Multiplex Paper-Based Colorimetric DNA Sensor Using Pyrrolidinyl Peptide Nucleic Acid-Induced AgNPs Aggregation for Detecting MERS-CoV, MTB, and HPV Oligonucleotides

Abstract

The development of simple fluorescent and colorimetric assays that enable point-of-care DNA and RNA detection has been a topic of significant research because of the utility of such assays in resource limited settings. The most common motifs utilize hybridization to a complementary detection strand coupled with a sensitive reporter molecule. Here, a paper-based colorimetric assay for DNA detection based on pyrrolidinyl peptide nucleic acid (acpcPNA)-induced nanoparticle aggregation is reported as an alternative to traditional colorimetric approaches. PNA probes are an attractive alternative to DNA and RNA probes because they are chemically and biologically stable, easily synthesized, and hybridize efficiently with the complementary DNA strands. The acpcPNA probe contains a single positive charge from the lysine at C-terminus and causes aggregation of citrate anion-stabilized silver nanoparticles (AgNPs) in the absence of complementary DNA. In the presence of target DNA, formation of the anionic DNA-acpcPNA duplex results in dispersion of the AgNPs as a result of electrostatic repulsion, giving rise to a detectable color change. Factors affecting the sensitivity and selectivity of this assay were investigated, including ionic strength, AgNP concentration, PNA concentration, and DNA strand mismatches. The method was used for screening of synthetic Middle East respiratory syndrome coronavirus (MERS-CoV), Mycobacterium tuberculosis (MTB), and human papillomavirus (HPV) DNA based on a colorimetric paper-based analytical device developed using the aforementioned principle. The oligonucleotide targets were detected by measuring the color change of AgNPs, giving detection limits of 1.53 (MERS-CoV), 1.27 (MTB), and 1.03 nM (HPV). The acpcPNA probe exhibited high selectivity for the complementary oligonucleotides over single-base-mismatch, two-base-mismatch, and noncomplementary DNA targets. The proposed paper-based colorimetric DNA sensor has potential to be an alternative approach for simple, rapid, sensitive, and selective DNA detection.

Scheme 1. (A) Design and (B) Operation of Multiplex Paper-Based Colorimetric Device

Scheme 2. Process of acpcPNA-Induced AgNP Aggregation in the Presence of DNA_com and DNA_nc

Figure 1

Photograph of visual color changes obtained from detection of MERS-CoV, MTB, and HPV in the presence of DNA_com.

Figure 2

Influence of (A) AgNPs/PBS ratio and (B) acpcPNA probe concentration on color intensity for MERS-CoV, MTB, and HPV detection. The error bars represent one standard deviation (SD) obtained from three independent measurements (n = 3).

Figure 3

Color intensity of (A) MERS-CoV, (B) MTB, and (C) HPV detection after hybridization of DNA_m1, DNA_m2, and DNA_nc. The error bars represent one standard deviation (SD) obtained from three independent measurements (n = 3).

Figure 4

Change of probe color intensity vs DNA target concentration (ΔI) and calibration graph between ΔI and log DNA target concentration (inset) for (A) MERS-CoV, (B) MTB, and (C) HPV detection. The error bars represent standard deviation (SD) obtained from three independent measurement (n = 3).

Figure 5

Selectivity of 100 nM MERS-CoV, MTB, and HPV detection using multiplex colorimetric PAD (1, C₁= AgNPs + MERS-CoV acpcPNA probe; 2, C₂ = AgNPs + MTB acpcPNA probe; 3, C₃ = AgNPs + HPV acpcPNA probe).