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.

To the best of our knowledge, this is the first report on paper-based devices for automating the sequential multistep procedures of a sandwich-type enzyme-linked immunosorbent assay (ELISA) that require only a single-step application of the sample solution. The device was based on a piece of nitrocellulose (NC) membrane with specially designed channels, where all the reagents are applied at different locations in order to control the fluid travel to the detection region. The inkjet printing method, a simple and low-cost process, was used to create the flow channel and device barrier patterns. The fabricated barrier was found to be an efficient boundary for the liquid along the printed design in the NC membrane, enabling direct control of the reagent flow time. ELISA results were obtained with a single-step sample application. The developed devices (so-called automated paper-based devices) provided a simple procedure for the sandwich ELISA, while reducing assay time and reagent consumption. Colorimetric results were measured using digital camera imaging with software processing. The capability of the method developed herein was successfully used to determine the levels of human chorionic gonadotropin (hCG) by ELISA.