A SAW-Based Programmable Controlled RNA Detecting Device: Rapid In Situ Cytolysis-RNA Capture-RNA Release-PCR in One Mini Chamber

Abstract

Viral RNA detection is crucial in preventing and treating early infectious diseases. Traditional methods of RNA detection require a large amount of equipment and technical personnel. In this study, proposed a programmable controlled surface acoustic wave (SAW)-based RNA detecting device has been proposed. The proposed device can perform the entire viral RNA detection process, including cell lysis by cell-microparticle collision through SAW-induced liquid whirling, RNA capture by SAW-suspended magnetic beads, RNA elution through SAW-induced high streaming force, and PCR thermal cycling through SAW-generated heat. The device has completed all RNA detection steps in one mini chamber, requiring only 489 µl reagents for RNA extraction, much smaller than the amount used in manual RNA extraction (2065 µl). The experimental results have shown that PCR results from the device are comparable to those achieved via commercial qPCR instrumental detection. This work has demonstrated the potential of SAW-based lab-on-a-chip devices for point-of-care testing and provided a novel approach for rapidly detecting infectious diseases.

Keywords: RNA detection; microfluidic; surface acoustic wave.

Figure 1

The concept of on‐chip RNA testing utilizing the mechanical and thermal effects of the SAW.

Figure 2

a) Three Layers of the designed microfluidic chip. b) The microfluidic chip assembly. c) Microscopic imaging of UIDT Structure 1, UIDT Structure 2, and Bidirectional IDT. d) The schematics of the fabrication process of IDT layer and microchannel layer. The detailed process can be found in Experimental Section.

Figure 3

a–c) The evaluation of SAW chip input power and lysis time in cell lysis by cell‐microparticle collisions. a) The RNA concentration obtained with three input powers and three reaction periods (1, 5, and 8 min). b) The RNA concentration trend with different input parameters. c) The concentration of RNA extraction at different time points from 1–8 min with an input power of ‐10 dBm. d) RNA concentration and OD 260/280 of different IDTs. With an input power of ‐10 dBm. e) Energy transmission of two types of IDTs: The energy of bidirectional IDT was completely released into the reaction chamber. The energy generated at the rear end of the UIDT(Structure 1 and Structure 2) was absorbed by PDMS. f) Gel electrophoresis: Experimental group (EG): RNA extracted by IDT 3; Control group (CG): RNA extracted by manual operation.

Figure 4

Fluorescence microscopy with AOPI staining was used to observe the extent of Hela cell lysis by SAW in the presence or absence of magnetic beads. a) Cells showed fluorescent green without SAW or magnetic beads. b) Cells remained fluorescent green with SAW but without magnetic beads. c) Cells showed fluorescent green without SAW but with magnetic beads. d) Cells turned fluorescent red with SAW and magnetic beads.

Figure 5

a) Force analysis of a magmatic bead. b) Schematic drawing of simulation of magnetic beads in the reaction chamber with the SAW on and off. c) Image of magnetic beads in a rectangular chamber with the SAW on and off. Simulation results can be found in Figure S6 (Supporting Information).

Figure 6

a–c) Fluorescent staining of RNA and magnetic beads. RNA and magnetic beads show red and yellow fluorescence at 490 nm excitation wavelength, respectively. a) Yellow fluorescent magnetic beads without RNA adsorption under a 490 nm excitation wavelength. b) The fluorescent staining of a combination process of RNA and the magnetic beads without SAW. c) The fluorescent staining of a combination process of RNA and the magnetic beads with ‐15 dBm input SAW. d) RNA concentration captured by magnetic beads with different input powers. e) RNA concentration versus elution time with different input powers.

Figure 7

a) PCR circulation temperature curve based on SAW. b) Schematics of temperature control, and the detailed process can be found in the Experimental Section. c) Infrared thermal imaging of SAW heating in the reaction chamber.

Figure 8

Workflow of the automatic fluidic control process. a) Cell lysis by SAW‐induced magnetic bead collisions. b) Washing off the impurities with the washing solution. c) Washing away DNA and proteins with a purified solution. d) Repeating the washing process for a second time. e) Filling eluate into the reaction chamber. f) Separating magnetic beads with a magnetic field. g) Mixing RNA with the PCR premix. h) Blotting off excess RNA samples. i) SAW‐induced PCR thermal cycling. The more information can be found in Video S1 (Supporting Information).

Figure 9

Fluorescence sensor positioned above microfluidic chip to collect fluorescence signal.

Figure 10

a) PCR amplification result of T2A infected sample. The mean squared error of the SAW PCR group (MSE‐S) was 0.63, the mean squared error of the control (MSE‐C) was 0.28. b) PCR amplification result of GIPC1 infected sample. MSE‐S:0.035; MSE‐C:0.011. c) PCR amplification result of CMTM3 infected sample. MSE‐S:0.031; MSE‐C:0.007. d) PCR amplification result of the negative test sample. MSE‐S: 0.063; MSE‐C: 7.7x10⁻⁶. e) Gel electrophoresis analysis of the amplicons. The original full image of gel electrophoresis analysis can be found in Figure S7 (Supporting Information).