DNA-templated silver nanoclusters (DNA-AgNCs) are fluorescent molecules containing few-atom clusters of silver stabilized by a short strand of DNA. They fluoresce at wavelengths in the visible spectrum with emission colors tuned by varying the sequence of the DNA used for their synthesis. The combination of their small size, tunable spectra and biocompatibility opens exciting, new possibilities for their use in chemical sensing, biological labeling and imaging, genetic mutation detection and the development of self-assembled DNA nanotechnologies.

A detailed understanding of the structure of DNA-AgNCs is vital for precisely engineering their properties for future applications. Specifically, changes in the arrangement and composition of bases surrounding a AgNC can strongly influence its fluorescence. Although this sensitivity has already been leveraged for the development of novel chemical sensors, a better understanding of the mechanism is needed to improve performance and broaden the applicability of DNA stabilized AgNCs. In this work, we use high-resolution microfluidic capillary electrophoresis to show that, although AgNC depend on single-stranded DNA for their stabilization, changes made to bases in the double-stranded stem region of a DNA hairpin can perturb their structure, leading to differences in fluorescence emission.

We also explore ways in which AgNC can be robust to DNA sequence changes. We document one DNA-AgNC in which the DNA adopts different conformations, or shapes, that yield clusters with the same emission color. We also show that poly-thymidine regions, which are know to bind silver poorly,can act as convenient handles to adjust the electrophoretic mobility of DNA-AgNCs without affecting their fluorescence.

Using poly-thymidine appendages, we demonstrate a way to tune the electrophoretic mobility of a AgNC-based DNA probe for use in a microfluidic assay. These label-free probes are composed of a DNA hairpin that generates a fluorescent silver nanocluster only after binding to a specific target DNA sequence. By tuning the mobility of probes designed to bind different targets, we demonstrate a rapid microfluidic separation assay for the multiplexed fluorescent detection of nucleic acid targets for Hepatitis A, B and C.

The probe design initially used for these studies suffered from several shortcomings. Most significantly, the probe DNA failed to generate a fluorescent AgNC for some binding domain sequences. To overcome this, as well as provide added functionality, we engineer a new DNA-AgNC based sensor that supports ratiometric fluorescence measurements for the sensitive, specific and low-cost detection of DNA. Probes based on our new design generate a green emitting AgNC in its hairpin state, and a red emitting AgNC after binding their target. The ratiometric fluorescence provides a stable signal and rapid quantification of DNA concentration regardless of the choice of target, a dramatic improvement over similar turn-on fluorescent probes.