This research was supported by National Institutes of Health (NIH) grant R01 EB008268.

1. Chip Fabrication12
<ol>
 <li>Make two plaques from Zeonex 690R pellets: distribute 8-9 grams Zeonex pellets evenly in the center of a metal plate, preheat on the heated press at 198 &deg;C for 5 min, and then apply pressure slowly to 2,500 psi for another 5 min. To complete this step, we used a Carver hot press.</li>
 <li>Emboss the microfluidic channel in the plaque with an epoxy mold. Details on the mold fabrication are outlined elsewhere12 (channel design in Figure 2b</str...

<ol>
	<li>Nicholls, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pandemic+Influenza:+The+Inside+Story.">Pandemic Influenza: The Inside Story.</a> PLoS Biol. 4, (2006).</li><li>Wenzel, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+performance+determination+and+clinical+validation+of+the+novel+roche+realtime+ready+influenza+A/H1N1+detection+set.">Analytical performance determination and clinical validation of the novel roche realtime ready influenza A/H1N1 detection set.</a> Journal of Clinical Microbiology. 48, 3088-3094 (2010).</li><li>Chan, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+sensitivity+of+rapid+influenza+antigen+detection+tests+for+swine-origin+influenza+virus+(H1N1.">Analytical sensitivity of rapid influenza antigen detection tests for swine-origin influenza virus (H1N1.</a> Journal of Clinical Virology: the. 45, 205-207 (2009).</li><li>Ginocchio, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+multiple+test+methods+for+the+detection+of+the+novel+2009+influenza+A+(H1N1)+during+the+New+York+City+outbreak.">Evaluation of multiple test methods for the detection of the novel 2009 influenza A (H1N1) during the New York City outbreak.</a> Journal of. 45, 191-195 (2009).</li><li>Aeron, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+influenza+rapid+point-of-care+tests+in+the+detection+of+swine+lineage+A(H1N1)+influenza+viruses.">Performance of influenza rapid point-of-care tests in the detection of swine lineage A(H1N1) influenza viruses.</a> Influenza and Other Respiratory Viruses. 3, 171-176 (2009).</li><li>Takahashi, H., Otsuka, Y., Patterson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+tests+for+influenza+and+other+respiratory+viruses:+determining+performance+specifications+based+on+clinical+setting.">Diagnostic tests for influenza and other respiratory viruses: determining performance specifications based on clinical setting.</a> Journal of Infection and Chemotherapy. 16, 155-161 (2010).</li><li>Selvaraju, S. B., Selvarangan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Three+Influenza+A+and+B+Real-Time+Reverse+Transcription-PCR+Assays+and+a+New+2009+H1N1+Assay+for+Detection+of+Influenza+Viruses.">Evaluation of Three Influenza A and B Real-Time Reverse Transcription-PCR Assays and a New 2009 H1N1 Assay for Detection of Influenza Viruses.</a> Journal of Clinical Microbiology. 48, 3870-3875 (2009).</li><li>Northrup, M. A., Ching, M. T., White, R. M., Watson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transducer'93, seventh international conference on solid state sensors and actuators. , 924-926 (1993).</li><li>Bhattacharyya, A., Klapperich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoplastic+microfluidic+device+for+on-chip+purification+of+nucleic+acids+for+disposable+diagnostics.">Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics.</a> Analytical Chemistry. 78, 788-792 (2006).</li><li>Cao, Q., Kim, M. -. C., Klapperich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastic+microfluidic+chip+for+continuous-flow+polymerase+chain+reaction:+Simulations+and+experiments.">Plastic microfluidic chip for continuous-flow polymerase chain reaction: Simulations and experiments.</a> Biotechnology Journal. 6, 177-184 (1002).</li><li>Cao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Chip+for+Molecular+Amplification+of+Influenza+A+RNA+in+Human+Respiratory+Specimens.">Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens.</a> PLoS ONE. 7, e33176 (2012).</li><li>R&#229;dstr&#246;m, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+Characterization+of+PCR-Inhibitory+Components+in+Blood+Cells.">Purification and Characterization of PCR-Inhibitory Components in Blood Cells.</a> J. Clin. Microbiol. 39, 485-493 (2001).</li><li>Boddinghaus, B., Wichelhaus, T. A., Brade, V., Bittner, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+PCR+Inhibitors+by+Silica+Membranes:+Evaluating+the+Amplicor+Mycobacterium+tuberculosis+Kit.">Removal of PCR Inhibitors by Silica Membranes: Evaluating the Amplicor Mycobacterium tuberculosis Kit.</a> J. Clin. Microbiol. 39, 3750-3752 (2001).</li><li>Andreasen, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+microRNA+quantification+in+total+RNA+from+clinical+samples.">Improved microRNA quantification in total RNA from clinical samples.</a> Methods. 50, S6-S9 (2010).</li></ol>

The diagnostic method presented here demonstrated the ability of an integrated microfluidic plastic chip to amplify influenza A RNA from patient specimens with high specificity and a low detection limit.13 We designed this chip for potential point of care testing: (a) the temperature and fluidic control were simplified, (b) the chip is low cost and suitable for high throughput fabrication using injection molding, and (c) the chip is disposable and intended for one time use, thus reducing the concern of s...

Millions of deaths have been reported during the three influenza pandemics of the 20th century1. Moreover, the most recent influenza pandemic was declared by World Health Organization (WHO) 2 in 2009, and as of August 1, 2010, 18,449 deaths were reported by WHO3. This pandemic demonstrated again the high burden of infectious disease, and the need for rapid and accurate detection of influenza to enable fast disease confirmation, appropriate public health response and effective treatment4.
There are several methods widely used for diagnosing influenza, these include rapid immunoassays, direct fluorescent antigen testing (DFA) and viral culture methods. Rapid immunoassays dramatically lack sensitivity5-8, while the other two methods are labor-intensive and time consuming9. Molecular tests offer multiple advantages including a short turn-around time, high sensitivity, and higher specificity. Several commercial entities have been working towards fast molecular tests (also called nucleic acid tests or NATs) for infectious diseases, and several have influenza assays in their pipelines. However most of them require off-chip sample preparation. None of the Clinical Laboratory Improvement Amendments (CLIA) waived molecular tests incorporate sample preparation into the assay cartridge or module.
Lab-on-a-chip technology plays an important role in the development of point-of-care testing devices. After the introduction of the first PCR chip in 199310, numerous efforts have been put into developing nucleic acid test chips. However, only a few of these have integrated crude sample preparation with downstream amplification.
We have previously demonstrated the miniaturization of a solid phase extraction column (SPE) into a plastic microfluidic chip11 and the development and optimization of a continuous flow PCR chip12. Here, we extend the previous work to integrate the SPE with RT and PCR steps into a single chip for clinical diagnostics and demonstrate its capability to amplify nucleic acids from patient nasopharyngeal (NP) swabs and aspirates.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>1-dodecanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>443816-500G</td>
 </tr>
 <tr>
 <td>2,2-Dimethoxy-2-phenylacetophenone</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>196118-50G</td>
 </tr>
 <tr>
 <td>2100 Bioanalyzer</td>
 <td>Agilent Technologies, Santa Clara, CA</td>
 <td>G2943CA</td>
 </tr>
 <tr>
 <td>2-Propanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>19516</td>
 </tr>
 <tr>
 <td>Benzophenone</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>239852-50G</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Thermo Fisher Scientific,pittsburge, PA</td>
 <td>A7979-50ML</td>
 </tr>
 <tr>
 <td>Butyl methacrylate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>235865-100 ml</td>
 </tr>
 <tr>
 <td>Carrier RNA</td>
 <td>Qiagen, Valencia, CA</td>
 <td>1017647</td>
 </tr>
 <tr>
 <td>Cyclohexanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>105899-1L</td>
 </tr>
 <tr>
 <td>Ethanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>E7023</td>
 </tr>
 <tr>
 <td>Ethylene dimethacrylate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>335861</td>
 </tr>
 <tr>
 <td>Ethylene glycol dimethacrylate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>335681-100ML</td>
 </tr>
 <tr>
 <td>Glass syringe 250 &mu;l</td>
 <td>Hamilton, Reno, NV </td>
 <td>81127</td>
 </tr>
 <tr>
 <td>Guanidine thiocyanate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>50981</td>
 </tr>
 <tr>
 <td>High Sensitivity DNA Kit</td>
 <td>Agilent Technologies, Santa Clara, CA</td>
 <td>5067-4626 </td>
 </tr>
 <tr>
 <td>Hot press</td>
 <td>Carver,Wabash, IN</td>
 <td>4386</td>
 </tr>
 <tr>
 <td>J-B Weld Epoxies</td>
 <td>Mcmaster-Carr,Elmhurst, IL </td>
 <td>7605A11</td>
 </tr>
 <tr>
 <td>Luer-Lok syringes</td>
 <td>BD-Medical, Franklin Lakes, NJ</td>
 <td>309628</td>
 </tr>
 <tr>
 <td>Magnesium Chloride</td>
 <td>Thermo Fisher Scientific,pittsburge, PA</td>
 <td>AB-0359</td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>494437</td>
 </tr>
 <tr>
 <td>Methyl methacrylate</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>M55909</td>
 </tr>
 <tr>
 <td>Nanoport</td>
 <td>Upchurch Scientific</td>
 <td>N-333-01</td>
 </tr>
 <tr>
 <td>Nanoport Fitting</td>
 <td>Upchurch Scientific</td>
 <td>F-120x</td>
 </tr>
 <tr>
 <td>Nuclease free water</td>
 <td>Thermo Fisher Scientific,pittsburge, PA</td>
 <td>PR-P1193</td>
 </tr>
 <tr>
 <td>OneStep RT-PCR kit</td>
 <td>Qiagen, Valencia, CA</td>
 <td>210210</td>
 </tr>
 <tr>
 <td>PEG8000</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>41009</td>
 </tr>
 <tr>
 <td>Power supply</td>
 <td>VWR,Radnor, PA</td>
 <td>300V</td>
 </tr>
 <tr>
 <td>RNAse Away</td>
 <td>Sigma-Aldrich, St. Louis, MO</td>
 <td>83931-250ML</td>
 </tr>
 <tr>
 <td>RNASecure</td>
 <td>Applied Biosystems, Foster City, CA</td>
 <td>AM7005</td>
 </tr>
 <tr>
 <td>Silica microspheres</td>
 <td>Polysciences,Warrington, PA</td>
 <td>24324-15</td>
 </tr>
 <tr>
 <td>Syringe pump </td>
 <td>Harvard Apparatus,Holliston, MA</td>
 <td>HA2000P/10</td>
 </tr>
 <tr>
 <td>Thermally Conductive Tape</td>
 <td>Mcmaster-Carr,Elmhurst, IL </td>
 <td>6838A11</td>
 </tr>
 <tr>
 <td>Thermocouple</td>
 <td>Omega Engineering, Stamford, CT</td>
 <td>5SRTC-TT-J-40-36</td>
 </tr>
 <tr>
 <td>Thin-film Heaters</td>
 <td>Minco,Minneapolis, MN </td>
 <td>HK5166R529L12A</td>
 </tr>
 <tr>
 <td>Ultraviolet Crosslinker</td>
 <td>UPV, Upland, CA</td>
 <td>CL-1000</td>
 </tr>
 <tr>
 <td>Zeonex</td>
 <td>Zeon Chemicals, Louisville, KY</td>
 <td>690R</td>
 </tr>
 </tbody>
</table>

microfluidic chip fabrication and method to detect influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

A typical result is shown in Figure 3 for an influenza A infected nasopharyngeal wash specimen. Due to the different amounts of influenza virus in each patient specimen, the final concentration of PCR product will vary. A good result should have low noise, two clear ladder peaks (35 and 10380 bp) and a single product peak at the designed product size (107 bp) for the positive sample. While the product peak should theoretically be absent for negative controls, we did observe spurious PCR peaks near...

Watch this Scientific Journal Video about Microfluidic Chip Fabrication and Method to Detect Influenza at JoVE.com

Microfluidic Chip Fabrication and Method to Detect Influenza

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

microfluidic-chip-fabrication-and-method-to-detect-influenza

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Bioengineering

14.8K 次观看.  Boston University. 一个集成的微流体热塑性芯片已被开发用于作为一种分子诊断。该芯片进行核酸提取，逆转录和PCR。用于制造和运行芯片的方法中描述。