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Edited by Robert H. Austin, Princeton University, Princeton, NJ, and approved October 16, 2006 (received for review June 5, 2006)
We describe a microfluidic genetic analysis system that represents (ME) amplicon separation and detection is completed in ⬍30
a previously undescribed integrated microfluidic device capable of min. This represents a previously undescribed integrated mi-
accepting whole blood as a crude biological sample with the crofluidic system that can accept biological samples as crude as
endpoint generation of a genetic profile. Upon loading the sample, whole blood, extract high-purity nucleic acids, and generate a
the glass microfluidic genetic analysis system device carries out PCR-targeted amplicon that can be characterized to provide a
on-chip DNA purification and PCR-based amplification, followed by genotypic readout.
separation and detection in a manner that allows for microliter
samples to be screened for infectious pathogens with sample-in– Results
answer-out results in <30 min. A single syringe pump delivers Microdevice Design. The MGA system has a microchannel archi-
sample/reagents to the chip for nucleic acid purification from a tecture with three distinct functional domains, two for sample
biological sample. Elastomeric membrane valving isolates each preparation (SPE and PCR) and one for analysis (ME) (Fig. 1).
distinct functional region of the device and, together with resistive A total of five elastomeric normally closed valves (8) direct flow
flow, directs purified DNA and PCR reagents from the extraction from a single syringe pump and localize the chemistries and
domain into a 550-nl chamber for rapid target sequence PCR reaction conditions that exist (Fig. 1b). The reagents used for
amplification. Repeated pressure-based injections of nanoliter ali- DNA extraction in the SPE domain were isolated from the PCR
quots of amplicon (along with the DNA sizing standard) allow chamber (valve V1), because these are known PCR inhibitors.
electrophoretic separation and detection to provide DNA fragment The PCR domain, gated from the ME domain by two valves (V3
size information. The presence of Bacillus anthracis (anthrax) in 750 and V4), must be passivated to avoid protein fouling and
nl of whole blood from living asymptomatic infected mice and of deactivation of the Taq polymerase. Valves V3 and V4 function
Bordetella pertussis in 1 l of nasal aspirate from a patient to gate the two domains and/or pump amplicon from the PCR
suspected of having whooping cough are confirmed by the result- chamber, whereas the DNA standard from the marker reservoir
ant genetic profile. is injected with valves V2 and V5, respectively. The sample is
mobilized across the analysis channel for injection, after which
full integration 兩 micro total analysis system 兩 microdevice 兩 pumping 兩 the components are separated and detected by laser-induced
valving fluorescence.
Easley et al. PNAS 兩 December 19, 2006 兩 vol. 103 兩 no. 51 兩 19273
tions (1.5 l) were collected from the SPE bed outlet during
extraction and evaluated for nucleic acids by fluorescence or for
PCR-amplifiable DNA by quantitative PCR (qPCR). The flu-
orescence assay was used to determine the timing needed for
valve V1 opening to allow eluted nucleic acids to be transferred
to the PCR chamber; however, qPCR revealed that the fractions
with the largest mass of DNA did not contain the most PCR-
amplifiable DNA. This trend is likely the result of PCR inhibition
because of residual isopropanol contamination (11). Fig. 2b
details the qPCR analysis with replicate DNA extractions from
human whole blood. The majority of DNA was eluted in 2–5 l,
and fraction 2 consistently provided the most PCR-amplifiable
DNA, thereby defining the timing for valve V1. SPE capacity was
determined by flowing human genomic DNA through the bed
and measuring the breakthrough volume (Fig. 2b Inset), reveal-
ing a capacity of 3.3 ng for a whole blood lysate, a mass sufficient
for downstream DNA amplification. After completion of SPE,
flow control for the remainder of the analysis was maintained by
using elastomeric valving/pumping. The valves (8) were used to
isolate the purified DNA in the PCR domain during amplifica-
tion, then to pump from the PCR domain to the ME domain for
injection and analysis as described (12, 13).
Discussion
The advantages of the MGA system are obvious: rapid turn-
APPLIED BIOLOGICAL
around time, decreased reagent consumption per test, decreased
SCIENCES
operator variability (human error factor), and improved opera-
tor safety. The comparisons in Fig. 4 b and c showcase the
capabilities of a MGA system with respect to reduction of volume
analysis time. Fig. 4c compares the turnaround time of the MGA
system for detecting B. pertussis from a sample, relative to
conventional molecular-, serologic-, and culture-based methods.
The 24-min turnaround time compares favorably with ⬎2 h for
analysis using conventional methods, a minimum of 24 h for
PCR-based analysis in a clinical microbiological testing lab , and
⬎48 h for serology and/or culturing of the organism (20). Fig. 4c
Fig. 4. Fully integrated microchip detection of B. pertussis from a human Inset highlights the comparison of the MGA system with con-
nasal aspirate in only 24 min. One microliter of human nasal aspirate was
ventional methods for extraction (green), amplification (blue),
extracted, PCR was performed on the purified DNA, and products were
pressure-injected and electrophoresed. (a) The ME trace was plotted alone to
and detection (black), assuming standard laboratory instrumen-
show the separation of the coinjected DNA sizing standard (peak sizes labeled tation used by the same operators, with no lost time between
in number of base pairs) with the PCR amplicon for product verification. The processes, and does not take into account ‘‘batching-related’’
amplicon (red) migrates between the expected size standards, and sequencing delays. Although not insignificant, the 5-fold reduction in anal-
analysis was used to further verify the product (see SI Supporting Text). (b) ysis time is outweighed by the potential for automation of the
Volumes for SPE (green) and PCR (blue) are compared for MGA and Conv., integrated analysis, which will further decrease technician labor
showing a significant reduction for both processes. (c) Total analysis times for time and isolate the operator from the analysis. Finally, Fig. 4b
crude biological samples of the MGA device (from a), conventional analysis highlights the value of a microfluidic system with respect to
performed in the research lab (Conv.) and a clinical lab (Clin.), and analysis by
reduced consumption of reagents for DNA extraction and
serology/cell culture. Analysis times for MGA and Conv. are shown in (Inset),
with SPE (green), PCR (blue), and ME (black) denoted.
amplification. Microfluidic devices are expected to inherently
scale reduction to the analytical system and, consistent with the
other elegant microfluidic developments from various (2–4)
direct analysis of a blood sample to genetically verify the groups, the MGA system allows for submicroliter PCR. This
presence of a pathogen in ⬍25 min. Because the early detection reduced size not only enhances amplification speed but also
of anthrax is critical to the survival of the host by early provides a 50-fold reduction in PCR volume. Consuming less
recognition and administration of antibiotics with postexposure Taq polymerase, the most costly reagent in this molecular
vaccination, the MGA system and its integrated methods provide analysis, yields the potential to dramatically decrease the cost per
a microfluidic path to improving biodefense surveillance mea- test. Concordantly, the ⬇25-fold reduction in volume of reagents
used for DNA extraction reduces the hazardous waste that must
sures.
be disposed of. The microfluidic nature of the MGA system [like
To demonstrate the broader utility of the MGA system, a
microdevices (2–4)] distinguishes it from larger-volume com-
different sample and nucleic acid target was evaluated. A nasal mercial systems (21–23) that do not reap the benefits of submi-
aspirate was obtained from a human patient symptomatic of croliter fluid manipulation.
whooping cough, a respiratory infection caused by the Gram- Although a comprehensive evaluation of device sensitivity for
negative bacterium Bordetella pertussis, which can be isolated these two diverse sample matrices is ongoing, the proof-of-
from the mouth, nose, and throat (19, 20). This infection is principle experimentation accomplished with anthrax-infected
characterized by severe spasms of coughing that can last several murine blood suggests the following regarding detection levels
weeks or months and, although not particularly threatening to with the MGA system. The average day 2 serum level of anthrax
those beyond their first year, it can lead to serious complications in immunoprecipitation-challenged mice was determined to be
or fatality in infants (19, 20). Using the same method described 2.5 (⫾1) ⫻ 106 cfu/ml. Of the total blood drawn, only 0.75 l of
above, a volume equivalent to 1 l of nasal aspirate was prepared blood was loaded onto the silica bed (purposefully overloaded to
in lysis buffer and loaded into the MGA device, with DNA ensure saturation of the phase for the purification), representing
Easley et al. PNAS 兩 December 19, 2006 兩 vol. 103 兩 no. 51 兩 19275
the equivalent of 1,500–2,000 cfu, which, in this case, is equiv- (PDMS) membrane (HT-6240, Bisco Silicones, Rogers, Carol
alent to the number of starting copies of amplifiable DNA. Stream, CT), with a thickness of 254 m. This unpatterned layer
Having demonstrated the amplification from ⬍10 DNA starting was irreversibly sealed by plasma oxidation (PDC-32G plasma
copies with the IR-PCR system used in the MGA device (24), cleaner, Harrick Scientific, Pleasantville, NY) to a fourth glass
sensitivity on the order of a few hundred starting copies is layer, previously drilled and patterned with valve control chan-
plausible with the MGA system, but this will only be established nels. These third and fourth layers were aligned, then pressed to
with certainty when serial dilution studies are completed. seal against the thermally bonded glass microchip, with the third
Although the MGA device shares similarities with other (PDMS) layer in contact with the drilled access holes of the
microfluidic devices reported in the literature (3, 17, 25, 26), it second layer to form pneumatically addressable valve seats in a
is important to define the distinguishing characteristics of this normally closed configuration (8, 26).
system. First and foremost, in contrast with other systems, the
incorporation of a purification step with downstream analytical Device Preparation. The glass microchips were cleaned before
processing allows for the removal of inhibiting chemical com- each experiment (before addition of the valve layer), to regen-
pounds, enabling the input of complex biological samples such as erate the surface (28). The PCR and ME domains were exposed
blood, a key requirement of a genetic -TAS (1). This MGA to a 1:1 methanol:HCl solution for 30 min, rinsed with ddH2O,
system displays a previously undescribed integration of DNA and exposed to concentrated H2SO4 for 30 min. The SPE domain
extraction from whole blood with multiple downstream pro- was cleaned with 2 M HCl for a total of 1 h. The entire device
cesses (PCR and electrophoretic analysis) on the same micro- was then rinsed thoroughly with ddH2O and the PCR and SPE
device. The second distinction is the simplistic design of this glass domains dried with nitrogen. The SPE and PCR domains, along
MGA device, which avoids costly and time-consuming metalli- with the syringe used to deliver master mix, were silanized by
zation steps. Circumventing the need to fabricate heaters and/or using Sigmacote (Sigma-Aldrich, St. Louis, MO). After silaniza-
temperature sensors (2, 3, 17) into the PCR system enhances cost tion, the SPE and PCR domains, as well as the syringe, were
effectiveness so that single-use disposability becomes a realistic rinsed with water and dried under nitrogen.
possibility.
The addition of DNA purification for the removal of inter- Macro-to-Micro Interfacing. After conditioning, the device was
fering species to already established microfluidic technology for loaded into a Plexiglas cartridge for interfacing (see Fig. 1c). The
PCR amplification, separation, and detection completes the cartridge consisted of two machined layers between which the
genetic analysis system and allows relevant genetic profiling for device was sandwiched. Buna-N O-rings were used for fluidic
a variety of applications. Through the integration of sample (004) and pneumatic (001) seals, with the device held in place by
pretreatment with analytical processing for the analysis of bio- using stainless steel knurled-head screws. The cartridge was
logical samples presented here, the goal of the -TAS described machined with access holes and fluidic reservoirs, interconnects
by Manz et al. (1) a decade ago has been realized. In an era for pneumatic control, and openings for IR heating and fluo-
witnessing a shift toward point-of-care testing and personalized rescence excitation and emission.
medicine, the MGA system presented here provides sample-in–
answer-out genetic testing. Its virtues are simplicity in function SPE. For all extractions using the MGA system, silica beads (5–30
and fabrication, combined with the possibility for turnkey mi- m) were packed in the SPE domain against the etched weir by
crofluidic detection systems for screening a panel of pathogens. using applied vacuum and replaced before each analysis. Flow
With whole-blood and nasal-aspirate analyses demonstrated, it is rates used for all extractions were 4.16 l䡠min⫺1 (29). The
clear that a variety of representative candidate samples, includ- extraction protocol used for all experiments was adapted from
ing body fluids (urine, blood, semen, etc.), nasal swabs, and fecal Legendre et al. (11); a more detailed description of the protocols
matter, could be analyzed in a microfluidic system designed for used can be found in SI Supporting Text.
use in emergency rooms, primary care clinics, and forensic labs. For generating the real-time qPCR elution profile, a sample
An analytical platform that utilizes disposable, cost-effective consisting of 4 l of human whole blood, lysed in a solution of
microfluidic chips reduces reagent consumption by orders of 5 l of proteinase K and 91 l of 6 M guanidine䡠HCl, was
magnitude, provides turnaround times of 30 min or less, and prepared. The lysed sample was loaded for 6 min and the bed
offers the potential of rapid inexpensive on-site screening. It is washed with 80% isopropanol (80/20, vol/vol 2-propanol/dd
reasonable to expect that compact portable instrumentation can H2O) for 5 min, with secondary flow of ddH2O through the
be assembled around the small disposable microfluidic device sidearm to imitate a fully integrated analysis. Finally, water was
described here to generate a portable and eventually handheld passed through the bed, and 13 1.5-l fractions of eluate were
system, applicable in a number of different clinical, biohazard- collected for subsequent qPCR amplification (n ⫽ 2) of the
ous, and forensic contexts. human thyroid peroxidase gene by Taqman chemistry, following
the protocol developed by Horsman et al. (30).
Methods To generate replicate breakthrough profiles, the same con-
Microchip Fabrication. All glass microchips were fabricated as centration of lysed blood sample as described above was used for
described (27) by using borofloat glass slides (127 ⫻ 127 ⫻ 0.7 consecutive breakthrough plots (n ⫽ 3), with the silica bed
mm) purchased from Telic (Valencia, CA). Differential etch removed and replaced between each run. The sample was flowed
depths were achieved by using hydrofluonic acid (HF) resistant through the SPE bed as described, whereas 10 1.5-l fractions
dicing tape (Semiconductor Equipment Corporation, Moorpark, were collected at the SPE outlet. These fractions were fluores-
CA), patterned manually. Dimensions of the device and channel cently assayed for DNA concentration (31) using the Picogreen
design, as well as device fabrication, are more fully detailed in assay (Invitrogen–Molecular Probes, Eugene, OR) according to
supporting information (SI) Supporting Text. the manufacturer’s instructions.
The four-layer integrated devices (30.0 ⫻ 63.5 mm) were For the integrated experiments, real clinical samples were
assembled as follows. The bottom two glass fluidic layers were used to show the versatility of the device for handling multiple
etched as described, with access holes drilled into the patterned sample types and applications. The first sample evaluated was
layer prebonding. After thermal bonding, glass was selectively the detection of anthrax in mouse blood. The C57BL/6 mice were
removed from around the PCR chamber by etching with 49% injected with 1 ⫻ 109 spores (B. anthracis strain 7702) in 100 l
HF, using HF-resistant tape as a mask. The third (valve) layer of water. Typically, mice challenged in this manner succumb 5–6
consisted of a commercially available poly(dimethyl siloxane) days post-challenge. All of the mice used in this experiment were
APPLIED BIOLOGICAL
Bridgewater, CT) through a 515-nm bandpass filter (Omega
PCR Amplification. For fully integrated analysis, the PCR master
Optical, Brattleboro, VT). The instrument and data acquisition
SCIENCES
mixture was made with the following final concentrations: 20 were controlled through a LabVIEW application written in-
mM Tris/100 mM KCl (pH 8.3)/6 mM MgCl2/0.8 M of each house.
primer/0.4 mM dNTP/0.5 units/l Taq polymerase. The thermal
cycling protocols used were 95°C for 30 sec (initial denatur- We gratefully acknowledge the pioneering developments of Drs. A.
ation), then 30 cycles of 95°C for 2 sec, 62°C/55°C for 3 sec (for Huhmer, B. C. Giordano, and Q. Wu, who laid the foundation for this
B. anthracis/B. pertussis, respectively), and 72°C for 5 sec, fol- work. We also thank the Mathies Laboratory at the University of
lowed by a single final extension for 1 min at 72°C after the 30 California, Berkeley, CA, for sharing their valving innovations early on.
cycles were completed. The primers for B. pertussis amplification Funding was provided by the National Institutes of Health/National
were adapted from Loeffelholz et al. (32). The primers used in Human Genome Research Institute through Grants R01 HG001832 and
the B. anthracis amplification were 5⬘-CAAATCAGCTC- R01 HG002613 (to J.P.L.). Funding for C.J.E. was provided by the
American Chemical Society, Division of Analytical Chemistry, and Eli
GAAAGTTAGGA (for) and 5⬘-CAGTAACTGTTCAGAAG- Lilly and Co. M.A.H., E.L.H., and T.J.M. acknowledge funding from the
GTACATCTGA (rev) for the amplification of a 211-bp frag- National Institutes of Health National Institute of Allergy and Infectious
ment of the virulence B gene on pXO1 and were designed Dieseases Mid-Atlantic Regional Center of Excellence for Biodefense
in-house (33). The noncontact thermal cycling PCR system (see (Grant U54 AI057168).
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