes (OLED) in the display industry has

led to the development of flexible displays and flexible electronics [MacDonald

2003]. The substrate materials for such displays require a high transparency with a

typical light transmission over 85 percent at wavelengths between 400 and 800

manometer. Again, thermoplastic materials like PET and PC are very suitable for such

applications. Other, more exotic, materials reported for this purpose are

polyethersulfone (PES) and polycyclic olefin (PCO). Although these materials are

highly transparent, they cannot be processed at elevated temperatures. Instead,

processes like solvent casting are required.

Less frequently, other standard polymeric materials have successfully been used in

microtechnology, mainly in a research environment outside the miniaturised

analytical domain. Those polymers include polyethylene (PE), polyetheretherketone

(PEEK), polystyrene (PS), polyamide (PA), polyetherimide (PEI), liquid crystal

polymer (LCP), polypropylene (PP), polybutyleneterephthalate (PBT),

polyoxymethylene (POM), polyphenylene ether (PPE), polysulfone (PSU) [Becker

2000]. In general, each of these materials fails to meet at least one of the essential

requirements to qualify as suitable material in miniaturized analytical devices, e.g.

because of a poor temperature stability or limited optical properties.


3.2.2 Membrane Materials

3.2.2.1 Actuation

Transportation of fluids is an important function in microfluidic devices. Fluid

transport is often realised by fluid displacement techniques. To achieve displacement,

some form of actuation is required. Actuation can take place either externally to a

disposable (e.g. in a host device using syringes) or internally. Internal methods are

preferable, particularly when reaction solutions such as buffers are stored on the

disposable. Additionally, processes such as polymerase chain reaction require cycling

of fluids within the disposable device, leaving external actuation less effective.

Different actuation methods have been demonstrated in microfluidics including

thermal, piezoelectric and pneumatic actuation [Auroux 2002]. Electric driven

actuation methods are rather expensive and therefore not suitable for integration into a

disposable unit. Pneumatic actuation on the other hand can be a cost-effective method

[see also Schuenemann 2004].

Flexible structures such as membranes are required to utilise pneumatic actuation for

fluid displacement. The membrane is actuated by a pneumatic housing that forms an

integral part of a multilayered polymer fluidic chip. A set of three chambers forms the

base for a peristaltic pump. Pneumatic actuation of the membrane above each of these

chambers in the right sequence displaces the fluid through the disposable (see also

figure 3.2.1). The membrane is a key material in this actuation method. A careful

selection of a suitable material that fulfils the mechanical pumping requirements as

well the bio-analytical requirements is essential.

fl_hole

fl_pipemembranepn_pipepn_linepn_hole

Microfluidic circuit

Pneumatic distribution Figure 3.2.1: Pneumatic actuation of a flexible membrane for peristaltic pumping


3.2.2.2 Requirements for Polymeric Membrane Materials

Analogous to polymeric substrate materials there are only limited materials that can

be applied as membrane materials in analytical microdevices. The requirements for

membrane materials are identical when looking at optical, thermal, biological and

environmental properties and availability. The mechanical requirements on the other

hand are distinctly different, as the film should be elastic to achieve a suitable

deflection. A high elasticity implies the need for a low Young’s modulus. This leads

to the need for elastomeric materials rather than thermoset or thermoplastic polymers.

3.2.2.3 Polymeric Membrane Materials in Microtechnology

Generally, membranes in microfluidic devices are related to pumps and valves. The

vast majority of reports discussing pumps and valves in microfluidics are based

around silicon and glass substrates. A few reports describe the use of silicone rubber

(MRTV) on a silicon substrate to form an elastomeric film [Grosjean 1999]. This

material is suitable when regarding the elastic properties. A major disadvantage of

silicones is their high permeability to water. Grosjean et al. solved this problem by

applying parylene as barrier layer to seal the silicone while maintaining its elastic

properties. This combination of materials in microanalytical devices is very

challenging as a result of processing limitations in the parylene film.

Another silicone rubber that is widely used in microdevices is PDMS (see also chapter

3.2.1.2). PDMS has been applied to construct valves where the membrane was filled

with Permalloy strips. Actuation was subsequently obtained by applying a magnetic

force, deflecting the membrane [Khoo 2001]. PDMS, however, suffers from the same

problems as the MRTV silicone. Due to a high permeability to water, this material is

not very attractive for applications in microfluidic devices. Similar to MRTV silicone,

a barrier layer was created using parylene to resolve this problem [Shin 2003].

As well as forming barrier layers, parylene has been applied as stand-alone membrane

material to form valves. These valves were formed by the deposition of parylene on

silicon wafers [Xie 2001]. Other reports describe the use of parylene to form flexible

microfluidic tubing [Feng 2003]. Although parylene is mechanically a very suitable


membrane material, the fabrication is not compatible with a continuous process.

Parylene (poly-para-xylylene) is only available using the chemical vapour deposition

technique (CVD). No suppliers could be found that offer parylene as film.

It can be concluded that elastomeric membranes have seen very limited applications

in microfluidic devices. Particularly the application of membranes in polymer based

microdevices is still a very immature field as evidenced by the few publications

describing this work. As both PDMS and parylene have limitations, either property or

fabrication related, more elastomeric materials need investigating.


3.3 Bonding and Sealing

3.3.1 General Bonding Theory

Several theories have been proposed in an attempt to provide an explanation for

adhesion phenomena. However, no single theory explains the mechanism of adhesion

in a general, comprehensive way. This has resulted in a number of fundamental

adhesion mechanisms that can be categorised into six different theoretical models

[Pizzi 1994].

3.3.2 Mechanical Interlocking

The mechanical interlocking model uses the interlocking of adhesive into cavities,

pores or surface roughness of the material surface to provide the bonding strength (see

figure 3.3.1) [McBain 1925]. This method of adhesion has been demonstrated several

decades ago by bonding textile fibres to rubber [Boroff 1949]. The penetration of the

fibre ends into the rubber has been the predominant factor in this type of joint.

Another example is the common rubber tyre repair kit that is based on this principle.

However, as this theory does not apply to smooth surfaces, it cannot be considered to

be universal.


(Copyright) Figure 3.3.1: Mechanical interlocking of adhesives (Source: SpecialChem S. A.)

To address this limitation it was proposed that the joint strength consists of two

components, the mechanical keying component and the thermodynamic interfacial

interactions component [Wake 1982]. A high level of adhesion could then be achieved

by generating both a good surface morphology and physicochemical surface

properties. This basically means that the enhancement of mechanical interlocking can

be obtained by increasing the interfacial area due to surface roughness as long as the

wetting conditions are fulfilled to allow for penetration of adhesives into pores and

cavities.


More recently this theory received further support when polyethylene fibres were

plasma treated before bonding with an epoxy resin. The plasma treatment resulted in a

pitted structure on the polyethylene surface, which can be filled with ease by the

epoxy resin [Ladizesky 1989].

It has been suggested, however, that the enhanced adhesion might not be achieved

from mechanical keying, but that the surface roughness can increase the viscoelastic

or plastic dissipation of energy around the crack tip and in the bulk of the substrate

during joint failure. This energy loss is known to be the major component of adhesive

strength [Hine 1984].

The theory above suggests that the bonding of polymer films enhances when the

surface roughness increases. A smooth surface that often can be seen on polymer

films is therefore less suitable to form a strong bond. To improve this, surface

treatment of the adhesion side of the polymer should be considered. Obviously, a

rougher surface does increase the effective surface area to bond, leading to a stronger

joint.

Good wetting is another important requirement. Untreated polymers generally have a

low surface energy and prevent good wetting of the adhesive with the surface to

occur. The wetting has to be improved by increasing the free energy on the surface of

the substrate. Surface treatment is therefore beneficial to both interlocking and good

wetting.

3.3.3 Electronic Theory

The electronic theory suggests that a significant contribution to the bond strength lies

in the electronic transfer mechanism between the substrate and adhesive [Deryaguin

1978]. The mechanism suggests equalising of Fermi levels of the different electronic

band structures on each surface that could induce the formation of a double electrical

layer at the interface (see figure 3.3.2). The resulting electrostatic forces can make a

significant contribution to the bond strength. This behaviour can be regarded as


equivalent to the principle of a capacitor. An example of this type of bond is the

sticking of a non conductive material to a rubbed balloon.


(Copyright)

Figure 3.3.2: The formation of a double electrical layer that induces electrostatic forces, contributing to the bond strength (Source: SpecialChem S. A.)

Although playing a major role when measuring adhesion, similar to the interlocking

theory, the viscoelastic or plastic dissipation of energy is not included in this theory.

A clear correlation between electronic interfacial parameters and adhesion is not

usually found in experiments. It was even shown that the electrostatic contribution to

peel strength could be neglected for a glass based systems with deposited layers of

gold, silver or copper [Von Harrach 1972]. Furthermore it can be remarked that it is

not clear whether the electronic mechanism is the consequence or the cause of the

high bond strength [Pizzi 1994].

Crucial element for the electronic effect in the bonding of polymeric layers in

microdevices is the choice of materials. The dielectric constant plays a determining

role in the bond strength. Still, considering the controversial results in the experiments

by Von Harrach et al., the electronic effect proves not the critical contribution in bond

strength and will be regarded as less significant in this work.

3.3.4 Theory of Weak Boundary Layers and Interfaces

Alterations and modifications of the adhesive can be observed in the area around the

interface of a substrate. This leads to the formation of an interfacial zone that has

different properties than the bulk of the material. This zone has been described as a

weak boundary layer [Bikerman 1961]. According to this theory, the cohesive

strength of this layer is the main factor when determining the adhesion strength


between two layers, even when an interfacial failure occurs. Therefore the adhesion

energy is considered always equal to the cohesive energy of the weaker boundary

layer on the interface. Bikerman proposed several weak boundary layers like the

presence of impurities or short polymer chains at the interface.

Several reports have opposed this theory. Firstly is there plenty of experimental

evidence showing clearly a failure purely on the interface rather than the weak

boundary layer. Furthermore bond failure is not a result of the existence of a weak

boundary layer, even when this failure is cohesive in the vicinity of the interface of

the material in contact. The stress distribution in the materials and the stress

concentration in the vicinity of the crack imply indeed that the failure occurs very

close to the interface, but not at the interface [Bascom 1975, Good 1972].

However, the existence of interfacial layers has received a lot of attention over the

years and has led to the concept of “thick interfaces” or “interphases” in adhesion

science [Sharpe 1971]. Such interphases have typical thicknesses varying between a

few Ǻngström and a few micrometers. Many physical, physicochemical and chemical

phenomena are responsible for the formation of these interphases [Schultz 1992].

Boundary layers, or interphases, will be present when laminating microfluidic

polymer devices. Indeed, such interphases can strongly alter the strength of multi-

layered structures. Schultz reported different possible mechanisms responsible for

interphases leading to weaker bonds or even failure that include:

-Orientation of chemical groups or over-concentration of chain ends to

minimize the free energy of the interface;

-Migration towards the interface of additives or low-molecular-weight fraction

-Growth of a transcrystalline structure;

-Formation of a pseudo glassy zone resulting from a deduction in chain

mobility through strong interactions with the substrate;

-Modification of the thermodynamics/kinetics of the polymerisation or cross-

linking reaction at the interface through preferential adsorption of reaction

species or catalytical effects.


All of these effects point to a potential problem in respect to the bonding of

microanalytical devices. The phenomena above are rather a cause of a weak bond than

a consequence. It is therefore important to take these into account when analysing

empirical data obtained for different bonding techniques.

3.3.5 Adsorption Theory

The most widely accepted model of adhesion at present is the thermodynamic theory.

The model suggests that the adhesive will adhere to the substrate material as a result

of interatomic and intermolecular forces established at the interface, as long as an

intimate contact occurs [Sharpe 1963]. The most common of these forces are Lewis

acid-base interactions and Van der Waals forces with the magnitude generally being

related to surface free energies of both adhesive and substrate (see figure 3.3.3). An

intimate contact between the substrate and adhesive is essential to obtain a good

adsorption such that Van der Waals interaction or the acid-base interaction or both

take place.


(Copyright)

Figure 3.3.3: Adhesion according to the thermodynamic model (Source: SpecialChem S. A.)

The adsorption theory assumes the application of an adhesive to obtain a secondary

bond between different substrates. An intimate contact is required for Van der Waal

or Lewis acid-base bonds to form. In the case of a direct joint between a substrate and

another substrate without the use of an adhesive, this type of bond is not very likely to

occur. Voids, contaminations and other irregularities avoid the formation of enough

secondary bonds to contribute to a strong joint.


3.3.6 Diffusion Theory

The theory suggesting adhesion by diffusion is based on the mobility of

macromolecular chains or segments of those chains on the interface of a polymer

[Voyutskii 1963]. An interphase region is created if those chains are mutually soluble

enough to diffuse across the interface (see figure 3.3.5). The adhesion strength highly

depends on a number of factors, including contact time, temperature, nature and

molecular weight of polymers and contact pressure.


(Copyright)

Figure 3.3.5: Interdiffusion of molecular chains across the surfaces of a substrate and adhesive

Documents

Design and production of polymer based miniaturised bio …€¦ · 3.4.1 Introduction to Surface Modification 43 3.4.2 Surface modification for bonding 44 3.4.2.1 Adhesion Promoting