An inexpensive, portable, refrigeration-free, ready-to-use microfluidic device for real-time multiplexed molecular...

Human immunodeficiency virus (HIV), Hepatitis B virus (HBV), and Hepatitis C virus (HCV) have infected or co-infected more than 400 million people worldwide and are becoming one of the most significant challenges for global healthcare. A simple, rapid, point-of-care diagnosis is playing a key role in controlling the spreading of these diseases and providing proper care for each patient. In this study, we have developed an inexpensive, portable, refrigeration-free, ready-to-use microfluidic device for multiplexed detection of HIV, HBV, and HCV, which is amenable to point-of-care application when combined with a simple upstream sample-preparation step. All reagents and essential components for nucleic acid amplification are fully dried on chip and can last for at least 10 weeks being exposed to the atmosphere at room temperature. The device operation only involves the manipulation of target-containing sample solutions without any need for chemical handling operated by skilled personnel. Built-in capillary circuits provide auto-distribution of sample solution and perform auto-sealing with a phase-change material. The chip consists of four reaction chambers for HIV, HBV, HCV, and one negative control, respectively. Each reaction chamber can release both colorimetric and fluorometric signals during amplification with the presence of target nucleic acids. The amplifications on chip were monitored in real time, and the device detected down to 24 copies of HBV DNA and 76 copies of HCV RNA during the 60-min on-chip amplification. All procedures were conducted using a portable custom processor comprising an electric resistance heater and a USB camera, which can be readily reproduced in resource-limited settings.