Rapid molecular diagnosis of infectious viruses in microfluidics using DNA hydrogel formation

Abstract

There has been an urgent need to quickly screen and isolate patients with viral infections from patients with similar symptoms at point-of-care. In this study, we introduce a new microfluidic method for detection of various viruses using rolling circle amplification (RCA) of pathogens on the surface of thousands of microbeads packed in microchannels. When a targeted pathogen meets the corresponding particular template, the DNAs are rapidly amplified into a specific dumbbell shape through the RCA process, forming a DNA hydrogel and blocking the flow path formed between the beads. Due to the significant increase in reaction surface area, the detection time was shortened to less than 15 min and the detection limit of various pathogens has been reached to 0.1 pM. By injecting the stained liquid, the existence of the target pathogens in a sample fluid can be determined with the naked eye. Furthermore, by integrating multi-channel design, simultaneous phenotyping of various infective pathogens (i.e., Ebola, Middle East respiratory syndrome (MERS), and others) in biological specimens can be performed at a point-of-care.

Keywords: DNA; Hydrogel; Microbead; Microfluidics; Molecular diagnosis; RCA.

Fig. 1

(a) A photograph of the experimental apparatus, (b) schematic illustration of the bead-packed microchannel, and (c) microscopic images of the bead-packed microchannel.

Fig. 2

Schematic of DNA hydrogel formation through rolling circle amplification using agarose-based microbeads. (a) The templates are self-assembled to form an asymmetric dumbbell shape. And the primers are immobilized on the microbeads. (b) The templates are hybridized with primers immobilized on microbeads surface. (c) When the template on the microbead hybridized with a target pathogen, (d) the template can be ligated to form a closed-loop template. (e) RCA products are elongated by Phi29 polymerase. (f) The dumbbell-shaped long DNAs are aggregated with neighbor DNAs and form a DNA gel in bead voids.

Fig. 3

Validation of primer immobilization on polystyrene (PS) and Sepharose beads. Schematic of immobilized primers on (a) PS beads and (b) Sepharose beads hybridized with fluorescence probe FAM. Fluorescence microscopic images of (c) PS beads and (d) Sepharose beads, and side view of DNA hydrogel using (e) PS beads and (f) Sepharose beads.

Fig. 4

Applied pressure with respect to (a) pathogen DNA concentrations of 0, 0.01, 0.1, 1, 100, and 10,000 pM (relative standard deviation (RSD) of each value were 12.8%, 17.4%, 26.6%, 5.2%, 5.8%, and 6.4%, respectively) and (b) incubation time of 0, 5, 10, 15, 20, 25, and 30 min (RSD of each value were 12.8%, 12.8%, 17.4%, 23.3%, 9.2%, 6.0%, and 4.9%, respectively). (*: p < 0.05, **: p < 0.001).

Fig. 5

The results of multiple detection of (a) sample 1 (Dengue and MERS) and (b) sample 2 (Ebola and Zika) using microchip.