Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single-nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal-transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem-loop structure holds the fluorescence-donor and fluorescence-acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein-DNA interaction studies, and protein recognition.

We have designed a novel molecular assembly of quencher molecules to form Superquenchers with excellent quenching efficiency. The Superquencher can be engineered as desired by assembling different types and different numbers of quencher molecules. By labeling a Superquencher to a molecular beacon, a 320 folds enhancement of fluorescent signal was achieved, compared to about 14-fold from a molecular beacon prepared with the same monomer quencher. Our molecular assembly approach can effectively improve the sensitivity of a variety of fluorescent assays and can be widely useful for molecular interaction studiesWe have designed a molecular assembly of superquenchers (SQs) for molecular interaction studies and for ultrasensitive bioanalysis. Signaling biomolecular interactions such as DNA/ RNA hybridization and protein interactions is critically important in areas such as medical diagnosis, disease prevention, and drug discovery. While selectivity in biomolecular recognition can be achieved by capitalizing on highly selective molecular interactions such as antibody-antigen binding and DNA base paring, the effectiveness of a molecular probe is highly dependent on the scheme and the ability to transduce a target recognition event into a measurable signal. Of many approaches, fluorescence is one of the most effective ways to signal molecular interaction and recognition. For instance, fluorogenic probes, such as Taqman, 1 molecular beacons (MB), 2;3 and protease probes 4 have been widely used for biotechnological research and development. The interaction of these molecular probes with target molecules renders unquenching of the fluorophores, yielding detectable fluorescence signal change. While exploited in broad areas such as real-time PCR monitoring, DNA assays, and protein studies, these probes have limited increment of signal change upon interacting with their targets, mainly due to an unquenched high background signal from the probe itself. Strategies for improving signal-to-background ratio of molecular probes promise higher assay sensitivity as well as better reproducibility. There has been encouraging progress in attempts at introducing novel signaling schemes, 5;6 exploring nanocomposites 7;8 , as well as making better quenchers using rational molecular design coupled with sophisticated synthesis. 9-11 Herein, we have molecularly assembled an array of quencher molecules to produce SQs for use in engineering fluorogenic molecular probes with high sensitivity and specificity. We believe that using multiple quenchers to pair with one fluorophore provides better quenching efficiency because of the improved absorption efficiency and the increased probability of dipole-dipole coupling between the quenchers and the fluorophore because of a collective quenching effect of these multiple quenchers. MB was used as a model here to test the performance of the SQs. An excellent example of fluorogenic probe, a MB 2;3;12 is a hairpin shaped DNA with a self-complementary stem that brings terminal labeled fluorophore and ...

Due to its large enhancement effect, nanostructure-based surface-enhanced Raman scattering (SERS) technology had been widely applied for bioanalysis and cell imaging. However, most SERS nanostructures suffer from poor signal reproducibility, which hinders the application of SERS nanostructures in quantitative detection. We report an etching-assisted approach to synthesize SERS-active plasmonic nanoparticles with 1 nm interior nanogap for multiplex quantitative detection and cancer cell imaging. Raman dyes and methoxy poly(ethylene glycol) thiol (mPEG-SH) were attached to gold nanoparticles (AuNPs) to prepare gold cores. Next, Ag atoms were deposited on gold cores in the presence of Pluronic F127 to form a Ag shell. HAuCl4 was used to etch the Ag shell and form an interior nanogap in Au@AgAuNPs, leading to increased Raman intensity of dyes. SERS intensity distribution of Au@AgAuNPs was found to be more uniform than that of aggregated AuNPs. Finally, Au@AgAuNPs were used for multiplex quantitative detection and cancer cell imaging. With the advantages of simple and rapid preparation of Au@AgAuNPs with highly uniform, stable, and reproducible Raman intensity, the method reported here will widen the applications of SERS-active nanoparticles in diagnostics and imaging.