Microsoft PowerPoint - EE-NFU-f-print_20091025.ppt []

~ Richard P. Feynman, 1959y ,
33Marc J. Marc J. MadouMadou, “Fundamentals of , “Fundamentals of MicrofabricationMicrofabrication: : The Science of Miniaturization, 2The Science of Miniaturization, 2ndnd Edition”, 2002Edition”, 2002



(Microarrays)
(L b O A Chi )(Lab-On-A-Chip)
(MEMS)(MEMS)
: (PCR) (PCR) (, , ) ( ): (CE)
77
19 bases
Time after injection (seconds)
99
PCR chamber (Dept. of ( p Biological Microsystem, IPHT Jena))
Chamber volume: 3 ~ 10 µl Heating/Cooling rate: 10 ~ g g 80 K/s Temperature/Heating control: thin-film transducers Thermal cycles: 30 PCR time: 10 ~ 30 mins
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Probe Molecule
Target Molecule
Probe Molecule
10 µm
1111
Synthesis Chips
Objective: Assembly a large DNA (~10,000 bps) within 24 hours
PCR ChamberPCR Chamber
Ligation/Fussion ChamberLigation/Fussion Chamber Check ValveCheck Valve
Cavity on Glass
Cavity on Glass
Channel On silicon Movable part of
the valve Diaphragm
valve close
Channel On silicon Movable part of
the valve Diaphragm
valve close
1212
Application: Detection of E. coli bacteriabacteria Functionalities
T t ll tTarget cell capture Cell pre-concentration and
ifi tipurification Cell lysis
iSize: 10cm x 6cm x 0.2cm Fabrication: Plastic
i f b i i R. Liu et al., “Self-Contained, Fully Integrated Biochips
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microfabrication R. Liu et al., Self Contained, Fully Integrated Biochips
for Sample Preparation, PCR Amplification, and DNA
Microarray Detection" Anal. Chem., 2004
: Microarrays
: :
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( b t t ) (substrate) DNA () () ()
(oDNA) (cDNA)
Important Features of DNA/Gene MicroarraysMicroarrays
Specificity (Accuracy): to ensure better p f y ( y) hybridization efficiency S iti it (D i R ) t d t tSensitivity (Dynamic Range): to detect gene expression differential levels Reproducibility (Spot-to-Spot & Array-to- Array CVs): to minimize technical errorsArray CVs): to minimize technical errors associated with technology platforms
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Specificity (Accuracy)
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Important Features of DNA/Gene Microarrays (Conti )Microarrays (Conti.)
Specificity (Accuracy): to ensure better p f y ( y) hybridization efficiency S iti it (D i R ) t d t tSensitivity (Dynamic Range): to detect gene expression differential levels
2020
Target Concentration (fM)
Linear dynamic range: 5 log
Important Features of DNA/Gene MicroarraysMicroarrays
Specificity (Accuracy): to ensure better p f y ( y) hybridization efficiency S iti it (D i R ) t d t tSensitivity (Dynamic Range): to detect gene expression differential levels Reproducibility (Spot-to-Spot & Array-to- Array CVs): to minimize technical errorsArray CVs): to minimize technical errors associated with technology platforms
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Spot-to-Spot Uniformity
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Spotted MicroarraysSpotted Microarrays Non-contact printing
Pi l i (P k d I• Piezoelectric pump (Packard Instrument, Incyte Pharmaceuticals)
• Syringe pump with solenoid valve• Syringe pump with solenoid valve (Cartesian Tech)
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Spotted MicroarraysSpotted Microarrays Contact printing
S li i (T l Ch I l )• Split-pin (TeleChem Intl.) • Pin-And-Ring (Genetic Microsystems)
ill i ( i )• Capillaries (Genomatrix)
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Hardware Print head Plate and substrate handlingPlate and substrate handling Positioning stage
i l C lEnvironmental Control Humidity (65% ~ 75%)y ( ) Dust (from ceiling tiles, ventilation systems, etc)etc)
Instrument Control Software
Sample Evaporation Solid PinSolid Pin
PinPin--AndAnd--Ring (PAR)Ring (PAR)
• Affymetrix (photolithography)• Affymetrix (photolithography) • Xeotron (digital photolithography)
XeoChipXeoChip GeneChipGeneChip
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N1
-OO-
Ni
O-P-amidite
Ni
DMT-O
N
OOR
N1
DeprotectionDeprotection 1 Detritylation1 Detritylation
DeprotectionDeprotection 1 Detritylation1 Detritylation
DeprotectionDeprotection 1 Detritylation1 Detritylation
DeprotectionDeprotection 1 Detritylation1 Detritylation
Repeat iRepeat i--1 times1 timesRepeat iRepeat i--1 times1 times O
1
N1
XEOTRONXEOTRON ConventionalConventional
Nucleotide MonomerNucleotide Monomer Nucleotide MonomerNucleotide Monomer Standard protecting groupStandard protecting group Photolabile protecting groupPhotolabile protecting group
NN NHROCH3
NN NHRO
O CH3
O NN
Chemistry not idealChemistry not ideal Very difficult for analogsVery difficult for analogs
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Automated Microarray Synthesis
O P
O P
O P
O P
O P
O P
O P
O P
O P
O P
O P
O P
P P PP P P P P PP P P
L
T
A C
G C
C G
GC
T T TC C C C C T T
L
T
A C
T T
In a stepwise synthesisIn a stepwise synthesisIn a stepwise synthesis, In a stepwise synthesis, one type of monomer is one type of monomer is coupled at a time to the coupled at a time to the predetermined sitespredetermined sitespredetermined sites.predetermined sites.
If the synthesis is done using photomask approach:If the synthesis is done using photomask approach:
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A 20A 20--mer oligo array could require 20x4 photomasks!mer oligo array could require 20x4 photomasks!
Digital Light Projector (DLP) for Microarray SynthesisMicroarray Synthesis
Texas Instruments DLP Xeotron DLP Process
3636
Xeotron’s Microfluidic Chip How it Works– How it Works
3737
H+
H+ H+
L L L L L L L L L L L L O P
O P
O P _
O P
O P
O P
O P
O P
O P
O P
O P
O P
_ _ _ _ _ _ _ _ _ _ _
L L L L L L L L L L L L O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
PGA Precursor
CH Cl
L L L L L L L L L L L L O H
O H
O H
O P
O P
O P
O H
O H
O H
O P
O P
O P
L
T
L L L L L L L L L L L
T T T T T
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
T
L L L L L L L L L L L
T T T T T
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
L
T
L L L L L L L L L L L
T T T T T
O P _
O P _
O P _
O P _
O P _
O P _
C-OP _
H+ H+
L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L L
P _
P _
P _
P _
P _
P _
P _
P _
P _
P _
P _
P _
T CT T T T TC C C C C
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
O P _
G C C G
PGA precursor
PG O _
3838
L L L L L L L L L L L L L T
L L L C
L L L L L L L L T T TC C C C CT TPGA
precursor T-O P _PG-O
lTest Cells disease …
Gene Expression Data
Sample A (Cy5) vs. Sample B (Cy3)Sample A (Cy5) vs. Sample B (Cy3)
Cl ster A Landmark of Gene E pression St d
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4141
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