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Cell
Sorting
Acoustic
Differential Extraction
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| This
project highlights the use of acoustic forces in a valveless
microfluidic device to trap sperm cells in the presence of female
epithelial DNA obtained from sexual assault evidence. The
device (Figure 1) is comprised of two layers: a printed circuit board
layer containing microtransducers, and a glass fluidic layer.
An ultrasonic frequency tuned specifically to the transducer
characteristics and channel dimensions is applied to the device, and an
acoustic standing wave is set up within the microchannel, producing a
trapping zone at a pressure node. This method exploits the
density, volume, and compressibility differences between sperm cells
and free DNA from epithelial cell lysate to create a force strong
enough to retain the sperm cells at these nodes, while allowing the
free DNA to pass through the device. Laminar flow valving is
implemented to direct the two fractions to separate outlets. |
(back to top)
Enhanced
Sperm Cell Recovery from Cotton Swabs for
Rape Kit Analysis
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| Comparison of sperm
cell recoveries from elutions using differential extraction
buffer and anionic detergents. Inset: Effect of
SDS concentration on the release of sperm cells from samples.1 |
Regardless of
the method
utilized for the separation of vaginal and sperm cell DNA, the overall
effectiveness of the procedure is ultimately dependent on the
efficiency with
which material can be eluted and recovered from a cotton swab. The
issue is
especially important with swab samples containing low numbers of sperm
cells,
where any loss makes it even more difficult to obtain a profile of the
perpetrator. This
project focuses on the
development of improved methods for cell elution from a cotton matrix.
Several alternative
methods of intact cell removal have been investigated; including the
use of
cellulase-based enzyme mixtures,1 as well as
the exclusive use of
detergents.2
These methods
can be utilized in conjunction with or to circumvent conventional
differential
extraction.
1. Norris,
J.V., Manning, K., Linke, S.J., Ferrance, J.P.,Landers, J.P. Expedited,
chemically-enhanced sperm cell
recovery from cotton swabs for rape kit analysis.
J Forensic Sci 2007 (in press).
2.
Voorhees,
J.C., Ferrance, J.P., Landers,
J.P.
Enhanced elution of sperm from cotton swabs via
enzymatic
digestion for
rape kit analysis. J Forensic
Sci 2006; 51(3):574-9.
Solid Phase Extraction (SPE)
New
Monolith Stationary Phase for Microfluidic DNA Purification
Today, solid-phase extraction (SPE) is the most popular preparation
method for the extraction and preconcentration of analytes. To obtain a
high loading capacity, a large surface area of the solid phase is
desired. Porous polymer monoliths are a new category of materials
developed during the last decade. These materials are prepared using a
very simple process in which a mixture of monomers and porogenic
solvent is polymerized within a closed tuber or other container under
carefully controlled conditions. Thus, the monoliths could be prepared
into any shape. The polymerization mixture typically contains monomers,
free-radical initiator, and porogenic solvent which affords macroporous
materials with both large through-pores with a pore size of 1 to 20
µm and small meso-pore in 100-1,000 nm size range. The pore
size can also be controlled over a broad range by different ratio of
porogenic solvents. Since all the mobile phase must flow through the
monolith, the mass transport within the monolith is dominated very much
by convection, and the monolithic materials performed very well at high
flow rates.
UV-induced photo-polymerization enables the accurate placement of
monolithic matrices within the architecture of microscale devices, and
the functional surface groups on the monomers allows for easy chemical
modification of the surface. These methods have been used to create a
high capacity, high efficiency silica-based DNA extraction monolithic
column within a microfluidic device.
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| Figure (A)
High
resolution of scanning electron micrograph of monolith internal
micro-structure. (B) A two parallel straight channel design illustrated
the accurate localization of solid phase within a channel. |
Microfluidic-based
Nucleic
Acid Purification in a Two-Stage, Dual-Phase Microchip
The need for
high DNA
binding capacity is important in many clinical applications that rely
on whole
blood as a source of genomic DNA. Lysed whole blood contains nucleic
acids,
proteins, lipids, metabolites, and inorganic ions, some of which are
known to
inhibit PCR, a technique used in almost all genetic analyses.
However,
the proteins in blood also bind to the solid phase limiting the DNA
binding
capacity. To solve this problem, a two-stage, dual phase
microdevice for
DNA extraction from whole blood has been developed. This
device captures
proteins using an in-line C18 phase, allowing the DNA to bind to the
DNA
extraction phase with significantly decreased protein
competition.
Successful PCR amplification following purification of DNA from human
whole
blood illustrated the effectiveness of the method. An added
benefit of
this method is the ability to remove the protein wash step normally
required to
remove proteins from the DNA extraction phase; this accelerates the
analytical
process, reduces the number of steps and eliminates potential sample
contamination that may occur from switching syringes or
tubing. With the
majority of the extracted DNA released in a small volume, this system
is ideal
for concentrating and purifying DNA from whole blood on integrated
microfluidic
devices.
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|
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Figure (A)
Integrated
protein capture/DNA extraction in
two-stage, dual-phase microdevice for DNA purification from
10 µL
human whole blood. Arrows from reservoir 1 to 2 show flow
through stage 1. Arrows
from reservoir
2
to 3 show
loading through stage 2. DNA elution was performed in the reverse
direction from reservoir 3 to 2. Masses and percentages of protein and
DNA at each stage were also incorporated to show the mass balance
through the system. (B) The channels are filled with dyes for
better visualization of the protein and nucliec acid capture phase
regions within the microchannel architecture. Chip
dimensions: 3 cm (length) x 2.5 cm (width). Reverse phase
(C18) packed bed length: 10 mm; monolith phase length: 4 mm; solid
phase volume: 0.4 µL. |
Large Volume Reduction
Solid Phase Extraction (vrSPE)
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The figure above shows the microdevice used for integrating vrSPE with subsequent SPE for the extraction of DNA.
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| Often
times in
forensic cases large volumes are required to
elute or solubilize a blood sample from various surfaces including
clothing and
walls. In contrast, microdevices are characterized by small sample
volumes. This
raises the need for a
sample processing step that would reduce the volume of the forensic
sample,
providing both a crude purification and sample concentration effect. My investigations focus on
a volume reduction
microdevice utilizing various solid phases including
chitosan-coated-silica
particles (charge switch binding) and magnetic silica particles
(hydrogen
bonding). A
comparison of the extraction
efficiency of the two phases will be investigated, in addition to
optimizing
the integration with a second, silica based SPE step.
Later work will also involve integration with
other processing steps including PCR, ME,
and fluorescence detection. |
Plastic
Solid Phase
Extraction (SPE) Microdevices
Silica or sol-gel
based solid phases in glass microdevices are
normally employed for DNA extractions but have several disadvantages
including
the difficulty in reproducibly packing the channel and the cost of the
glass
devices. Polymeric
devices provide for a
cost effective method to produce microdevices which could be
disposable, and
current fabrication methods allow for establishment of a polymeric
matrix
within a microchannel during the fabrication process.
This
project will investigate the designs utilized in generating a highly
reproducible
solid phase, and the derivatization of the phase for extraction of DNA
from
these polymeric devices. This
device
will be used to perform extractions of samples including blood, cells,
frozen
tissue, and laser-microdissected histological tissue which can play a
major
role in cancer research.
(back to top)
RNA
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Messenger
RNA expression analysis requires
isolation of RNA from
biological
samples, followed by reverse transcription-PCR (RT-PCR) amplification
and
separation of target amplicon to identify the sample. The
method is based
on the inherently variable mRNA expression from different cell types,
producing
gene-specific patterns which can be verified by the presence of a
unique mRNA
expression patterns. Recently, Juusola et al. [1]
described
a method using mRNA expression to identify specific body
fluids. To
obtain mRNA for the transcription and amplification necessary for gene
expression analysis, RNA must first be isolated and purified from the
biological source of interest. Consequently, a robust system
for purification
of RNA will be essential as the development of messenger RNA expression
analysis methods unfold. A closed microfluidic, silica-based
purification
system would represent a significant improvement to current
methodologies for
RNA extraction, which often involve time- and reagent-consuming organic
extractions, by decreasing the opportunity for introduction of
contaminants and
RNases, as well as reducing the amount of sample, reagents, and time
required
to perform this delicate isolation. The application of a
silica-based
microchip solid-phase extraction method for purification of RNA as a
precursor
to mRNA profiling would be also be a promising step toward simultaneous
DNA and
RNA purification technology, permitting a more comprehensive genetic
analysis
from a single source. In addition, development of a
single-process RNA
purification device represents the first step towards creation of an
integrated
micro-total analysis system (μ-TAS) capable of total mRNA
profiling. Work
with silica and alternative phases to silica for extraction of RNA is
underway
for use in genetic analysis as well as clinical diagnostics.
1. Juusola, J.; Ballantyne, J. Forensic Science
International 2003, 135,
85-96.
Testing
of new phases
A well-characterized method for the adsorption of
nucleic acids to
silica surfaces has been established and exploited for the purification
of DNA
and RNA from biological samples in commercially-available macroscale
systems. Using a chaotropic salt to promote adsorption of DNA
and RNA to
the silica surface, contaminating proteins can be removed through a
series of
washes and the purified nucleic acids recovered for downstream genetic
analysis. This method has been translated for use in
microfluidic systems
for DNA purification, where reduced sample and reagent consumption, as
well as
a reduction in analysis time has been achieved1-5.
Currently,
work is underway to explore alternative matrices for solid phase
extraction of
DNA from biological samples.
1. Breadmore, M. C.; Wolfe, K. A.; Arcibal, I. G.; Leung, W.
K.;
Dickson, D.; Giordano, B. C.; Power, M. E.; Ferrance, J. P.; Feldman,
S. H.;
Norris, P. M.; Landers, J. P. Analytical Chemistry 2003,
75,
1880-1886.
2. Easley, C. J.; Karlinsey, J. M.; Bienvenue, J. M.;
Legendre,
L. A.; Roper, M. G.; Feldman, S. H.; Hughes, M. A.; Hewlett, E. L.;
Merkel, T.
J.; Ferrance, J. P.; Landers, J. P. Proceedings of the
National Academy of
Sciences of the United States of America 2006,
103,
19272-19277.
3. Bienvenue, J. M.; Duncalf, N.; Marchiarullo, D.; Ferrance,
J.P.; and Landers, J.P. Journal of Forensic Sciences
2006, 51,
266-273.
4. Tian, H.; Huhmer, A. F. R.; Landers, J. P. Analytical
Biochemistry 2000, 283,
175-191.
5.
Wolfe,
K. A.; Breadmore, M. C.; Ferrance, J.
P.; Power, M. E.; Conroy, J. F.; Norris, P. M.; Landers, J. P. Electrophoresis
2002, 23,
727-733.
PCR
Microwave PCR
Dielectric
heating is commonly used, most notably in the
form of a microwave oven. Our group in collaboration with Dr. N. Scott
Barker
in UVA’s department of Electrical and Computer Engineering is
developing a
microfluidic microwave heating device for small volumes of solution.
Using
microstrip transmission lines focused microwave power can be delivered
directly
to a microliter chamber containing solution. Figure 1 shows an example
of our
chip-based microwave heating device. With about 1 W of non-resonant
microwave power
(for comparison a microwave oven uses ~ 1000 W) we rapidly heat buffer
to boiling
and can vary temperature by adjusting the microwave frequency, or
delivered
power. Figure 2 shows an example of temperature control by varying the
applied
microwave frequency. We are currently applying this technology to the
polymerase chain reaction which requires thermocycling between two or
three
temperatures for up to 30 cycles to amplify a desired DNA fragment.
Integrated
Integration
of Cell Sorting and
Solid Phase
Extraction
This
project focuses on the use of an integrated microdevice that combines
sedimentation-based
cell sorting and solid phase extraction (SPE) of DNA from the sorted
cells. The
microdevice (Figure 1), fabricated using standard photolithographic
techniques,
is designed with a domain for cell sorting and two separate SPE
regions. A
mixture of sperm and epithelial cells are separated according to their
physical
properties (Figure 2). Sperm cells are then lysed on-chip, followed by
isolation and purification of its DNA fractions. Following cell
separation and
SPE on the microdevice, DNA amplification and separation are performed
using
conventional laboratory methods. This work represents a major step
towards the
development of a fully integrated microdevice capable of genetic DNA
analysis.
DNA
Extraction and PCR Amplification Performed in a Single Microfluidic
Chamber
Integrated
microdevices provide the opportunity
to encompass multiple analytical steps on a single device, with the
possibility
of automating the entire sample analysis process.
Current work being developed involves a glass
microdevice that has been designed to perform solid phase extraction
(SPE) of
DNA and IR-PCR (infrared-mediated) amplification in a novel format
– with both
processes in the same chamber. This
provides an inherent advantage over any microchip-based DNA extraction
described previously,[1] in that all of the sample DNA is used for
nanoliter
amplification, improving detection limits by 1-2 orders of magnitude. A
novel
solid phase, chitosan-coated magnetic beads, has been developed that
has high DNA
recoveries (72% ± 6%) using a pH-induced DNA release
technique,[2] while
eliminating the use of high salt solutions and organics (both potent
PCR
inhibitors). A
simple external magnet is
used to control the location of the beads in the chamber, removing the
need for
etching structures (such as weirs or pillars) into the channels,
increasing the
simplicity of device design and fabrication.
While previous SPE phases have been composed of
materials incompatible
with PCR[1] (due to high protein adsorption) the chitosan coating does
not
affect the efficiency of PCR, allowing the beads to remain in the very
same
chamber used for PCR.
[1]
Legendre, L. A., Bienvenue, J. M., Roper, M.
R., Ferrance, J. P., Landers, J. P.
Analytical
Chemistry, 2006, 78, 1444.
[2]
Cao, W., Easley, C. J., Ferrance, J. P.,
Landers, J. P. Analytical Chemistry, 2006,
78, 7222-7228.
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