Capillary flow velocity-based length identification of PCR and RPA products on paper microfluidic chips

Abstract

This work demonstrates a novel, non-fluorescence approach to the length identification of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products, utilizing capillary flow velocities on paper microfluidic chips. It required only a blank paper chip, aminated microspheres, and a smartphone, with a rapid assay time and under ambient lighting. A smartphone evaluated the initial capillary flow velocities on the paper chips for the PCR and RPA products from various bacterial samples, where the pre-loaded aminated microspheres differentiated their flow velocities. Flow velocities were analyzed at different time frames and compared with the instantaneous flow velocities and interfacial tension (γ_LV) data. Subsequent error analysis justified the use of the initial time frames. A robust linear relationship could be established between the initial flow velocities against the square root of the product lengths, with R² values of 0.981 for PCR and 0.993 for RPA. The assay seemed not to have a significant dependency on the cycle numbers and initial target concentrations. This novel method can be potentially used with various paper microfluidic methods of nucleic acid amplification tests towards rapid and handheld assays.

Keywords: Capillary action; Microspheres; Polymerase chain reaction; Recombinase polymerase amplification; Smartphone.

Figure 1.. Representative results with PCR samples.

(A) A smartphone captures a video clip of capillary action. Aminated microspheres at the optimized concentration of 0.1 ng/μL are pre-loaded one-third of the way down the channel before loading the PCR product sample. (B) Representative flow velocity profiles of 416 bp PCR products with 0.5 μm (left) or 1.0 μm (right) microspheres. Pixel numbers (representing the flow distance $l$) are plotted against the square root of the frame number (representing $\sqrt{t}$). Three to four replicates per sample. (C) The same is plotted against the frame number $t$.

Figure 2.. Time Optimization with PCR samples.

(A) Initial slopes of capillary flow velocities were evaluated for the first 10, 20, 30, or 40 frames with various PCR products and 0.5 μm microspheres. The 10- and 20-frame results showed an increasing trend, except for the 682 bp and 1121 bp products. (B) Graphical illustrations show the lowering of interfacial tension (${\gamma }_{LV}$) by the microspheres at the moving front (liquid-vapor interface) (left), regaining of interfacial tension (${\gamma }_{LV}$) due to the neutralization of the microspheres via amplicon binding (middle), and limited binding of longer targets (682 bp and 1121 bp) to the microspheres. (C) Initial slopes with various PCR products and 1.0 μm microspheres, showing the best results with 20 frames and an increasing trend, including 682 bp and 1121 bp products. (D) Graphical illustrations show the capability of the longer (682 bp and 1121 bp) PCR products’ binding to larger microspheres.

Figure 3.. Linearity of Standard Curves and Slope Fluctuations over Time.

(A) Initial slopes of capillary flow velocities were plotted against the square root of amplicon length to construct standard curves. R2 values of such standard curves were summarized with varying frame numbers (10, 20, 30, 40, 50, 100, 200, 300, 400, and 500) for 1.0 μm aminated microspheres. (B) The optimized standard curve with the initial 20-frame slopes. $R^2$ value was 0.981. (C) Instantaneous slopes (K) were plotted against $\sqrt{t}$, representing the fluctuations in the capillary flow velocities. Relatively smaller fluctuations could be found until $\sqrt{t}=4.5$ ($t=20$ frames), as indicated by an arrow. (D) Pendant droplet analysis of PCR products with 0.5 μm and 1.0 μm microspheres (N = 3). Interfacial tension (${\gamma }_{LV}$) was observed to increase with amplicon length.

Figure 4.. Representative results with RPA samples.

(A) The assay procedure is the same as that with PCR samples. (B) Representative flow velocity profiles ($l$ against $\sqrt{t}$) of 416 bp RPA products with 0.5 μm (left) or 1.0 μm microspheres. Four replicates per sample. (C) The same is plotted against $t$.

Figure 5.. Time Optimization with RPA samples.

(A) Initial slopes of capillary flow velocities were evaluated for the first 20, 100, 200, or 300 frames with various RPA products and 0.5 μm microspheres. 100, 200, and 300 frame results displayed an increasing trend, except for the 682 bp product. (B) Graphical illustrations show the neutralization of the microspheres via amplicon binding and the limited binding of longer targets (682 bp) to the microspheres. (C) Initial slopes with various RPA products and 1.0 μm microspheres, showing the best results with 300 frames and an increasing trend including 682 bp product. (D) Graphical illustrations show the capability of the longer (682 bp) RPA products’ binding to larger microspheres.

Figure 6.. Linearity of Standard Curves and Slope Fluctuations over Time.

(A) Initial slopes of capillary flow velocities were plotted against the square root of amplicon length to construct standard curves. $R^2$ values of standard curves with varying frame numbers (20, 100, 200, 300, 400, and 500) for 1.0 μm aminated microspheres. (B) The optimized standard curve with the initial 300-frame slopes. $R^2$ value was 0.993. (C) Instantaneous slopes ($K$) were plotted against $\sqrt{t}$, representing the fluctuations in the capillary flow velocities. Relatively smaller fluctuations could be found until $\sqrt{t}=17$ ($t=300$ frames), as indicated by an arrow. (D) Pendant droplet analysis of RPA products with 0.5 μm and 1.0 μm microspheres (N = 3). Interfacial tension (${\gamma }_{LV}$) was observed to increase with amplicon length.