Fully Dried Two-Dimensional Paper Network for Enzymatically Enhanced Detection of Nucleic Acid Amplicons

Abstract

Two-dimensional paper networks (2DPNs) have enabled the use of paper-based platforms to perform multistep immunoassays for detection of pathogenic diseases at the point-of-care. To date, however, detection has required the user to provide multiple signal enhancement solutions and been limited to protein targets. We solve these challenges by using mathematical equations to guide the device design of a novel 2DPN, which leverages multiple fluidic inputs to apply fully dried solutions of hydrogen peroxide, diaminobenzidine, and horseradish peroxidase signal enhancement reagents to enhance the limit-of-detection of numerous nucleic acid products. Upon rehydration in our unique 2DPN design, the dried signal enhancement solution reduces the limit-of-detection (LOD) of the device to 5 × 10¹¹ nucleic acid copies/mL without increasing false positive detection. Our easy-to-use device retains activity after 28 days of dry storage and produces reliable signal enhancement 40 min after sample application. The fully integrated device demonstrated versatility in its ability to detect double-stranded and single-stranded DNA samples, as well as peptide nucleic acids.

Figure 1

(A) Schematic of fully assembled 2DPN and location of dried reagent pads. (B) Target binding: fresh nucleic acid samples with fluorescein isothiocyanate (FITC) and biotin tags applied to the dried SA–HRP–AuNP pad. Nucleic acid binding to SA–HRP–AuNPs occurs, followed by capture at the test line in the main lateral flow channel. Unbound AuNPs bind to the biotin-tagged control line. Signal enhancement: DAB and hydrogen peroxide solution flows from the signal enhancement leg to the main lateral flow channel and is oxidized by AuNP-bound poly-HRP at test and control lines, producing a visually darkened (enhanced) signal.

Figure 2

2DPN flow optimization. (A, B) Experimental data (circles) fit with eq 1 (line). k refers to the parameter from eq 1 and R² is the coefficient of determination for the fit of eq 1 to the data shown. (A) AuNP solution velocity over distance in a 5 mm wide channel. (B) DAB solution velocity over distance in a 2 mm wide channel. (C) Proof-of-concept 2DPN flow with food coloring in PBST demonstrating the sequential delivery of reagents to the detection region (boxed).

Figure 3

Signal development over time. (A) Signal intensity of the test line over time for 5 × 10¹² and 5 × 10¹¹ copies of ssDNA/mL. Dashed lines indicate time points when the AuNP signal and the DAB-enhanced signal data are compared. (B) Detection region of 2DPN imaged at 10 min (AuNP signal) and at 60 min (DAB-enhanced signal). (C) Normalized test line intensities at 10 min (AuNP signal) and at 60 min (DAB-enhanced signal) for varying ssDNA concentrations. The visible threshold at 0.02 is indicated by a dashed line. (Dunnett’s multiple comparison vs 0 copies/mL, *p < 0.05, ***p < 0.001, ****p < 0.0001, n = 3, replicates shown in Figure S5).

Figure 4

Quantification of test line intensity. Background-subtracted grayscale test line intensity for hybridized DNA samples run on fully dried and assembled 2DPNs imaged after 60 min. The visible threshold at 0.02 is indicated by a dashed line. (n = 6, **p < 0.01 in Dunnett’s multiple comparison with 1 day dried storage 2DPN as the control).

Figure 5

Results of TARA sample (1:10 dilution in PBST) on a dried 2DPN. (A) Scans taken of a representative test at 10 and 60 min. (B) Test line signal intensity analysis of the TARA sample on 2DPNs before and after signal enhancement (paired t-test, *p < 0.05, n = 3, replicates shown in Figure S6).