Naked Eye Y Amelogenin Gene Fragment Detection Using DNAzymes on a Paper-based Device

Abstract

Nowadays, there are strong efforts in developing new technology for rapid detection of specific DNA sequences for environmental monitoring, forensic analysis and rapid biomedical diagnosis applications. That is where microfluidic paper-based analytical devices are positioned as suitable platforms for the development of point of care analytical devices, due to their simple fabrication protocols, ease of use and low cost. Herein, a methodology for in situ single strand DNA detection by using a colorimetric assay based on the formation of a DNAzyme within a paper substrate was developed. A DNAzyme that could only be formed in the presence of a specific sequence of the Y human amelogenin gene was designed. The performance of the DNAzyme was followed colorimetrically first in solution and then in paper substrates. The reaction was found to be specific to the Y fragment selected as analyte. The DNAzyme reaction on paper enabled the unequivocal colorimetric identification of the Y single strand DNA fragment both qualitatively, with the naked eye (143 ng), and quantitatively by image analysis (45.7 ng). As a proof of concept, a microfluidic paper-based device, pre-loaded with all DNAzyme reagents, was characterized and implemented for the simultaneous detection of X and Y single strand DNA fragments.