dr michael loughran team leader biophotonics & microfluidics research integration of silicon and...

Dr Michael Loughran Team Leader Biophotonics & Microfluidics Research

Integration of silicon and glass processing for lab on a chip development

• Dr Mike Loughran

• Tyndall National Institute

• Cork, Ireland


Overview

• Introduction Mike Loughran, Tyndall National Institute• Lab on a chip research development• Choice of substrate and fabrication techniques• Fluidic Inter-connects• Microchip HPLC Development on silicon substrates• Capillary Gel Electrophoresis in Single Glass Microchip• Processing of Glass Microchips • Electrowetting (microfluidic transport• Encoded silicon microbead technology for lab on a chip• Integration of VSCEL (optical light source for microbead detection)• Development of customised reaction chamber for micro bead

functionalization• Chemiluminescent allergen detection• Acknowledgements


PhD Biotechnology Cranfield University 1995

Senior Research Fellow AIST Tsukuba Japan 2002-2004

Dr Mike Loughran Brief Introduction

JSPS Research Fellow Tokyo University of Fisheries from 1995-1997

Research Fellow University of Manchester, U.K , 1999-2001

Visiting Associate Professor Tokyo University 2001

Team Leader Biophotonics & Microfluidics Research Tyndall National Institute 2004-2006

Research Fellow Dublin City University, 1997-1999


Lab on Chip Research Development

Micro-reactor Surface coating

Micro-sensor Optical/ Electrochemical

Micropump/valveLiquid transport

Fabrication techniques must be cost-effective Economical precise control of channel dimensions/geometry Accuratepreferably made on large scale wafers Mass production

Intrinsic Advantages of -tas lab on chip systemsHigh through put, rapid analysis, reduced reagent/sample consumption

Continuing challengesSample transport/inter-connects from bench to micro-chipReproducibility (feature size) genuine versatile platformObscure terminology “nano” or micro,

top down, bottom up


Choice of substratePolymer micro-chips: replication technology, embossing, imprintingIn expensive, rapid proto-typing, non clean room processingSolvent in-compatibility, channels not uniform, temp defects, opaque, hydrophobic Examples Polycarbonate, PMMA, SU-8, PDMS

Silicon Micro-machiningPhoto-lithography, wet etching, dry etching, anodic bonding, dicingSystem integration: electrodes, micro-channels Not suitable for high voltage capillary electrophoresis separations silicon breaks down at voltages > 1000 V

Glass processingPlanar technology, transparent, surface properties well characterised, amenable for bio-conjugation,self assembly, facilitate high voltage separation > 50kV, clean room and no clean room processing,

Flexible processing photo-lithography, wet etching, dry etching, anodic bonding, dicing

But bonding with UV curable adhesives not always provide permanent seal

Fusion bonding (high temperature 650oC above glass transition phase) more reliable seal provided micro channel alignment can be confirmed


Micro chip fabrication techniques


Fabrication Feature Size

simple process for wet anisotropic channel etching with a controlled depth up to 500 nm, an accuracy of a few percent and etch roughness less than 0.5 nm

In photolithography ultraviolet light is used (typically 250 nm wavelength)

fabrication of spacings < 125 nm causes blurred features, can melt together. technical improvements enable structural resolutions ca. 70 nm in experimental setups and ca. 100 nm for mass production

Lithography technologies based on focused beams are an alternativee.g. Electron beam lithography (EBL) and focused-ion beam (FIB) lithography (feature size 10 nm) to create nanochannels for DNA separation


Fluidic Inter-connects


Rheodyne Valve for Fluidic Switching

Fluidic Inter-connects for microchip HPLC


SU-8 microfluidic Chip rapid processing

• Chip designs made from low cost acetate masks

• SU-8 lithography in the plating lab (process developed by D. Hoffman)

• Access holes drilled manually in lab 1.13

Fluidic interconnects for allergen microarray


Preliminary packaging of allergen microarray

User-friendly chip interface

•Initial prototype chip development:

Covalent binding of PDMS on glass with oxygen plasma treatment (in plating lab)

• Plastic chip holder:

More stable solution

Holder fabricated by J. Rea

Sealing techniques (minimize dead volume)


Integrated access holes for sample introduction

Glass microfluidic Chip processing

• Chip designs realised on chromium masks (to withstand HF etching)

•Customised Casper Process Development(

-> integration of access holes in the clean room fabrication process by deep(200 um) HF etching (better alignment, less fragile)

-> Glass channel lithography and wet etching (100 um) in a clean room environment


Customized microchip holder

Plastic chip holder


Packaging of HPLC Microchip

Figure 9: Problems with packaging. (a) Dead volume at the end of the optical fibre and air bubbles existing in the glue possibly cause the leakage. (b) Dead volume at the end of the fused silica capillary. (c) Blockage caused at the end of the fused silica capillary

UV glueChannel

CapillarySilicon

With UV glue

Without UV glue (Dead volume)

ChannelCapillary

Capillary end

SiliconSilicon

Capillary

Optical fibre

Dead volume Air bubble(a)

(b)

(c)


Final Mask Design with Integrated HPLC Chips


HPLC Chip Fabrication A fabricated micro HPLC chip

Injection channel

Micropillar (5×5 µm)

UV detection

Injection

Separation

Figure 7


Glass Processing:

Capillary Gel Electrophoresis

Rapid microchannel fabrication


Separation capillary length 60 mm, diameter 500 μm, depth 300 μm.

Magnified view of self-contained stacking capillary

Length 12 mm,

diameter 1 mm,

depth of 300 μm.

Sep

aration

cap

illary

Stacking capillary

SDS/Native Capillary Gel Electrophoresis in Single Glass Microchip

Shou-Wen Tsai , Michael Loughran, Hiroaki Suzuki & Isao Karube, “Native and SDS Capillary Gel Electrophoresis of Proteins on a Single Microchip -” Electrophoresis (2004)25:494-501


Simultaneous separation of both native and SDS marker proteins in assembled chip

Experimental conditionsconstant current of 2 mA, 10 minutes after sample pre-concentration at 50V

Simultaneous SDS Native Electrophoresis In Multiple Capillaries

Shou-Wen Tsai , Michael Loughran, Hiroaki Suzuki & Isao Karube, “Native and SDS Capillary Gel Electrophoresis of Proteins on a Single Microchip -” Electrophoresis (2004)25:494-501


Wet etching of microfluidic structures in glass using photo-definable epoxy (SU-8) as an etching mask

• Rapid generation of microfluidic structures in glass • Using an epoxy based polymer (SU-8) as an etching mask • 49% concentrated hydrofluoric acid as the glass etchant• Generation of microfluidic structures with a maximum etch depth of 100 µm• The glass material used was Borofloat33• The wafers were 600 µm thick and non-polished (both sides)• This type of glass can also be used for anodic bonding to silicon substrates

– Fabricated microfluidic glass chips were etched for 10 minutes

– Resulting channel depth is about 70 µm


Bonding and sealing of fabricated microfluidic glass chips

• PMMA to glass dircect bonding• PMMA/glass to glass bonding via PDMS interface layer

2 methods were applied to seal the microfluidic glass chips

Both types of bonded glass chips were tested with a fluorescent dyed liquid at different flow rates

• Maximum flow rate tested: 417 µl/s• Resutling average velocity: 15 m/s• Resulting pressure: 250kPa 36 psi• No leakage observed for both methods• No delamination observed for both

methods

Sufficient sealing for most applications


Test of fabricated microfluidic glass chips

InletOutlet

Sequence 1 Sequence 2 Sequence 3


Observed advantages of glass processing

• Successful implementation of a microfluidic chip manufacturing technology where microchannels are defined in glass

• Offers a very good alternative to microfluidic chips with microchannel structures defined in SU-8

• Offers alternative bonding methods avoiding the use of UV glue or SU-8 bonding techniques

• The bond is clean and of high quality in terms of uniformity and tightness

• Microfluidic glass chip is completely visualisable as both substrate and superstrate are transparent

• DNA was successfully hybridised with probe DNA prior immobilised into the channel structure


Electrowetting


PDMS

W.E.

hydrophobic

hydrophobic

hydrophilic

hydrophilic

OFFOFF

Electrolyte

ONON OFFOFF ONON

hydrophilic

hydrophilic

hydrophilic

hydrophilic

ONONhydrop

hobichydrop

hobicOFFOFF

Principle of Electrowetting


setup

CCD camera

potentiostat

Control PC

Microfluidic chip

Electrowetting droplet transport recorded in dark room

Experimental setup


PDMS substrateFlow channel Reservoir

Glass substrateA.E. (Pt)R.E. (Ag/AgCl)

W.E. (Au)

WorkingElectrode

Flow channelElectrolyte

300 m

40 m

Insulating layer

PDMS electrowetting micro-fluidic chip


12.5

mm

23 mm

15 m

m23 mm

100 or 300 m

Wettability of glass more uniform than PDMS.Diameter of microchannel reduced from 500 to 100 mm

Electro-wetting: glass microchip fabrication


• Materials• Ceramic plate (Al2O3), weight, oven, pyrex glass,

• Glass cleaning

• acetone, isopropanol, deionized water, acid cleaning (H2SO4:H2O2=9:1), deionized water, drying (N2 gas)

• Ceramic plate cleaning

• Isopropanol

glass

Ceramic plate

weightSetup

Glass flow channel was bonded byFusion bonding.Alexandra helped me to completefusion bonding.

Fusion bonding: to seal glass electrowetting micro-chip


Leakage

Sometimes glass/glass bonding is not uniform due to presence of air in capillary channel during wafer alignment.Use of a vacuum oven may minimize this problem in future

Images of glass microchip


Encoded Silicon Microbead Technology


Results:

Encoded Silicon Microbeads - Design and fabrication

Optically encoded

No photobleaching

Material Silicon/Silicon Oxide

High chemical stability

Design is mask programmable

Variety of shapes

Large range of sizes

High member library

Compatible with standard MEMS fabrication processes


Multiple microbead injections: Approach I

Cartridge Development

• Loading Mechanism

• Injection Mechanism

Image sequence showing successive injection of two microbeads

System Integration - Microbead Injection

Problem: Repeatability of precise injection of individual microbeads


Characterisation – Identification System

Implementation of optical detection system

[1]

[5]

[2]

[3]

[4]

[4] Focusing Lens - Aplanatic Doublet (f - 6 mm)

[5] Photo Detector - Silicon Phototransistor

[1] Laser Diode and Collimator - AlGaInP, 635 nm, 3 mW

[3] Beam Splitter - 50:50 Cube

[2] Aperture - 3mm

[D] Encoded microbead

1 cm


Silicon Photo-Detector

Type B Microbead

IntegratedVCSEL

Polymer Suberstrate with Microchannel

Polymer Substrate with Cavity

Slit

Encoded Silicon Microbeads - Optical Detection

Integrated detection system: Principle of operation Detection of microbeads with through-hole barcode pattern


PMMA (Substrate - Cavities)

PMMA (Superstrate - Microchannels)

Casting Resin (Master - Microchannels)

Silicon (Master - Cavities)

SU-8

VCSEL Package

Process Flow - Cavities Process Flow - Microchannels

1a

2

3

1b

4

5


Integrated detection system: Illustration of fabrication process



Integrated detection system: Experimental set-up and results


System Integration - Reaction chamber

Integrated detection system: Illustration of fabrication process


Experimental setup:

Application – Instrumentation

Temperature Control

Electronics

Peltier Element

Temperature Feed back

loopCurrent Control

PC

Syringe Pumps

Temperature Monitoring

Syringe Control

Flow Control

Microfluidic Reaction Chamber

Temperature Sensor


Temperature Controller – Technical Description

Temperature Controller

Electronics

Temperature Measurement

Electronics

Temperature Monitoring with

Labview

DAQ

PC

Peltier Element

Microfluidic Flowcell

Temperature Sensors

Requirements:

•Temperature range 35°C ... 45°C•Stability ± 0.5°C over a period of 1h•Control offset less than 0.2°C at 40°C

Temperature Feed back

Current Control


-15V

┴

+15V

DAQ

4.5V

┴

Set-pointer

Switch

PT100

PT100

Peltier


Device simulation: Results

Application – set-up

To be finalised!


+15V

-15V

+5V

DAQ

Laser Diode HL4314MG

+2.7 V, 30 mA

Silicon PhototransistorSD3443

Battery

I/O Connector

DAQ Card PC

Channel 3


Experimental Evaluation

DNA hybridisation at microbead surface in 4 x 4 array

Accepted for Lab on a chip: December 2006 in press


Chemiluminescent Allergen Detection


Principle of Chemiluminescent allergen detection

h h

HRP HRP

PDMS PDMS

Alergen immobilisation

Microfluidic environment

Reaction Chamber

UCBL Lyon, France

Tyndall, IrelandGlass Superstrate (SU-8 on superstrate)

PDMS Substrate

SU-8 SU-8


Allergen deposition by piezo-electric spotter

•Matrix of alergen probes•Simultaneous detection of 48 probes•Incubation with serum of target patient


Optimization of Fluidics by Coventor Simulation

Coventor simulations

With MEMs CFD package

Steady and dynamic flow simulations as a function of :• Microfluidic design• Chip dimension• Experimental conditions (flow rate, etc..)

Allow us to see the distribution of flow velocities, or of the filling of a liquid in the microchannel

Compared various geometries of microfluidic system and flow cell design


Chemi-luminescent allergen detection

Interaction with UCBL (C. Marquette, K. Heyries, Lyon)

They perform: * immobilization of the proteins by spotting technique on PDMS * antigen/antibody assays with our microfluidic chip

• Assay requires uniform reagent distribution -> flow cell with optimized geometry (flow simulations using COVENTOR)

• Chip processing -> SU-8 -> Glass channels

• Need friendly chip-user interface, enabling reproducible measurements -> Chip holder


Acknowledgements

• I appreciate cooperation of all members of Biophotonics and Microfluidics Research Team

• Tyndall CFF Fabrication engineers and management team for their support.

• Jenny Patterson and Intel for finance of fabrication and processing costs (EI Intel Innovation Fund)

• Wataru Satoh JSPS Research Fellowships sponsored by Japan Society for the Promotion of Science

• Dr Miloslav Pravda Dept UCC Chemistry

dr michael loughran team leader biophotonics & microfluidics research integration of silicon and...

Documents

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tyndall national institute

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silicon microbead technology

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