Microfluidics for DNA Analysis

EE C245

Dr. Thara Srinivasan

Lecture 19
Picture credit: Nanogen

Lecture Outline
Reading from reader
Mastrangelo, C. H. Microfabricated Devices for Genetic
Diagnostics, (1998) pp. 1769-87.
Khandurina, J. et al., Bioanalysis in Microfluidic Devices,
(2002) pp. 159-83.
Zhang, L., et al., Microchip Electrophoresis-Based
Separation of DNA, (2003) pp. 1645-54.

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Todays Lecture
DNA and Analysis Methods
Scaling in Microfluidics
Survey of Microfabricated Chips

U. Srinivasan

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P

Genetic information is stored in chromosomes as

long strings of DNA grouped as genes

DNA

In humans, 46 chromosomes are 50 - 400 106

DNA units long (compared to 4 106 for E. coli)

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Units of DNA are nucleotides, consisting of:

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A base, a sugar and a phosphate bridge

Sugar linkage has directionality, 5 and 3 ends
Four bases: adenine, thymine, guanine, and
cytosine
Bases hydrophobic, backbone hydrophilic
Single-stranded DNA attaches to complementary
strand (G-C, A-T)

DNA Analysis
DNA is extracted from cell nucleus and purified
Break cell membranes using detergent
Remove cell debris, proteins, enzymes

DNA assays
Detect specific fragments in fingerprint pattern-matching mode
Sequence DNA fragment for base pair order of fragment

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Analysis tools

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Chemical amplification
Restriction digestion
Electrophoretic separation
Sanger sequencing process
Hybridization
Fluorescence visualization
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Amplification

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Polymerase chain reaction

Double-stranded DNA
denatured, 95C
Primers attach (anneal) to
strands, flanking section to
be amplified, 50-65C
Taq enzymes attach to
primer sites and synthesize
new strands from bases in
solution, 72C
Repeat cycle 20-30 times
to get effective
amplification
Macroscopic thermal
cyclers need 90 min per
amplification

Animations at: http://www.dnalc.org/resources/BiologyAnimationLibrary.htm

http://bldg6.arsusda.gov/pberkum/Public/sarl/cregan/pcr.gif

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Detection
Fluorescent labeling with molecules
which emit light when excited allows
extremely sensitive visualization of
fragment

Excitation maximum
Emission maximum

Intercalating dye: ethidium bromide

Single fluorophore: fluorescein

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Excitation
With UV laser-induced fluorescence,
emission signal must be separated
from excitation; requires confocal
microscope
Electrochemiluminescence (ECL)
uses Ru(bpy)2+3 end label, can be
detected with conventional CCD

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Cutting
Restriction digestion is
fragmentation of DNA

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Use restriction nuclease enzymes

to cleave DNA at specific locations
(can recognize specific sequences
of 4-8 bases)
Size distribution of restriction
fragments can fingerprint DNA
molecule

Molecular Biology of the Cell

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and Pasting

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Hybridization is hydrogen bonding

of two complementary single
strands of DNA
Occurs at specific T and salinity
conditions
In analyses, known strand is probe,
other is unknown and binding
indicates match
Recognition not perfect, single base
mismatches occur
DNA probes immobilized on surface
using linker make pixels for
microarrays
Microarray pattern matching

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ACGTA

TCGTA
AGCAT

CCGTA

GCGTA
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Separation
Electrophoresis to separate DNA fragments based on size

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Mobility EP depends on fragment size and charge and mobile phase

DNA fragments in solution are (-) charged and have constant charge
to length ratio
Additional molecular sieving matrixes are needed to separate DNA
based on length.
Fragments drift in race track where separation is L = EP Et
Separation resolution important

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Macroscale Separations
Macroscale gels
Thin multilane slabs; preparation is laborintensive
V up to 2 kV over 20-100 cm
Joule heating limits E to 5-40 V/cm
Good separation may require hours

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Capillary electrophoresis
Capillaries 10-300 m in diameter, 50 cm
long
Increased surface to volume ratio and
faster heat dissipation permits higher field
use (up to 1.2 kV/cm)
Good separation in < 1 hour
Use of confocal laser-induced
fluorescence

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Agilent
Technologies

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Sequencing

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Sanger method
Combine PCR and
electrophoretic separations
Duplication of DNA
fragment starts at primer
location, as in PCR
But in addition to
nucleotides in solution, also
add small amount of
dideoxy nucleotides
(ddNTPs) of one type (ddA,
ddC, ddG, or ddT).
When ddNTP is captured,
growing strand terminates,
resulting in
complementary strand
fragments terminated at all
possible positions for each
base
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Sequencing
Four-color sequencing

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Carry out four separate reactions, one for each base.

Electrophoretically separate each sample
Superimpose results to read out fragment sequence

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Todays Lecture

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DNA and Analysis Methods

Scaling in Microfluidics
Survey of Microfabricated Chips

13

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Miniaturization Benefits
Benefits
Reagent consumption ~ [s3]
Miniscule reaction volumes reduce reagent cost.

Heat transfer

~ [s2]

Surface phenomena

Mass transfer ~ [s2]

Reduced analysis times, with minimum assay time limited by
speed of enzyme (30-100 bp/s)

Flow is laminar

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Electroosmotic flow for valveless systems ~ [s2]

Capillary flow ~ [s1]

Separation efficiency ~ [s-2]

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Injection volume well-defined

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Miniaturization Issues
Issues
Detection limit ~ [s3],
S/N degraded as [s3] unless detector area scales with
sample [s1]

Pressure flows ~ [h3]

Other surface phenomena ~ [s2], [s1]

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Wall adsorption effects and sample evaporation [s2], capillary

forces [s1]

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Microfluidics Fabrication
Fabrication
Batch fabrication
Microchip cost ~ [s2], but limited by package cost

Parallelization to arrays easy

Portability increased

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Less need for external pumps, detection equipment

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Scaling and Microfluidics

Mastrangelo

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Scaling and Mixing by Diffusion

Mixing by diffusion
For channels 1 mm wide and flow velocities 1 cm/s, Re
is low and flow is laminar
Time required to travel distance x by diffusion is x2/2D
For channel width of 70 m and velocity 1 cm/s,
fluorescein (D = 3 10-6 cm2/s) will take 2 s to mix over
channel length of 2 mm

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Upper limit of 100 m width for channels

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Scaling and Diffusion Effects

While being carried by electroosmosis and drifted by
electrophoresis, a sample slab can spread out in width due to
diffusion
Ls
U0

in time transit =

LS

U 0 , infinitesimal slab grows to width

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Wmin =

D transit

DL S
DL S
=
U0
w Ex L D

If initial slab is smaller than Wmin, separation is limited only by

diffusion
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Scaling and Separation Efficiency

Separation efficiency
Number of theoretical plates, N, per unit time

N=

L2

2
x

x = 2 Dt =

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L
L2
t= =
,
U i EKV

2DL
,
EK Ex
t [Ld ],

Resolution parameter, R
Peak capacity, n
Signal to noise ratio, SNR

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N=

EKV
2D

L
V
d

N
1
2 = [ s 2 ]
t
d

R = l
n ~ L l

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Todays Lecture

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DNA analysis methods

Scaling in microfluidics
Survey of microfabricated devices

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Chip Electrophoresis
Capillary electrophoresis (CE) onchip
First demonstrations in 90s, Manz
group (Imperial College, London) and
Harrison group (Univ. of Alberta)
Separation 100 faster than slab
gels, 10 faster than CE

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CE chips

Caliper Technologies

Material ~ glass or plastic

Electrodes ~ metal pins inserted into
wells or patterned conductive layer
Separation medium ~ chips filled with
unpolymerized liquids are reusable
Layout ~ offset double-T
Detection ~ confocal fluorescence
microscope focused at single spot

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Array CE

Parallelization of CE
using arrays for high
throughput
384-channels radial
microplate for
genotyping
analyses in <7 min
with >98% success

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96-channel
wafer,
Mathies group
UCB
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Developments
Injection schemes to give
thinner plug
Higher resolution
separations

Santiago group, Stanford

For 10-1000 improved sensitivity, increase sample concentration by

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Sample stacking
Solid-phase extraction

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de Rooij group, University of Neuchtel

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Commercial CE Chips

Caliper Technologies and Agilent

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Sequencing On-Chip
DNA sequencing on a microchip
First demonstrated in 1995 by Mathies group, UCB:
150 bases in 540 s with 97% accuracy
In 2002, 96-channel plate demonstrated:
430 bases read in parallel at average rate of 1.7 kb/min
with >99% accuracy

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Mathies group

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Integrated Microfluidics
Mathies et al.,
Hilton Head 2002

Mathies group microfabricated 96channel CE plate with integrated:

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Pneumatic valves and pumps ~ PDMS

Resistive heaters and temperature
sensors ~ Ti/Pt
Photodiode detectors ~ amorphous Si

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PCR On-Chip
First on-chip device:
50 L microwell formed in Si
substrate with anisotropic etching
Bottom of well is SiN membrane with
poly-Si heaters on underside
Cover glass bonded to top
sandwiches tubing
Heating rate 15C/sec, cycle time
1 min

Northrup et al., LLNL Labs

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Recent developments:
Array detection of multiple DNA
fragments
Photodiodes integrated in microwells
to detect PCR products by
electrochemiluminescence
Reagent loading with inkjet
technology

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Gender determination with

CE-PCR, Mathies group

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PCR Devices
Woolley, Mathies and
Northrup et al., 1996

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Cepheid,
Sunnyvale CA
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Microarrays
Fabrication using lithography
and combinatorial chemistry
Fodor et al., 1991

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Glass coated with linker

molecule with photoremovable
protective group
UV light through mask removes
protective group selectively
Nucleoside with protected 5
end bonds to deprotected
linkers
Process repeated one base at
a time to give oligonucleotides
of arbitary length
Array of 1024 peptides in 10

steps (210 ), 100 m probe

patches

McGall et al., 1996, showed

technique which uses polyimide
photoresist as protective layer

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Affymetrix process
Basic microarray today

50-200 m patches on 1 cm2 chip

Up to 40,000 different probes
Possible oligonucleotides for 15-mer is 415
~ 109

Finished chip in flow-cell package

Detection mainly by fluorescent labeling 30

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How Microarrays Work

Investigate gene
expression in
healthy and
diseased cell
populations by
monitoring
messenger RNA in
cell nuclei.
mRNAs are
extracted, reversetranscribed into
complementary
DNA, and
fluorescently labeled
cDNA is hybridized
to microarray.

untreated

treated

Scientific American, Feb 2002

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Gene expression
profiling

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Developments
Affymetrixs GeneChip
Application-specific microarrays
Human set > 33,000 human genes ~ $800/2 chips

Workstation where hybridization, analysis and

data mining are performed

Issues

Molecular Probes

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Detection time is diffusion-controlled and slow

Nanogen uses electric fields to direct sample to
probes 25 quicker
By reversing direction of field, can denature
incorrectly bonded strands
Single base pair mismatches (SBPMs) denature 4
faster than exact matches
Need high-density electrically addressable circuit
plane

Cheaper detection in the works, i.e. electrochemiluminescent labeling

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Affymetrix

Nanogen32

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