Integrated microfluidic systems with sample preparation and nucleic acid amplification

Abstract

Rapid, efficient and accurate nucleic acid molecule detection is important in the screening of diseases and pathogens, yet remains a limiting factor at point of care (POC) treatment. Microfluidic systems are characterized by fast, integrated, miniaturized features which provide an effective platform for qualitative and quantitative detection of nucleic acid molecules. The nucleic acid detection process mainly includes sample preparation and target molecule amplification. Given the advancements in theoretical research and technological innovations to date, nucleic acid extraction and amplification integrated with microfluidic systems has advanced rapidly. The primary goal of this review is to outline current approaches used for nucleic acid detection in the context of microfluidic systems. The secondary goal is to identify new approaches that will help shape future trends at the intersection of nucleic acid detection and microfluidics, particularly with regard to increasing disease and pathogen detection for improved diagnosis and treatment.

Figure 1. Conceptual integration of nucleic acid isolation, amplification and the microfluidics platform.

Improved technologies that promote efficient and clean nucleic acid extraction will improve the quality and time cost associated with nucleic acid amplification. Together, these approaches applied to the flexibility associated with the microfluidics platform can increase the sensitivity, improve the accuracy, reduce time to results, minimize required technical training, and improve the development of POC. The end goal is increased treatment efficiency and improved care.

Figure 2. Magnetic beads-based microfluidic nucleic acid extraction chip.

(A) Overview of the TREDA system (Shi et al., 2015) (a) schematic of the TREDA chip where (b) controls the dispersion, aggregation, and movement of magnetic beads. Adapted from Ref. with permission from the Royal Society of Chemistry. (B) Schematic of the DNA extraction process showing (Mosley et al., 2016) (a) Sample loading and lysis. (b) Mixing of superparamagnetic particles (PMPs) to combine with DNA. During the process, the magnet can control the movement of the magnetic beads, so the magnetic beads are in an aggregate state. This process can be accelerated by controlling the movement of the magnetic beads. (c) Transfer of PMPs through the immiscible phase for washing, (d) elution of DNA from the PMPs and collection of the nucleic acid for off-chip analysis is shown. Adapted from Ref. with permission from the Royal Society of Chemistry.

Figure 3. Silica pillar-based nucleic acid extraction method.

(A) Silica-coated pillar arrays on microchips for DNA extraction (Petralia et al., 2017). The chip is composed of a 6-layer structure and the size of silicon pillars array is 5 × 1.8 mm². Adapted from Ref. with permission from the Royal Society of Chemistry. (B) Silicon bead-silicon beads nucleic acid extraction method. Schematic illustration of the integrated rotary microdevice for the DNA extraction, the Loop-mediated isothermal amplification (LAMP) reaction, and the lateral flow strip detection are shown (Park et al., 2017). Nucleic acid extraction is based on the Silica microbead-bed channel, which serves as a solid phase matrix. DNA extraction is achieved by controlling different speeds and the extraction efficiency can be up to 80% in the microdevice. Adapted from Ref. with permission from Elsevier.

Figure 4. Chitosan-modified Fusion 5 filter paper and DNA capture mechanism

(Gan et al., 2017). (A) 3-mm-diameter discs of chitosan-modified Fusion 5 filter paper. Schematic and scanning electron microscope image of the fiber matrix coated with chitosan polymers. (B) Schematic of the DNA capture mechanism. At a pH around 5, DNA molecules are “actively” adsorbed onto the chitosan-modified fibers. Once DNA is on the fibers, the physical entanglement of the long-chain molecules with the fiber matrix can also assist the capture. At a pH of 9, although DNA is not “actively” absorbed onto the fiber, DNA molecules remain bound due to the physical trapping of these long-chain DNA molecules within the fiber matrix against washing and elution. Adapted from Ref. with permission from ACS Publications.

Figure 5. Schematic of DMA chip and DMP chip for nucleic acid extraction.

(A) DMA chip for RNA isolation. a:Cell lysis. Different components including DNA, RNA and protein are released. b:on-chip RNA isolation. RNA is bound and eluted by controlling pH. Adapted from Ref. with permission from the Elsevier. (B) DMP chip for DNA and RNA isolation. (a) Chemical structure of DMP and schematic drawing for assembling of a plastic type microfluidic cartridge with a 3D disposable chip. (b) schematic and photograph workflows for the DMP system for RNA (b) and DNA (c) extraction. Adapted from Ref. with permission from ACS Publications.

Figure 6. Schematic drawing of the fractal branching microchannel net chip

(Zhu et al., 2017). (A) Schematic diagram of the chip that has 4096 microwells for dPCR reaction. (B) Diagram of the details of the chip design. (C) Photograph of the chip. (D) The scalability of the chip with 16384 microwells in each reaction panel. (E) The principle and operation procedure of the microfluidic device: (a) the chip is degassed in a vacuum pump and then adhesive tape is attached to seal the top surface of the chip after the degassing step; (b) the adhesive tape is punctured, and the reagent can be dispensed into the inlet, while the degassing-drive flow primes the sample into the microwells quickly; (c) the oil is then dispensed into the inlet, and the oil phase is self-primed into the channels; (d) all the sample solutions are partitioned into each microwell by the oil, and no sample is wasted. Finally, the chip is sealed using a coverslip to run PCR amplification. Adapted from Ref. with permission from the Royal Society of Chemistry.

Figure 7. Technical variance for the INEAD and SIDAO systems.

(A) The paper-based INEAD system (Connelly et al., 2015) is shown for comparison with the (B) centrifugal microfluidic that integrates the nucleic acid extraction with LAMP (Loo et al., 2016) and (C) the capillary-based INEAD system (Liu et al., 2013). “A” has the advantage of low cost while “B” and “C” can be automated. Despite the different integration options of the microfluidic chip, the systems can be simple and fast to achieve “sample-in-answer-out”. (D) The magnetic bead-based system combines nucleic acid extraction with a digital Recombinase Polymerase Amplification (RPA) chip (Yang et al., 2018). “D” can automatically achieve “sample-in-digital-answer-out”. Figure A and C are adapted from Ref. and Ref. with permission from ACS Publications, Figure B is adapted from Ref. with permission from Elsevier, and Figure D is adapted from Ref. with permission from Springer.