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
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