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