(559d) A Gold Nanostructure-Dynamic Light Scattering Tandem for the Detection of Transcription Factors and Micro RNA

Gold nanoparticles (AuNP) functionalized with DNA has shown to be versatile building blocks for the formation of nanostructures, which function as intermediate checkpoints or end-point readouts in diagnostic platforms. The affinity of thiolated DNA for citrate-capped AuNP not just allows their attachment onto AuNPs, but also creates conjugates that have unique physical properties. Through the use of techniques such as electrophoretic mobility gel shift assays, conjugates bearing distinct numbers of DNA can be recovered, and used as probes for further detection.

We have designed conjugates that are complementary to specific miRNA targets, whereupon binding nanostructures are formed. Due to the control of the DNA loading on AuNP, dimers are primarily formed, which is unlike typical aggregation techniques that is without control and lacks a clearly defined structure. This dimer formation process also leads to distinct physical changes such as potential plasmon coupling of the constituent AuNPs, and more directly, an overall increase in the size of the system. To present the successful dimer formation process, we queried the as-formed nanostructures on the dynamic light scattering (DLS) platform. DLS has been well utilized in the characterization of nanoparticles and polymers, and we seek to leverage the DLS readout to show the formation of nanostructures in the presence of the miRNA target. Given the large scattering cross section of AuNPs, and enhanced by the coupled particles in a dimeric arrangement, DLS can distinctly show the increase in the size of the particles in the system, which reflect the binding and detection of the target.  The size increase from the nanostructure formation also leads to even more intense signals on the DLS, and this amplification further enhances the sensing capabilities of the technique. With miRNA as the model of study, we present AuNP probes that have successfully detected and distinguished members (let7a, 7f and 7g) of the let7 miRNA family, in a significantly selective and sensitive manner, that is also rapid (DLS readout presented in 5 minutes). Optimization works have also been done to enhance the binding process and stability of the readout. With more works reporting on the role of miRNAs in the pathogenesis of diseases and control of cellular machinery, the detection and study of miRNA has taken on increasing importance, and a simple and direct screening of miRNA in a quantitative manner as shown in our technique presents a viable alternative for miRNA sensing.

In addition, we further developed the nanostructure platform such that dimers are not just endpoints but probes for the detection of transcription factors. With the estrogen receptor (ER) being strongly implicated in breast cancer and treatment considerations, there is strong motivation to study these transcription factors. We had fabricated dimers containing the ER-binding site - ERE, localised within the DNA duplex holding the two AuNPs together. The dimers purified and recovered through gel elecctrophoresis presented a single distinct peak on the DLS. When ER is added, an additional complex peak was observed. And this was observed only for ER on dimers with ERE. When a different DNA sequence was used, or a different protein was queried, little or no complex peak was observed, which validated the selectivity of the technique. A pM detection limit is also an improvement over other AuNP methods.