Post on 03-Aug-2020
Biomedical Applicationsof MEMS
Dr. Bruce K. GaleFundamentals of Micromachining
Biochip Technology
with special thanks to Xiaolian Gao, University of Houston
Genetic Database• Challenges
– Function must be assigned to gene (discovery)– Location of gene determined (mapping)– How often is gene used (expression)– How do these genes differ between individuals
(genetic variation)
DNA Hybridization Arrays• High density arrays of polynucleotide
probes• Used for genetic sequence analysis• Why do we care?
– New targets for drugs or other therapeutic intervention
– Diagnostic markers for disease– Development of improved agricultural products
ACGT
Probe (immobilized on a surface)Probe (immobilized on a surface)
Target (in solution)Target (in solution)
hybridizationhybridization
Basic Principles of DNA Basic Principles of DNA MicroarraysMicroarrays How Important is the Sequence ?How Important is the Sequence ?Biological relevance of SNPs (Single Nucleotide Polymorphism)
1 acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcacc 61 tgactcctgX ggagaagtct gccgttactg ccctgtgggg caaggtgaac gtggatgaag 121 ttggtggtga ggccctgggc aggctgctgg tggtctaccc ttggacccag aggttctttg 181 agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag gtgaaggctc 241 atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac aacctcaagg 301 gcacctttgc cacactgagt gagctgcact gtgacaagct gcacgtggat cctgagaact 361 tcaggctcct gggcaacgtg ctggtctgtg tgctggccca tcactttggc aaagaattca 421 ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat gccctggccc 481 acaagtatca ctagctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc 541 taagtccaac tactaaactg ggggatatta tgaagggcct tgagcatctg gattctgcct 601 aataaaaaac atttattttc attgc
Hemoglobin B Hemoglobin B
SequenceSequence
If X = A, normal
If X = T
Sickle Cell Anemia
•• Fatigue, paleness, and shortness of breathFatigue, paleness, and shortness of breath•• PainPain•• Eye problems (can cause blindness)Eye problems (can cause blindness)•• Delayed growthDelayed growth•• InfectionsInfections•• StrokeStroke•• Acute chest syndromeAcute chest syndrome•• HandHand--foot syndromefoot syndrome•• Yellowing of the skin and eyes.Yellowing of the skin and eyes.
Biochip Biochip -- the beginning and terminologythe beginning and terminology
•• Southern, Southern, EkinsEkins, , Drmanc,andDrmanc,and MirzabekovMirzabekov, Affymetrix pioneered , Affymetrix pioneered (~1989)(~1989)
•• 22--D array of DNA molecules (D array of DNA molecules (probesprobes))
•• Probes are anchored to a glass substrateProbes are anchored to a glass substrate
•• When the array is exposed to other molecules (When the array is exposed to other molecules (targetstargets) ) carrying luminescence tags, the tags light up at the sites carrying luminescence tags, the tags light up at the sites where binding occurswhere binding occurs
•• The emitted intensity provides The emitted intensity provides qualitative and quantitative qualitative and quantitative information information -- reporting what reporting what molecules are in a sample, etc.molecules are in a sample, etc.
Manufacturing Methods• Photolithography
– Affymetrix chips
– Advantages• Precise• Small spot size• Control
– Disadvantages• Lower yield• Cost
• Mechanical printing (spotting)– Used by biologists– Soft lithography– Ink jet– Pins– Advantages
• Cheap• Longer chains
– Disadvantages• Less specificity• Lower density
Stanford Chips- Spotting• Use robot to spot glass slides at precise
points with complete gene sequences• Used to measure qualitative relative
expression levels of genes– Differential expression by means of
simultaneous two-color hybridisation
www.genomics.stanford.edu
Photolithographic Design• Smaller dimensions
allow higher density analysis
• Signal drops with sample size
• Longer probes require more steps– 4 steps per layer
• Number of probes (and masks) goes up at 4n
O
N1
DMT-O
O
N1
HO
2 Coupling2 Coupling2 Coupling
O-P-amidite
Ni
DMT-O
3 Oxidation3 Oxidation3 Oxidation
O
N1
OO- -
Ni
DMT-O OPOR
=-
O
N1
-OO-
Ni
DMT-O POR
-
Repeat i-1 timesRepeat iRepeat i--1 times1 times
O
N1
OO- -
N2
OPOR
=-
O- -O
NiOPOR
=-
OO- -
N3OPOR
=-
OO- -
N4OPOR
=-
HO
O
N1
AcO FailureFailurefragmentsfragments
4 Capping4 Capping4 Capping
Deprotection1 Detritylation
CCl3CO2-H+
DeprotectionDeprotection1 Detritylation1 Detritylation
CClCCl33COCO22--HH++
Standard Conventional DNA Oligonucleotide SynthesisStandard Conventional DNA Oligonucleotide Synthesis
O
N1
-OO-
Ni
HOPOR
-
L L L L L L L L L L L LOP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
hv hv
L L L L L L L L L L L LOP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
PGA Precursor
CH2Cl2
L L L L L L L L L L L LOH
OH
OH
OP
OP
OP
OH
OH
OH
OP
OP
OP
T-OP_
L
T
L L L L L L L L L L L
T T T T T
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
PGA precursor L
T
L L L L L L L L L L L
T T T T T
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
L
T
L L L L L L L L L L L
T T T T T
OP_
OP_
OP_
OP_
OP_
OP_
OOO OOOH HH H HH
C-OP_
L
T
L L L
C
L L L L L L L L
T T T T TC C C C C
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
LT
L L LC
L L L L L L L L
A CT T T
A CC C C C C
G CC G
AT A
TC
AT
C
AT
C
GA
C
GA
ACT
ACT
AGC
AGC
T T
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
OP_
PGA precursor
PGA precursor
_A-O P
PC-O _
T-O P_PG-O
_
hv hv
HHHH++++HHHH++++HHHH++++ HHHH++++
HHHH++++HHHH++++
LightLight--directed Parallel Synthesis of oligodirected Parallel Synthesis of oligo--DNADNAUsing AcidUsing Acid--labile Groups Protected Phosphoramiditeslabile Groups Protected Phosphoramidites
HHHH++++HHHH++++HHHH++++ HHHH++++
HHHH++++HHHH++++
Basic Fabrication• A, C, G, and T bases
applied to each layer– Each has its own
protective block
• Photolithography or printing used to define location by removing block
• Multiple methods
Photoresist Method• Single layer and
bilayer methods• Bilayer method
provides better chemistry for nucleotides
Digital Light Projection Digital Light Projection -- A solution for A solution for flexibility, simplicity and reduced costflexibility, simplicity and reduced cost
Digital Light Projector from Digital Light Projector from Texas InstrumentsTexas Instruments Chip Projector at XeotronChip Projector at Xeotron
PCR• Technique used to produce a large number of
copies from a target DNA sequence• Repetitive 3 step process
– Denaturation (~95ºC)– Annealing (~55ºC)– Chain Extension (~ 72ºC)
• Creates 2n copies– Typically 30 cycles
• Typical molecular analysis problems require statistically significant quantities and must pass detection limits on the order of millions and billions of molecules
How PCR Works• Basic PCR Reagents
– Template DNA– Complementary Primers (~20
nucleotides)– Thermostable Polymerase
Enzyme (TAQ)– Single nucleotides (A,C,G,T)– Buffers (pH and ionic
concentrations)
• High temp to split strands• Low temperature to anneal• Medium temp to extend• Repeat
Why Apply Micromachining?• Small reagent costs• Fast cycling time
– Low thermal mass– High surface to volume ratio
• System integration– Electrophoresis– Point of care system
• Low cost
Design Considerations• Biocompatibility• Chamber volume• Control system• Bulk or surface
micromachining• Bonding method (if
necessary)• Move the fluid or
cycle in position• External equipment
• Components– Heater– Chamber– Reagent mixing– Temperature control– Feedback– Detection?
Temperature Control• Heaters
– Boron doped regions– Metallization– Other?
• Temperature measurement– Thermistor– Thermopile