Improvement of Seed-Mediated Growth of Gold Nanoparticle Labels for DNA Membrane-Based Assays

Gold nanoparticles (AuNPs) are popular labels for colorimetric detection of various analytes, involving proteins, nucleic acids, viruses, and whole cells because of their outstanding optical properties, inertness, and modification variability. In this work, we present an improved approach for enhancement of color intensity for DNA membrane microarrays based on seed-mediated growth of AuNP labels. Biotin-labeled DNA is hybridized with capture oligonucleotide probes immobilized on the microarrays. Then biotin is revealed by a streptavidin-AuNP conjugate followed by the detection of AuNPs. Optimization of seed-mediated enlargement of AuNPs by the reduction of tetrachloroauric acid with hydroxylamine made it possible to change the coloring of specific spots on the microarrays from pink to a more contrasting black with minor background staining. Mean size of the resulting AuNPs was four times larger than before the enhancement. Adjusting the pH of HAuCl₄ solution to 3.5 and use of a large excess of hydroxylamine increased the signal/background ratio by several times. The method's applicability was demonstrated for quantification of a short oligonucleotide of 19 bases and full-length TEM-type β-lactamase genes of 860 bp responsible for the development of bacterial resistance against β-lactam antibiotics. Improved protocol for AuNP enlargement may be further transferred to any other membrane-based assays of nucleic acids with both instrumental and visual colorimetric detection.