Transcript Dr Michael Loughran Team Leader Biophotonics

Integration of silicon and glass
processing for lab on a chip development
• Dr Mike Loughran
• Tyndall National Institute
• Cork, Ireland
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Overview
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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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Dr Mike Loughran Brief Introduction
PhD Biotechnology Cranfield University 1995
JSPS Research Fellow Tokyo University of Fisheries from 1995-1997
Research Fellow Dublin City University, 1997-1999
Research Fellow University of Manchester, U.K , 1999-2001
Visiting Associate Professor Tokyo University 2001
Senior Research Fellow AIST Tsukuba Japan 2002-2004
Team Leader Biophotonics & Microfluidics Research
Tyndall National Institute 2004-2006
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Lab on Chip Research Development
Micro-reactor Surface coating
Micro-sensor Optical/ Electrochemical
Micropump/valve
Liquid transport
Intrinsic Advantages of m-tas lab on chip systems
High through put, rapid analysis, reduced reagent/sample consumption
Fabrication techniques
must be cost-effective
Economical
precise control of channel dimensions/geometry Accurate
preferably made on large scale wafers
Mass production
Continuing challenges
Sample transport/inter-connects
from bench to micro-chip
Reproducibility (feature size)
genuine versatile platform
Obscure terminology
“nano” or micro,
top down, bottom up
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Choice of substrate
Polymer micro-chips: replication technology, embossing, imprinting
In expensive, rapid proto-typing, non clean room processing
Solvent in-compatibility, channels not uniform, temp defects, opaque, hydrophobic
Examples Polycarbonate, PMMA, SU-8, PDMS
Silicon Micro-machining
Photo-lithography, wet etching, dry etching, anodic bonding, dicing
System integration: electrodes, micro-channels
Not suitable for high voltage capillary electrophoresis separations silicon breaks down at voltages > 1000 V
Glass processing
Planar 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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Micro chip fabrication techniques
(6) Strip photoresist Mask 2
(1) Wet oxidation
20µm
(2) Pattern photoreisst Mask 1 and etch
oxide to open sharrow etch windows
Alignment marks for mask layer 2
(7) Shallow etch silicon to 20 µm
20µm
(8) Etch wet oxide
(3) Strip photoresist Mask 1
(4) Pattern photoresist Mask 2
to open deep etch windows
200µm
Silicon (4 ")
Wet dioxide (0.5 µm)
(5) Deep etch silicon to 200µm
Dr Michael Loughran
(9) Anodic bonding
Photoresist 1 (Mask 1)
Photoresist 2 (Mask 2)
Pyrex glass lid
Team Leader Biophotonics & Microfluidics Research
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 alternative
e.g. Electron beam lithography (EBL) and focused-ion beam (FIB) lithography
(feature size 10 nm) to create nanochannels for DNA separation
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Fluidic Inter-connects
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Fluidic Inter-connects for microchip HPLC
Rheodyne Valve for Fluidic Switching
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Fluidic interconnects for allergen microarray
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
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)
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Customized microchip holder
Plastic chip holder
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Packaging of HPLC Microchip
Capillary
Without UV glue (Dead volume)
Silicon
Silicon
Channel
Capillary
Optical fibre
With UV glue
Dead volume
Capillary end
Air bubble
(a)
(b)
Silicon
Capillary
Channel
UV glue
(c)
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Final Mask Design with Integrated HPLC Chips
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
HPLC Chip Fabrication
A fabricated micro HPLC chip
Micropillar (5×5 µm)
UV detection
Separation
Injection
Injection channel
Figure 7
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Glass Processing:
Capillary Gel Electrophoresis
Rapid microchannel fabrication
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
SDS/Native Capillary Gel Electrophoresis in Single Glass
Microchip
Separation capillary
Stacking
capillary
Magnified view of
self-contained
stacking capillary
Length 12 mm,
diameter 1 mm,
depth of 300 μm.
Separation capillary length 60 mm, diameter 500 μm, depth 300 μm.
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Simultaneous SDS Native Electrophoresis In Multiple
Capillaries
Simultaneous
separation of both
native and SDS
marker proteins in
assembled chip
Experimental
conditions
constant current of
2 mA, 10 minutes
after sample preconcentration at 50V
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Wet etching of microfluidic structures in glass using photodefinable 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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Bonding and sealing of fabricated microfluidic glass chips
2 methods were applied to seal the microfluidic glass chips
• PMMA to glass dircect bonding
• PMMA/glass to glass bonding via PDMS interface layer
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Test of fabricated microfluidic glass chips
Outlet
Sequence 1
Dr Michael Loughran
Inlet
Sequence 2
Sequence 3
Team Leader Biophotonics & Microfluidics Research
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Electrowetting
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Principle of Electrowetting
Electrolyte
W.E.
PDMS
ON
OFF
OFF
OFF
Team Leader Biophotonics & Microfluidics Research
ON
hydrophilic
hydrophobic
hydrophobic
hydrophilic
hydrophilic
Dr Michael Loughran
ON
Experimental setup
setup
Control PC
CCD camera
potentiostat
Electrowetting droplet transport
recorded in dark room
Microfluidic chip
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
PDMS electrowetting micro-fluidic chip
Flow channel
Electrolyte
40 mm
Flow channel Reservoir
PDMS substrate
Working
Electrode
Insulating
layer
W.E.
(Au)
R.E. (Ag/AgCl)
300 mm
A.E. (Pt) Glass substrate
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
100 or 300 mm
15 mm
12.5 mm
Electro-wetting: glass microchip
fabrication
23 mm
23 mm
Wettability of glass more uniform than PDMS.
Diameter of microchannel reduced from 500 to 100 mm
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Fusion bonding: to seal glass electrowetting
micro-chip
• Materials
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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
weight
Setup
Ceramic plate
Glass flow channel was bonded by
Fusion bonding.
Alexandra helped me to complete
fusion bonding.
glass
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Images of glass microchip
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Encoded Silicon Microbead
Technology
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Encoded Silicon Microbeads - Design and fabrication
Results:
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
System Integration - Microbead Injection
Multiple microbead injections: Approach I
Cartridge Development
• Loading Mechanism
• Injection Mechanism
Image sequence showing successive injection of two microbeads
Problem: Repeatability of precise injection of individual microbeads
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Characterisation – Identification System
Implementation of optical detection system
[1] Laser Diode and Collimator - AlGaInP, 635 nm, 3 mW
1 cm
[1]
[2]
[3]
[5]
[2] Aperture - 3mm
[3] Beam Splitter - 50:50 Cube
[5] Photo Detector - Silicon Phototransistor
[4] Focusing Lens - Aplanatic Doublet (f - 6 mm)
[D] Encoded microbead
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
[4]
Encoded Silicon Microbeads - Optical Detection
Integrated detection system: Principle of operation
Detection of microbeads with through-hole barcode pattern
Slit
TypeB
M
icrobead
Integrated
VCSEL
Dr Michael Loughran
SiliconPhoto-Detector
Polym
erSuberstrate
w
ithM
icrochannel
Polym
erSubstrate
w
ithCavity
Team Leader Biophotonics & Microfluidics Research
Encoded Silicon Microbeads - Optical Detection
Integrated detection system: Illustration of fabrication process
ProcessFlow- Cavities
1a
ProcessFlow- Microchannels
1b
2
3
PMMA(Substrate - Cavities)
4
PMMA(Superstrate- Microchannels)
CastingResin(Master - Microchannels)
Silicon (Master - Cavities)
5
SU-8
VCSEL Package
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Encoded Silicon Microbeads - Optical Detection
Integrated detection system: Experimental set-up and results
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
System Integration - Reaction chamber
Integrated detection system: Illustration of fabrication process
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Application – Instrumentation
Experimental setup:
Temperature
Sensor
Microfluidic
Reaction Chamber
Flow
Control
Peltier Element
Temperature
Feed back
loop
Dr Michael Loughran
Syringe
Control
Current
Control
Temperature
Control
Electronics
Syringe Pumps
Temperature
Monitoring
PC
Team Leader Biophotonics & Microfluidics Research
Temperature Controller – Technical Description
Temperature Sensors
Temperature
Measurement
Electronics
Microfluidic Flowcell
DAQ
Peltier Element
Temperature
Feed back
Current
Control
Temperature
Controller
Electronics
PC
Temperature
Monitoring with
Labview
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
PT100
PT100
-15V
4.5V
┴
┴
+15V
Peltier
Switch
Dr Michael Loughran
Set-pointer
DAQ
Team Leader Biophotonics & Microfluidics Research
Application – set-up
Device simulation: Results
To be finalised!
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
DAQ Card PC
I/O Connector
+2.7 V, 30 mA
Laser Diode
HL4314MG
Battery
Channel 3

+5V

-15V
DAQ
Silicon Phototransistor
SD3443
Dr Michael Loughran
+15V
Team Leader Biophotonics & Microfluidics Research
DNA hybridisation at microbead surface in 4 x 4 array
Experimental Evaluation
Accepted for Lab on a chip: December 2006 in press
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Chemiluminescent Allergen
Detection
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Principle of Chemiluminescent allergen detection
Alergen immobilisation
hn
hn
HRP
PDMS
HRP
UCBL Lyon, France
PDMS
Reaction Chamber
Microfluidic environment
Glass Superstrate (SU-8 on superstrate)
Tyndall, Ireland
SU-8
SU-8
PDMS Substrate
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
Allergen deposition by piezo-electric spotter
•Matrix of alergen probes
•Simultaneous detection of 48 probes
•Incubation with serum of target patient
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research
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
Dr Michael Loughran
Team Leader Biophotonics & Microfluidics Research