The need for point-of-care (POC) devices has been urged by the emergence of a novel coronavirus, SARS-CoV-21. Microfluidic Lab-on-a-Chip (LOC) technologies have brought revolutionary changes and development in the POC platforms by providing easy-to-use, cost-effective miniaturised systems with faster analysis time2–4. POC systems have led to effective prevention and early detection of diseases which help deplete mortality drastically5–7.
Typical POC diagnostic devices, dipstick tests have been widely used for tests of pregnancy, influenza, cardiac markers and other infectious diseases7–10. This type of POC diagnostic system, however, has limitations to achieve higher accuracy. For enhancing detection accuracy, nucleic acids (DNA and RNA) are widely used as biomarkers to identify specific infectious diseases11–13. However, multi-step and complex fluid handling is essentially required for the workflow of molecular diagnostics10 (sample preparation, target amplification and signal read-out)7.
A microfluidic LOC system is the most suitable platform for POC diagnostic system. Bio-analytical devices integrated with the microfluidic component can be used for multi-step processes in a miniaturised system with fast analysis time, high sensitivity and specificity14,15. Especially, microfluidic components based polymeric materials have many advantages, including low-cost fabrication, duplicability and disposability. In a nucleic acids LOC system, the three main components that determine its cost, performance and practicability are the pumps/actuators, valves, and the process of nucleic acid amplification.
Microfluidic valves are critical driving components in the LOC system besides pumps and mixers16,17. Suitable micropumping methods for flow control represent a major technical hurdle in developing microfluidic systems for point-of-care testing (POCT). Passive pumping for LOC systems is essential in POC systems18 to avoid the need for cumbersome external equipment (i.e., syringe pumps and pressure pumps), which are still widely seen in microfluidic devices19,20. They control fluid flows by opening or closing fluidic passageways and are usually actuated by external power resulting in a bulky and complex design and limitation in POC testing applications. Various microvalves have been introduced based on their actuating principles, such as electrochemical, piezoelectric, magnetic, electromagnetic, pneumatic, thermopneumatic, shape memory alloy, surface acoustic wave21–28. Also, various kinds of powerless valves have been suggested for microfluidic devices29–31, together with the development of the powerless pump. Capillary force, gravitational force and finger pressure have been widely used as actuation sources to transport samples in POC diagnostic systems3,32−34. Three representative methods have a common problem: they are hard to control the flow rate of reagents and use precise control required reaction steps.
An original polymerase chain reaction (PCR) process was designed to amplify a portion of DNA, an essential step for nucleic acid analysis: temperature control is crucial for the procedure. Due to the commonplace features of the methods, PCR is evolved far beyond simple target DNA or RNA detection. Fu, Yayun, et al. describes a low-cost and straightforward self-priming compartmentalisation platform for PCR analysis35. The critical element of the platform is the degassed PDMS pump aligned with the outlet port of the chip. It creates the negative pressure in the channel, which automatically derives the sample and oil into a microchamber for self-partition. Therefore, the platform doesn’t require an external component that needs power for pumping. Salman, Abbas, et al. report a PCR microfluidic device by integrating three systems: microfluidic PCR chip, thermal cycler, and fluorescence detector36. The thin microfluidic layer of the chip facilitates rapid temperature change, allowing fast cycling times. In addition, the device incorporated with photodetector enables further analysis to monitor the PCR progress. But, both devices are not suitable for the POC diagnostic assays due to time demanding and complex external components that need power, respectively.
In addition, nucleic acid testings such as the reverse transcription-polymerase chain reaction (RT-PCR) have become the most reliable method for COVID-19 detection due to their accuracy and sensitiveness to viral genomes recognised as a gold standard by WHO for virus detection technique. Heating elements, however, is not avoidable due to the thermal cycling process of the PCR. Thus, the method heavily depends on the equipment, well-trained staff, and equipped laboratories37,38.
Unlike conventional PCR, isothermal amplification methods enable the amplification of DNA at one constant temperature. Loop-mediated isothermal amplification (LAMP), recombinant polymerase amplification (RPA), rolling circle amplification (RCA), and helicase dependent amplification (HDA) employ the isothermal method. They have been used for DNA-based POC diagnostic applications39–44. The RPA is one of the popular isothermal methods used in POC testing. Its isothermal amplification overcomes shortcomings in temperature control of PCR based tests while providing good sensitivity, low-cost, and fast detection with simple instruments such as paper-based microfluidic devices. It can achieve 109–1011 fold amplification of target DNA at the optimum temperature around 37–42 ◦C45. Although the RPA method would give more errors and contamination, the reverse-transcription RPA (RT-RPA) was successfully used as an isothermal alternative to RT-PCR for viral disease detection such as Ebola46.
Researchers have made enormous efforts to develop non-instrumented nucleic acid amplification devices with various isothermal amplification methods to realise equipment-free POC diagnostic systems. Liu et al. developed a self-heating cartridge and maintained a temperature within ±3°C while Huang et al. managed to keep the temperature within ±2°C 47,48. We believe the non-instrumented RPA device is the most suitable isothermal amplification method for POC applications due to the low operating temperature and less time. Therefore, it is worthy of designing a simple powerless controllable pump with a microvalve and non-instrumented RPA device.
This paper presents a non-instrumented DNA analysis system development that enables total analysis of DNA biomarkers from sample to results within 1 hour. This proof-of-concept study employs 1) a self-powered actuator (or pump) based on a hydration reaction and as a valve; the gas flow is controlled by attaching or detaching a cover film on the microchannels’ holes (inlets and outlets). Then 2) an exothermal RPA that amplifies DNA with self-powered heating. We evaluated the performance of these components individually and demonstrated the integrated non-instrumented DNA analysis system.