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Sensitive and specific visualization of cell surface biotin receptors (BRs) a class of clinically important biomarkers, remains a challenge. In this work, a dual-emission ratiometric fluorescent nanoprobe is developed for specific imaging of cell surface avidin, a subtype of BRs. The nanoprobe comprises a dual-emission quantum dot nanohybrid, wherein a silica-encapsulated red-emitting QD (rQD@SiO2) is used as the "core" and green-emitting QDs (gQDs) are used as "satellites", which are further decorated with a new "love-hate"-type BR ligand, a phenanthroline-biotin conjugate with an amino linker. The nanoprobe shows intense rQD emission but quenched gQD emission by the BR ligand. Upon imaging, the rQD emission stays constant and the gQD emission is restored as cell surface avidin accrues. Accordingly, the overlaid fluorescence color collected from red and green emission changes from red to yellow and then to green. We refer to such a color change as a traffic light pattern and the nanoprobe as a fluorescent traffic light nanoprobe. We demonstrate the application of our fluorescent traffic light nanoprobe to characterize cancer cells. By the traffic light pattern, cervical carcinoma and normal cells, as well as different-type cancer cells including BR-negative colon cancer cells, BR-positive hepatoma carcinoma cells, breast cancer cells, and their subtypes, have been visually differentiated. We further demonstrate a use of our nanoprobe to distinguish the G2 phase from other stages in a cell cycle. These applications provide new insights into visualizing cell surface biomarkers with remarkable imaging resolution and accuracy.

The combination of microbead array, isothermal amplification, and molecular signaling enables the continuous development of next-generation molecular diagnostic techniques. Herein we reported the implementation of nicking endonuclease-assisted strand displacement amplification reaction on quantum dots-encoded microbead (Qbead), and demonstrated its feasibility for multiplexed miRNA assay in real sample. The Qbead featured with well-defined core-shell superstructure with dual-colored quantum dots loaded in silica core and shell, respectively, exhibiting remarkably high optical encoding stability. Specially designed stem-loop-structured probes were immobilized onto the Qbead for specific target recognition and amplification. In the presence of low abundance of miRNA target, the target triggered exponential amplification, producing a large quantity of stem-G-quadruplexes, which could be selectively signaled by a fluorescent G-quadruplex intercalator. In one-step operation, the Qbead-based isothermal amplification and signaling generated emissive "core-shell-satellite" superstructure, changing the Qbead emission-color. The target abundance-dependent emission-color changes of the Qbead allowed direct, visual detection of specific miRNA target. This visualization method achieved limit of detection at the subfemtomolar level with a linear dynamic range of 4.5 logs, and point-mutation discrimination capability for precise miRNA analyses. The array of three encoded Qbeads could simultaneously quantify three miRNA biomarkers in ∼500 human hepatoma carcinoma cells. With the advancements in ease of operation, multiplexing, and visualization capabilities, the isothermal amplification-on-Qbead assay could potentially enable the development of point-of-care diagnostics.

By complexing a nonionic G-quadruplex ligand with hybrid dual-emission quantum dots (QDs), a ratiometric fluorescent nanoprobe is developed for G-quadruplex detection in a sensitive and specific manner. The QDs nanohybrid comprised of a green-emission QD (gQD) and multiple red-emission QDs (rQDs) inside and outside of a silica shell, respectively, is utilized as the signal displaying unit. Only the presence of G-quadruplex can displace the ligand from QDs, breaking up the QDs-ligand complexation, and inducing the restoration of the rQDs fluorescence. Since the fluorescence of embedded gQD stays constant, variations of the dual-emission intensity ratios display continuous color changes from green to bright orange, which can be clearly observed by the naked eye. Furthermore, by utilizing competitive binding of a cationic ligand versus the nonionic ligand toward G-quadruplex, the nanoprobe is demonstrated to be applicable for assessing the affinity of a G-quadruplex-targeted anticancer drug candidate, exhibiting ratiometric fluorescence signals (reverse of that for G-quadruplex detection). By making use of the specificity of the ligand binding with G-quadruplex against a double helix, this nanoprobe is also demonstrated to be capable of sensitive detection of one-base mutation, exhibiting sequence-specific ratiometric fluorescence signals. By functionalizing with a nuclear localization peptide, the nanoprobe can be used for visualization of G-quadruplex in the nucleus of human cells.

We have developed a proof-of-concept quantum dot-ligand (QD-L) system for visual selective detection of nucleic acids, in combination with a ratiometric fluorescence technique. This system comprises a dual-emission QDs nanohybrid formed by embedding a red-emission QD (rQD) in a silica nanoparticle and electrostatically assembling green-emission QDs (gQDs) onto the silica surface, as the signal displaying unit, and a hydrophobic compound, dipyrido[3,2-a:2',3'-c]phenazine (dppz), attached onto the gQDs surface via phase transfer, as the ligand as well as fluorescence quencher of gQDs. This system is successfully used for quantification of double-stranded DNA (dsDNA). Because of its avid binding with dppz, dsDNA can break up the QD-L system, displacing the dppz ligand from the gQDs surface and restoring the gQDs emission. Since the red emission of embedded rQDs stays constant, variations of the dual-emission intensity ratios display continuous color changes from orange to bright green, which can be clearly observed by the naked eye. More importantly, this system is advantageous in terms of specificity over a QD ionic conjugate, because the electrical neutrality of dppz excludes its nonspecific electrostatic association with dsDNA. The QD-L system also is capable of detecting single-nucleotide polymorphism, exhibiting sequence-specific ratiometric fluorescence as a QD-bioconjugate does, but possessing the obvious advantage in terms of low cost, with the avoidance of modification, labeling, and purification processes. Therefore, the QD-L system provides an extremely simple but general strategy for detecting nucleic acids in a facile, sensitive, and specific manner.