Biomedical Applications

Biochip Technology
of MEMS
Dr. Bruce K. Gale with special thanks to
Fundamentals of Micromachining Xiaolian Gao, University of Houston

Genetic Database DNA Hybridization Arrays

• 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)
• 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
 Basic Principles of DNA Microarrays How Important is the Sequence ?
Biological relevance of SNPs (Single Nucleotide Polymorphism)
1 acatttgctt ctgacacaac tgtgttcact agcaacctca aacagacacc atggtgcacc

Target (in solution) 61

121
tgactcctgX
ttggtggtga
ggagaagtct
ggccctgggc
gccgttactg
aggctgctgg
ccctgtgggg
tggtctaccc
caaggtgaac
ttggacccag
gtggatgaag
aggttctttg
Hemoglobin B 181 agtcctttgg ggatctgtcc actcctgatg ctgttatggg caaccctaag gtgaaggctc
241 atggcaagaa agtgctcggt gcctttagtg atggcctggc tcacctggac aacctcaagg

Sequence 301
361
gcacctttgc
tcaggctcct
cacactgagt
gggcaacgtg
gagctgcact
ctggtctgtg
gtgacaagct
tgctggccca
gcacgtggat
tcactttggc
cctgagaact
aaagaattca
421 ccccaccagt gcaggctgcc tatcagaaag tggtggctgg tgtggctaat gccctggccc
481 acaagtatca ctagctcgct ttcttgctgt ccaatttcta ttaaaggttc ctttgttccc

Probe (immobilized on a surface) 541

601
taagtccaac
aataaaaaac
tactaaactg
atttattttc
ggggatatta
attgc
tgaagggcct tgagcatctg gattctgcct

hybridization • Fatigue, paleness, and shortness of breath

• Pain If X = A, normal
• Eye problems (can cause blindness)
• Delayed growth
• Infections
• Stroke
• Acute chest syndrome If X = T
ACGT • Hand-
Hand-foot syndrome
• Yellowing of the skin and eyes. Sickle Cell Anemia

Biochip - the beginning and terminology Manufacturing Methods

• Southern, Ekins,
Ekins, Drmanc,and Mirzabekov,
Mirzabekov, Affymetrix pioneered
(~1989) • Photolithography • Mechanical printing
• 2-D array of DNA molecules (probes
(probes)) – Affymetrix chips (spotting)
– Used by biologists
• Probes are anchored to a glass substrate – Advantages – Soft lithography
• When the array is exposed to other molecules (targets
• Precise – Ink jet
(targets))
• Small spot size – Pins
carrying luminescence tags, the tags light up at the sites
where binding occurs • Control
– Advantages
– Disadvantages • Cheap
• The emitted intensity provides • Lower yield • Longer chains
qualitative and quantitative • Cost
information - reporting what – Disadvantages
molecules are in a sample, etc. • Less specificity
• Lower density
 Stanford Chips- Spotting Photolithographic Design
• Smaller dimensions
• Use robot to spot glass slides at precise allow higher
points with complete gene sequences density analysis
• Signal drops with
• Used to measure qualitative relative
sample size
expression levels of genes • Longer probes
– Differential expression by means of require more steps
simultaneous two-color hybridisation – 4 steps per layer
• Number of probes
(and masks) goes
up at 4n
www.genomics.stanford.edu

Standard Conventional DNA Oligonucleotide Synthesis Light-

Light-directed Parallel Synthesis of oligo-
oligo-DNA
Using Acid-
Acid-labile Groups Protected Phosphoramidites
Ni hv hv
N1
DMT-O H+ H+ H+ H+
O- P -O H+ H+
-

22 Coupling
Coupling Ni OR O 33 Oxidation
Oxidation
___ ___ ___ ___
PPP PPP PPP PPP
___ ___ ___ ___
PPP PPP PPP PPP HHH P P P HHH P P P
OOO OOO OOO OOO PGA OOO OOO OOO OOO OOO OOO OOO OOO _
DMT-O LLL LLL LLL LLL Precursor LLL LLL LLL LLL LLL LLL LLL LLL T-OP
O-P-amidite
CH2Cl2
Ni
Ni N1
N1 DMT-O O N1
HO
HO
- =

O- P -O
O-OP -O N1 hv hv
OR O
-

OR O ___ ___ _ _ _ H+ H+ ___ H+ H+ ___ ___

DMT-O 44 Capping
O Capping PPP
___
PPP
___
PPP H+ PPP
_H
+ PPP PPP
OOO OOO OOO _ _ _ OOO __ OOO OOO
PPP PPP PPP PPP H HH H HH
TTT OOO TTT OOO T T T OOO TTT OOO TTT OOO TTT OOO
Deprotection
Deprotection
PGA
precursor
_
C-OP
N1 LLL LLL LLL LLL LLL LLL LLL LLL LLL LLL LLL LLL
11 Detritylation
Detritylation
- + AcO
CCl
CCl33CO
CO22H- +
H O Failure
fragments
Repeat
Repeati-
i-i-1
1 times
times ___ ___ ___ ___
PPP PPP PPP PPP
OOO OOO OOO OOO
Ni ___ ___ ___ ___ _ CCC TTT GGG AAA
HO O N4 PPP PPP PPP PPP PGA A-OP
O N3
OOO OOO OOO OOO _ GGG CCC AAA TTT
- =

precursor
O- P-O
O N2
C-O P AAA AAA CCC CCC
- =

TTT CCC TTT CCC _

OR O- P -O O N1 TTT CCC TTT CCC
- =

O- PGA G-OP _
OR P -O LLL LLL LLL LLL LLL LLL LLL LLL
- =

precursor
OR O- P -O T-OP
OR O
 Basic Fabrication Photoresist Method
• A, C, G, and T bases • Single layer and
applied to each layer bilayer methods
– Each has its own • Bilayer method
protective block provides better
• Photolithography or chemistry for
printing used to define nucleotides
location by removing
block
• Multiple methods

Digital Light Projection - A solution for PCR

flexibility, simplicity and reduced cost
• 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

Digital Light Projector from

Chip Projector at Xeotron
• Typical molecular analysis problems require
Texas Instruments statistically significant quantities and must
pass detection limits on the order of millions
and billions of molecules
 How PCR Works Why Apply Micromachining?
• Basic PCR Reagents
– Template DNA • Small reagent costs
– Complementary Primers (~20
nucleotides)
• Fast cycling time
– Thermostable Polymerase – Low thermal mass
Enzyme (TAQ)
– Single nucleotides (A,C,G,T)
– High surface to volume ratio
– Buffers (pH and ionic • System integration
concentrations)
– Electrophoresis
• High temp to split strands – Point of care system
• Low temperature to anneal • Low cost
• Medium temp to extend
• Repeat

Design Considerations Temperature Control

• Biocompatibility • Components • Heaters
• Chamber volume – Heater – Boron doped regions
• Control system – Chamber
– Metallization
– Reagent mixing
• Bulk or surface – Other?
– Temperature control
micromachining
– Feedback • Temperature measurement
• Bonding method (if – Detection? – Thermistor
necessary)
– Thermopile
• Move the fluid or
cycle in position
• External equipment