Microfluidic Chip Fabrication and Method to Detect Influenza

Summary

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.

Abstract

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.

Introduction

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, dire....

Protocol

1. Chip Fabrication¹²

1. 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 °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.
2. Emboss the microfluidic channel in the plaque with an epoxy mold. Details on the mold fabrication are outlined elsewhere¹² (channel design in Figure 2b): put one plaque onto the epoxy mold; preheat them on the heated press at 157 °C for 10 min and then apply pressure slowly to 1,000 psi for....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalogue Number
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	Upchurch Scientific	N-333-01
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