Applications:

BioMEMS

CSE 495/595: Intro to Micro- and Nano- Embedded Systems

Prof. Darrin Hanna

Microfluidics

Reynolds number
Ratio of kinetic energy to rate of loss of energy due to
friction
vD/
is fluid density
v is the average velocity
D is the diameter of the channel
is the absolute viscosity
Below approx. 2,300 flow is laminar
slower at edges
Higher turbulent

Microfluidics

Reynolds number
microfluidics usually water-based fluids are used
approx. 1 gm/cm3
approx. 0.01 g / (cm s)
Example
D = 30 m
1 mm/s
Reynolds number = 0.03
usually below 1
No mixing
Joining streams flow side-by-side with only diffusion
special flow structures for mixing
increase the area of diffusive mixing

Microfluidics

Agilent Cell LabChip.

detects cells stained with fluorescent dyes
vacuum pulls separate flows of cells and buffer
Y-shaped junction

Microfluidics

flow of cells pushed to one side of the microchannel by the

flow of buffer
Individual stained cells are detected as they pass under an
excitation beam and fluoresce
If channels were same width as a cell the cell would clog

DNA

deoxyribonucleic acid
built from nucleotides
4 types of nucleotides
adenine, thymine, cytosine, guanine
A
T
C
G
Genes
sequences in the DNA encodes
functional product (i.e. protein)
Proteins
required for structure, function, and
regulation of cells, tissues, and organs
each protein has unique functions

A T
CG

DNA

Proteins
made up of amino acids
20 amino acids
Chromosome
self-replicating structure of cells
containing the cellular DNA that bears
in its nucleotide sequence the linear
array of genes

DNA

Human genome
23 separate pairs of chromosomes (46 chromosomes)
averaging 130 million base pairs in length each
= total of about three billion base pairs
genes that form the template for proteins are typically 27,000
base pairs long
only about 1,000 are used, rest is filler

DNA

Each nucleotide molecule has two ends

3 and 5
corresponds to hydroxyl and phosphate
groups attached to the 3 and 5 positions
of carbon atoms in the backbone
two strands joined together by weak
interactions
A T
CG

Copying DNA

amplification
Polymerase chain reaction (PCR)
1980 Kary Mullis
Awarded Nobel Prize Chemistry 1993
PCR
separate the two strands and use each as a template
replicate compliments

Copying DNA

Copying DNA

raise temperature of the DNA fragment to 95C

denature the two strands
Incubation occurs next at 60C
DNA polymerase such as Taq polymerase
ample supply of nucleotides (dNTPs)
two complementary primers
short chains of nucleotides that match up using
complementarity with a very small segment of the DNA
fragment
defines the starting point for the replication process

Copying DNA

raise temperature of the DNA fragment to 95C

denature the two strands

Copying DNA

DNA polymerase enzyme (Taq) catalyzes construction

begins from the position of the primer
proceeds 5 3 direction
Replication of a portion of the single strand is rapid
~ 50 bases per second
cycle ends with two
identical copies of only the
sections between (and
including) the primers
in addition to the
starting DNA template

Copying DNA

each cycle increases the number of identical copies with a

factor of 2n, n is the number of cycles
after 20 cycles, about one million copies have been created
efficiency drops after about 20 cycles
30 to 40 cycles are typically sufficient

Copying DNA

Copying DNA

SHOW PCR CLIP

Copying DNA

Mini-PCR
small chambers greater ratio of surface area to volume
surface area affects the rate of heat conduction
volume determines the amount of heat necessary for
a thermal cycle
enables faster thermal cycling in PCR
less sample and volume of expensive reagents
integrated system
detection scheme
electrophoretic separation
tagging
process is simplified, making it faster, less expensive,
and more repeatable

Copying DNA

PCR on a silicon chip ~ 1994

Lawrence Livermore National Laboratory (LLNL)
thermally cycle a solution between the denaturing and
incubation temperatures
approximately 95C and 60C
one chamber, with a volume of 25 to 100 l, is made of
two silicon chips with etched grooves
bonded together
SiN window
bare silicon inhibits PCR amplification
disposable polypropylene liner added to chamber
slows the heat/cool rate from an all-silicon
version to about 8C/s

Copying DNA

Copying DNA

polysilicon heater on a silicon nitride membrane for heating

the fluid inside the chamber with external sensor
platinum heater can do both heating and sensing operations
temperature variations as high as 10C across the chamber
temperature uniformity move heater away from the
membrane so that heat flows through the Si walls
fan can be added for more rapid cooling
modifications yield tighter closed-loop temperature control
enables faster cycling, from around 35s per cycle to as
little as 17s per cycle
Large-scale devices ~ 4 min per cycle!

Detection - dyes

add TaqMan dyes (probes)

link to certain sections of a DNA strand like the primers
do
results in fluorescence of green light from each
replicated DNA strand when excited by blue or ultraviolet
light
intensity of the fluorescence is proportional to the
number of replicated DNA matching strands
typically no detectable fluorescence signal for the first 2025
cycles
after cycling on the order of 515 minutes, the signal
appeared and rapidly grows if there is a match
simultaneous DNA amplification for multiple

DNA Sequencing

amplify DNA strand and chemically label the amplified

DNA fragments with specific fluorescent or radioactive tags
detect labeled DNA products
electrophoresis
separates DNA (charged), in suspension under the effect
of an electric field

DNA Sequencing

In solution, a hydrogen ion dissociates from DNA backbone

net negative charge on DNA strand
charge-to-mass ratio is approximately the same for strands of
different lengths
when driven with an electric field through a molecular
sieve, larger molecules move more slowly
groups of small molecules move farther than larger ones
over time

DNA Sequencing

gel electrophoresis
DNA products put in at edge of porous gelatinous sheet
20 to 100 cm long
electric field is limited to only 540 V/cm
heating problem
capillary electrophoresis
products are fed into thin capillary tube
10 to 300 m in diameter and ~ 50 cm long
applied electric field of up to 1,200 V/cm
higher fields can be used with smaller cross sections
due to the ability to remove heat more rapidly
tag DNA with tag to light up strands across gel
radioactive or florescence

DNA Sequencing

Sanger method of sequencing

for fragments up to about 1,000 bases long
many identical copies of single, denatured sections of
DNA
replication is started from the 5 end, just as in PCR
a small concentration of bases in the solution of one type
is altered so that the replication of that DNA strand stops
when the replication-halting base is used
results in copies of the original strands of varying length
that always end in a particular base

DNA Sequencing

Sanger method of sequencing (contd)

same is done in separate solutions with small
concentrations of replication-halting bases of the other
types
four groups of variable-length copies undergo
electrophoresis in four parallel channels
sequences of each length are separated for reading
results from the four channels are compared to infer the
entire sequence of the strand

DNA Sequencing

DNA Sequencing

Miniaturization
capillary electrophoresis
length of the sample emitted can be kept short
on the order of 100 m
reduces distance for the fragments of different
lengths to travel to separate
reduces length of the channel decreases the applied
voltage to maintain a high electric field
from few kilovolts down to hundreds of volts
faster separation with shorter distances
overall volume of DNA and reagents decreases
significantly to one microliter or less

DNA Sequencing

capillary electrophoresis on a chip ~ 1992

University of California, Berkeley
first in 1994 to demonstrate DNA sequencing by
capillary electrophoresis on a glass chip
two orthogonal channels etched with HF acid into a first
glass substrate
a short channel for injecting fluid and a long channel for
separating the DNA fragments. A second glass substrate
covers the channels

DNA Sequencing

secure second glass substrate to first substrate

adhesive or thermal bonding
holes etched or drilled with a diamond-core drill in the top
glass substrate
fluid access ports
both channels are typically 50 m wide and 8 m deep
can be as wide as 100 m and as deep as 16 m
separation channel is 3.5 cm long
surfaces of the channels have a coating to eliminate charging
prevent electroosmosis
injection and separation channels are filled with sieving matrix
of hydroxyethylcellulose by applying a vacuum to one end

DNA Sequencing

DNA Sequencing

fluid containing DNA fragments put into the injection channel

fragments are electrophoretically pumped by means of an
electric field of 170 V/cm
3060s
injection-channel loading time is critical
too short, more short DNA fragments are injected in
the next step
too long, the sample is biased toward longer fragments
applied voltage is switched to separation channel
applied electric field directs fragments from the
intersection of the two channels into the separation channel

DNA Sequencing

electric field of 180 V/cm

~ 2 min to complete the separation of the DNA fragment
compare to 8 to 10 hours to complete an equivalent
separation using conventional gel electrophoresis
compare to 1 to 2 hours with conventional capillary
electrophoresis
optical imaging of a fluorescent tag on each DNA fragment is
used to detect separated products inside the channel
up to 1,000 nucleotides long

DNA Sequencing

Agilent DNA 1000 LabChip

DNA concentration range of 0.550 ng/l
size range of 251,000 base pairs
size accuracy of 15%
resolution better than 10% over most of the range
sample volume is 1 l and takes 30 min to analyze

DNA Hybridization Arrays

preassembled nucleotides attached to a substrate

DNA sections to be identified are tagged with a fluorescent
dye at one end
lengths range of a hundred to thousands of bases
buffer solution on the substrate
sections of some of the unknowns hybridize to the
complementary sequences on the substrate

DNA Hybridization Arrays

substrate is then rinsed and illuminated

locations of fluorescence indicate hybridization and thus
which sequences are present
detection of specific gene mutations
search for known pathogens

DNA Hybridization Arrays

Using a standard photolithographic mask

UV light is shone through 20-m square openings
remove the protection groups
activating selected sites on the substrate

DNA Hybridization Arrays

A solution containing one type of nucleotide with a removable

protection group is flushed across the surface
nucleotides bond to activated sites in each square that was
exposed but not in the other areas

DNA Hybridization Arrays

process is repeated to start chains of other three-nucleotides

repeated exposure with different masks to remove the protection
groups and flushing with the four nucleotide solutions grow
DNA strands
typically 25 nucleotides long

DNA Microelectrodes

DNA Microelectrodes

DNA Microelectrodes