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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
• ρ∙v∙D/µ
• ρ is fluid density
• v is the average velocity
• D is the diameter of the channel
• µ 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
• ρ approx. 1 gm/cm3
• µ approx. 0.01 g / (cm ∙ s)
• Example
• D = 30 µm
• 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
C–G
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
C–G
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 95ºC
• denature the two strands
• Incubation occurs next at 60ºC
• 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 95ºC
• 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 95ºC and 60ºC
• one chamber, with a volume of 25 to 100 µl, 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 8°C/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 10°C 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 20–25
cycles
• after cycling on the order of 5–15 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 5–40 V/cm
• heating problem
• capillary electrophoresis
• products are fed into thin capillary tube
• 10 to 300 µm 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 (cont’d)
• 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 µm
• 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 µm wide and 8 µm deep
• can be as wide as 100 µm and as deep as 16 µm
• 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
• 30–60s
• 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 0.5–50 ng/µl
• size range of 25–1,000 base pairs
• size accuracy of ±15%
• resolution better than 10% over most of the range
• sample volume is 1 µl 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-µm 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