COMPLETE SAMPLE-TO-ANSWER GENETIC ANALYSIS OF INFLUENZA H1N1 VIA THE MAGNETIC INTEGRATED

COMPLETE SAMPLE-TO-ANSWER GENETIC ANALYSIS OF
INFLUENZA H1N1 VIA THE MAGNETIC INTEGRATED
MICROFLUIDIC ELECTROCHEMICAL DETECTOR (MIMED)
B.S. Ferguson,1 S.F. Buchsbaum,1 T.-T. Wu,2 K. Hsieh,1 R. Sun,2 and H.T. Soh1, 2*
1
University of California, Santa Barbara; 2University of California, Los Angeles, USA
ABSTRACT
We describe an integrated microfluidic system capable of sequence specific detection of H1N1 viruses directly from
swab samples with a detection limit of 10 TCID50/sample, which is significantly below the infectious dose of ~105
TCID50/mL. To achieve this performance, the Magnetic Integrated Microfluidic Electrochemical Detector (MIMED) device integrates 1) immunomagnetic capture and concentration, 2) RNA-nucleoprotein denaturation, 3) reversetranscriptase polymerase chain reaction (RT-PCR), 4) single-stranded DNA (ssDNA) generation, and 5) sequencespecific electrochemical DNA detection (E-DNA), in a single monolithic device.
KEYWORDS: Point of Care Diagnostics, Pathogen Detection, Influenza Detection, Microfluidics
INTRODUCTION
The capability to obtain sequence specific,
genetic information of rare target organisms (e.g.
viruses, bacteria or mammalian cells) directly from
complex mixtures, at the point of care (POC) would
have a broad impact in many areas of biotechnology
including clinical diagnostics, forensics, food
safety, and environmental monitoring. [1-3] In the
case of clinical diagnostics, the low titers of target
organisms, and complexity of clinical samples pose
significant technical challenges for POC
diagnostics.[4-5] For example, influenza specimens
from throat and nasopharyngeal swabs contain viral
loads of up to ~105 TCID50/mL in a background of
nucleases, PCR inhibitors, and aggregators.
In order to meet these demanding requirements,
ideally, an effective POC detection technology must
effectively combine the capability to purify and enrich the rare target organisms from the complex
mixture with methods of performing molecular amplification and detection within a low
cost/disposable device. Toward this end, we demonstrate the MIMED system, which integrates the
following functionalities in a single monolithic device (Fig 1): 1) immunomagnetic capture and concentration, 2) RNA-nucleoprotein denaturation, 3)
reverse-transcriptase polymerase chain reaction, 4)
single-stranded DNA generation, 5) electrochemical
DNA detection. Through integration, the system
bears the following advantages while maintaining a
simple architecture: universal target enrichment
from complex voluminous samples at high
efficiency and throughput; minimal sample loss and
contamination risk; sustained exponential target
amplification versus linear-growth asymmetric
assays; and exquisite specificity through immunomagnetic capture, PCR and gene-specific electrochemical readout.
The MIMED platform is general and can be used
for a wide range of viral and bacterial pathogens. As
a model, here we demonstrate the detection of Influenza H1N1 viruses from throat swabs, wherein we
obtained a detection limit of 10 TCID50/sample,
which is significantly below the infectious dose of
~105 TCID50/mL.
978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS
Figure 1: MIMED device and assay. (A) The 1 x 6 cm MIMED
device contains a PDMS channel between two glass wafers, containing 35 and 7 µL sample prep and electrochemical chambers.
(B) Virus and throat swab are added to stabilization medium.
(C) Antibody-coated magnetic beads are added and incubated
for 30 min. (D-E) Sample is pumped into the device; an external
magnet captures labeled virus on-chip; buffer is injected to
wash, followed by RT-PCR mix. (F-G) The chip is heated to
50ºC to denature the RNP and reverse-transcribe. (H-I) PCR is
performed (38 cycles) with phosphorylated reverse primers then
mixed with lambda exonuclease generating ssDNA. (J) Product
is mixed with high-salt buffer and pumped into the EDNA module where it hybridizes with the probe, repositioning a redox label; changes in faradic current are measured via ACV.
52
14th International Conference on
Miniaturized Systems for Chemistry and Life Sciences
3 - 7 October 2010, Groningen, The Netherlands
EXPERIMENTAL
The MIMED device measures 1 x 6 cm and
contains a 250 µm-thick polydimethylsiloxane
(PDMS) channel sandwiched between two SiO2coated borofloat substrates (Fig 2). Magnetic capture/RT-PCR/ssDNA process steps occur in the
sample-prep chamber (volume = 35 µL), and the
E-DNA detection is performed in the electrochemical cell (volume = 7 µL) containing working (WE), counter (CE) and reference (RE) electrodes. The thiolated probe DNA sequence is
immobilized on the gold WE in a manner similar
to our previous work [6], and is complimentary to
a sequence of genomic influenza RNA.
For the detection of Influenza A/PR/8/34
H1N1, the following viral genome sequence encoding for the matrix protein M1 is used: 5’- CCA
GCT CTA TGC TGA CAA AAT GAC CAT CGT CAG CAT
CCA CAG CAC TCT GCT GTT CCT TTC GA-3’. For
Figure 2: MIMED device fabrication. Two borofloat glass substrates are sputter-coated with 100 nm SiO2 to improve chip PCR
efficiency. The electrode substrate is photolithographically patterned to produce Pt counter and reference electrodes and Au
working electrodes. Metals are electron-beam evaporated at 200
nm with a 20 nm Ti adhesion layer. The via substrate is CNCdrilled to fabricate the fluidic ports. An electronic cutting plotter
is used to cut the chamber design into a 250 µm-thick PDMS sheet.
The PDMS layer is then treated with UV-ozone and bonded to the
via substrate. To assemble the chip, the PDMS film is UV-ozone
treated and then bonded to the electrode substrate. Fluidic ports
are affixed to the device. Finally, the E-DNA probes are selfassembled on the gold electrodes.
amplification, sense and antisense primer sequences were chosen as follows: 5’-/5Phos/-TCG
AAA GGA ACA GCA GAG TG-3’ and 5’-CCA GCT CTA
TGC TGA CAA AAT G-3’ (Integrated DNA Technologies, Coralville, IA). The E-DNA probe, synthesized by Biosearch Technolgies (Novato, CA),
was: 5’- HS-(CH2)11-GTG CAC GAA AGG AAC AGC
AGA GTG CAC- NH2-MB 3’ with an 18-nucleotide
sequence complimentary to the target (underlined).
Influenza stock titers were prepared at 107
TCID50/mL and stored at -80 ºC until use. Capture
beads were prepared with 10 µL of streptavidincoated magnetic beads (MyOne Streptavidin C1,
r = 0.5 µm, Invitrogen, Carlsbad, CA) and 2 µL of
biotinylated anti-Influenza A nucleoprotein antibody (Bioscience Research Reagents, Temecula,
CA).
To initiate the detection, a throat swab is collected from a healthy donor and added to stabilization medium followed by diluted virus and 107
antibody-coated beads. The sample was incubated
for 30 min at 4 ºC. Next, permanent magnets are
placed against the device, while sample solution
is pumped through the chip at 60 mL/hr. Magnetic
gradients generated by the external magnets trap
the beads and concentrate the viral particles in the
sample-prep chamber. The sample is washed by Figure 3: MIMED capture performance. (A) The vertical magnetic
flowing 1 mL of PBS through the chamber.
gradient is simulated along the capture channel in the vicinity of
RT-PCR is conducted by injecting reagents permanent magnets. Gradients >200 T/m saturate the superpar(OneStep RT-PCR kit, Qiagen, Valenica, CA) amagnetic beads. The magnetic force on the beads is then, F =
m
containing a standard reverse primer and phos(4/3)πr3M∇B and is balanced by Stokes drag, Fd = 6πηa(vf – vp). (B)
phorylated forward primer into the sample-prep
The capture efficiency is solved along the channel as uniformly
chamber. The chip, mounted on a PID-controlled
dispersed beads are injected. At 6 and 60 mL/hr, 100% of beads
thermoelectric cooler is heated to 50 ºC for 30
are captured, with ~75% at 600 mL/hr. (C) MIMED capture is exminutes to denature the ribonucleoprotein (RNP)
perimentally measured via flow cytometry, where 100% of beads
and reverse transcribe the target sequence. ssDNA
are captured at 6 and 60 mL/hr and 65% at 600 mL/hr.
generation was performed by injecting lambda
exonuclease enzyme (New England Biolabs, Ipswitch, MA) into the chamber and incubating for 20 min at 37 ºC.
Electrochemical measurements are performed in a manner similar to our previous work.[6] Voltammetric scans are
performed in the presence of high-salt buffer (HSB) to maintain consistent salt concentration and pH. Baseline signals
are established prior to sample injection. Subsequently, the PCR product is mixed with an equal volume of 2x HSB and
injected into the E-DNA chamber for a 30-min probe hybridization, after which AC voltammetry (ACV) signals are obtained. Finally, the E-DNA probe is regenerated by flushing with 50 mM NaOH and deionized (DI) water.
53
RESULTS AND DISCUSSION
The performances of major components of the
system were first measured independently. First, the
magnetic field gradients in the sample-prep chamber (Fig 3A), and the resulting efficiency of the
magnetic particle concentration were simulated (Fig
3B). Next, we experimentally verified that, due to
the large magnetic field gradients in the sampleprep chamber (>200 T/m), we achieve ~100% recovery of beads at 60 mL/hr, as measured by flowcytometry (Fig 3C). The efficiency of the on-chip
RNA-nucleoprotein denaturation, RT-PCR and
ssDNA reactions were similar to that of benchtop
controls, and zero-template negative controls produced no detectable product (data not shown). The
characterization of E-DNA sensors confirmed sequence specific DNA detection in the range of 10300 nM within 30 minutes, which corresponds to
viral loads of 10-1000 TCID50/sample.
The complete sample-to-answer test was performed with inactivated H1N1 viruses on clean
swabs in PBS (Fig 4). H1N1 samples at 10, 100,
1000 TCID50/sample resulted in peak faradic current suppression of 21, 29 and 31%, representing
unambiguous positive signals versus <1% change
from zero-virus negative controls. The sensor is regenerated with deionized water (DI) within 95% of
baseline, thus validating that the signal indeed
originated from the RT-PCR products.
Figure 4: Limit of detection of MIMED is below 10
TCID50/sample. In the absence of target DNA, the sensor reports a baseline current (red). In the presence of target, peak
faradic current is suppressed with respect to the baseline by
<1%, 21%, 29% and 31%, respectively by zero-virus control
and 10, 100, 1000 TCID50/sample (purple). The sensor is regenerated (via NaOH and DI water) to within 95% of the
baseline signal (dashed blue), validating specific detection.
CONCLUSION
We demonstrate a system that integrates high throughput immunomagnetic capture, viral RNP denaturation, symmetric RT-PCR, ssDNA generation, and sequence-specific electrochemical detection in a monolithic, disposable device. The
MIMED device unambiguously detected H1N1 loads at 10 TCID50/sample - significantly below clinical viral titers. We
believe the integration of sample preparation with genetic detection demonstrated here, offers an important strategy towards effective clinical diagnosis at the point-of-care.
ACKNOWLEDGEMENTS
We thank the K.W. Plaxco Lab, Jonathan Adams, Adriana Patterson, Ryan White, and Yi Xiao for valuable discussions and assistance. We are grateful of the financial support from the Institute of Collaborative Biotechnologies through
the Army Research Office, and the National Institute of Health. We also thank the UCSB Nanofabrication Facility.
REFERENCES
[1] J. Kling, Nat. Biotechnol., 24, 891, 2006
[2] C. A. Holland, F. L. Kiechle, Curr. Opin. Microbio., 8, 504, 2005
[3] J. G. E. Gardeniers, A. van den Berg, Anal. Bioanal. Chem., 378, 1700, 2004
[4] I.G. Wilson, Appl. Environ. Microb., 63, 3741-3751, 1997
[5] P. Yager, et al., Nature, 442, 412-418, 2006
[6] B.S. Ferguson et al., Anal. Chem., 81, 6503-6508, 2009
CONTACT
*H.T. Soh, tel: +1-805-893-7985; [email protected]
54