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1Department of Mechanical Engineering, Boston University, 2Department of Biomedical Engineering, Boston University
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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.
Keywords: Bioengineering, Issue 73, Biomedical Engineering, Infection, Infectious Diseases, Virology, Microbiology, Genetics, Molecular Biology, Biochemistry, Mechanical Engineering, Microfluidics, Virus, Diseases, Respiratory Tract Diseases, Diagnosis, Microfluidic chip, influenza virus, flu, solid phase extraction (SPE), reverse transcriptase polymerase chain reaction, RT-PCR, PCR, DNA, RNA, on chip, assay, clinical, diagnostics
Cao, Q., Fan, A., Klapperich, C. Microfluidic Chip Fabrication and Method to Detect Influenza. J. Vis. Exp. (73), e50325, doi:10.3791/50325 (2013).
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.
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.
1. Chip Fabrication12
2. Solid Phase Extraction Method
4. PCR Products Detection
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 the flu-specific locus from primer-dimers formation in some samples. Multiple product peaks reflect potential contamination of the test. If this occurs, another aliquot of the sample should be retested. Gel electrophoresis can be used for further verification of the test results13.
Figure 1. Simplified top down chip schematic. The red outlines show the placement of the contact heaters on the bottom of the chip.
Figure 2. The microfluidic assay flow13. (a) Image of two microfluidic chips with attached thin film heaters and two-barrel syringe pump. Glass syringes were connected to each chip with flexible tubing to load reagents and samples. Three ports were glued at the inlets of SPE channel and the waste port, and the outlet of the PCR channel. (b) Channel design with three sections: sample preparation (SPE), RT chamber and continuous flow PCR channel. Two fixed resistance heaters are attached via thermal tape to the bottom of the chip. Fluid flow between the channels was laminar, and changes in fluid resistance allowed for valve-less operation. The depth is 500 μm for SPE and RT channels, and the PCR channel is 100 μm deep. The widths are 500 μm for the SPE column, 1 mm for the RT chamber, and vary from 200 to 400 μm for the wide and narrow sections of the continuous flow PCR channel. The chip is 70 mm in length, 25 mm in width and 1.4 mm in height. (c) Microfluidic assay process flow. The nasopharyngeal sample is mixed with lysis buffer, applied to the chip, the chip is run, and the PCR products are read using a commercial capillary electrophoresis chip.13 Click here to view larger figure.
Figure 3. On chip end point diagnostic result. The PCR product peak is the middle peak at the size of 107 bp. Left (35 bp) and right peaks(10380 bp) are the control ladder peaks. The concentration of the products of each peak is the area under the curve. These are tabulated. (a) positive result without any noise peak. (b) positive result with primer-dimers at 42 bp. Click here to view larger figure.
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 specimen cross contamination.
Although our current on-chip runtime of 3 hr, our extraction platform contains ample room for process optimization. For instance, the wash and dry times right before elution could be reduced by a combination of increasing the flow rate and reducing the wash volumes. Furthermore, cost can be trimmed by streamlining the chip fabrication process and replacing expensive or cumbersome peripherals, such as the syringe pumps, power supply, with alternatives like disposable syringe infusion pumps and small battery packs Opening up the solid phase extraction column geometry and porosity will allow for significant reductions in the pump requirements. Also, once the chip design is standardized for high volume manufacturing, many of the thermocouples used in device development will be eliminated.
More so than other factors, the robustness of our on-chip influenza detection module depends on both good temperature control and appropriate sample handing. Slight offset from the desired on-chip temperature, as well as unintentional multiple freeze-thaw cycles of the influenza specimen, could both reduce the final PCR yield. Aside from these factors, the variations in our on-chip PCR results13 from different patient specimens could also be attributed to: Variation in viral load, different levels of contaminating human cells and blood; the presence of PCR inhibitors after nucleic acid extraction,14,15 and unanticipated chemical and/or physical reactions between the specimens and the test reagents or the device itself16.
In summary, we have demonstrated the feasibility of our microfluidic chip to amplify RNA from the influenza A virus in medically-relevant nasopharyngeal samples. The chip has the potential to be applied to other DNA or RNA targets.
The authors have declared no competing financial interests.
This research was supported by National Institutes of Health (NIH) grant R01 EB008268.
|1-dodecanol||Sigma-Aldrich, St. Louis, MO||443816-500G|
|2,2-Dimethoxy-2-phenylacetophenone||Sigma-Aldrich, St. Louis, MO||196118-50G|
|2100 Bioanalyzer||Agilent Technologies, Santa Clara, CA||G2943CA|
|2-Propanol||Sigma-Aldrich, St. Louis, MO||19516|
|Benzophenone||Sigma-Aldrich, St. Louis, MO||239852-50G|
|BSA||Thermo Fisher Scientific,pittsburge, PA||A7979-50ML|
|Butyl methacrylate||Sigma-Aldrich, St. Louis, MO||235865-100 ml|
|Carrier RNA||Qiagen, Valencia, CA||1017647|
|Cyclohexanol||Sigma-Aldrich, St. Louis, MO||105899-1L|
|Ethanol||Sigma-Aldrich, St. Louis, MO||E7023|
|Ethylene dimethacrylate||Sigma-Aldrich, St. Louis, MO||335861|
|Ethylene glycol dimethacrylate||Sigma-Aldrich, St. Louis, MO||335681-100ML|
|Glass syringe 250 μl||Hamilton, Reno, NV||81127|
|Guanidine thiocyanate||Sigma-Aldrich, St. Louis, MO||50981|
|High Sensitivity DNA Kit||Agilent Technologies, Santa Clara, CA||5067-4626|
|Hot press||Carver,Wabash, IN||4386|
|J-B Weld Epoxies||Mcmaster-Carr,Elmhurst, IL||7605A11|
|Luer-Lok syringes||BD-Medical, Franklin Lakes, NJ||309628|
|Magnesium Chloride||Thermo Fisher Scientific,pittsburge, PA||AB-0359|
|Methanol||Sigma-Aldrich, St. Louis, MO||494437|
|Methyl methacrylate||Sigma-Aldrich, St. Louis, MO||M55909|
|Nanoport Fitting||Upchurch Scientific||F-120x|
|Nuclease free water||Thermo Fisher Scientific,pittsburge, PA||PR-P1193|
|OneStep RT-PCR kit||Qiagen, Valencia, CA||210210|
|PEG8000||Sigma-Aldrich, St. Louis, MO||41009|
|Power supply||VWR,Radnor, PA||300V|
|RNAse Away||Sigma-Aldrich, St. Louis, MO||83931-250ML|
|RNASecure||Applied Biosystems, Foster City, CA||AM7005|
|Silica microspheres||Polysciences,Warrington, PA||24324-15|
|Syringe pump||Harvard Apparatus,Holliston, MA||HA2000P/10|
|Thermally Conductive Tape||Mcmaster-Carr,Elmhurst, IL||6838A11|
|Thermocouple||Omega Engineering, Stamford, CT||5SRTC-TT-J-40-36|
|Thin-film Heaters||Minco,Minneapolis, MN||HK5166R529L12A|
|Ultraviolet Crosslinker||UPV, Upland, CA||CL-1000|
|Zeonex||Zeon Chemicals, Louisville, KY||690R|
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