DNA‐Templated Assembly of a Heterobivalent Quantum Dot Nanoprobe For Extra‐ and Intracellular Dual‐Targeting and Imaging of Live Cancer Cells

Quantum dots (QDs) hold great promise for the molecular imaging of cancer because of their superior optical properties. Although cell‐surface biomarkers can be readily imaged with QDs, non‐invasive live‐cell imaging of critical intracellular cancer markers with QDs is a great challenge because of the difficulties in the automatic delivery of QD probes to the cytosol and the ambiguity of intracellular targeting signals. Herein, we report a new type of DNA‐templated heterobivalent QD nanoprobes with the ability to target and image two spatially isolated cancer markers (nucleolin and mRNA) present on the cell surface and in the cell cytosol. Bypassing endolysosomal sequestration, this type of QD nanoprobes undergo macropinocytosis following the nucleolin targeting and then translocate to the cytosol for mRNA targeting. Fluorescence resonance energy transfer (FRET) based confocal microscopy enables unambiguous signal deconvolution of mRNA‐targeted QD nanoprobes inside cancer cells.     

A new method for the detection of ATP using a quantum-dot-tagged aptamer

Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3′-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3′-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3′-biotin-modified DNA and 3′-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3′-biotin-modified DNA and 3′-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3′-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer–ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection. Zhang Chen and Guang Li contributed equally to this work.     

Nanoassemblies designed from semiconductor quantum dots and molecular arrays

The formation of nanoassemblies of CdSe/ZnS quantum dots (QD) and pyridyl-substituted free-base porphyrin (H(2)P) molecules has been spectroscopically identified by static and time-resolved techniques. The formation of nanoassemblies has been engineered by controlling the type and geometry of the H(2)P molecules. Pyridyl functionalization gives rise to a strong complex formation accompanied by QD photoluminescence (PL) quenching. For some of the systems, this quenching is partly related to fluorescence resonance energy transfer (FRET) from the QD to H(2)P and can be explained according to the F?rster model. The quantitative interpretation of PL quenching due to complexation reveals that (i) on average only about (1)/(5) of the H(2)P molecules at a given H(2)P/QD molar ratio are assembled on the QD and (ii) only a limited number of "vacancies" accessible for H(2)P attachment exist on the QD surface.     

Capillary electrophoretic separation and characterizations of CdSe quantum dots

We have developed a capillary electrophoresis method to characterize the QD surface ligand interactions with various surfactant systems. The method was demonstrated with 2–5 nm CdSe nanoparticles surface-passivated with trioctylphosphine oxide (TOPO). Water solubility was accomplished by surfactant-assisted phase transfer via an oil-in-water microemulsion using either cationic, anionic, or non-ionic surfactants. Interaction between the QD surface ligand (TOPO) and the alkyl chain of the surfactant molecule produces a complex and dynamic surface coating that can be characterized through manipulation of CE separation buffer composition and capillary surface modification. Additional characterization of the QD surface ligand interactions with surfactants was accomplished by UV-VIS spectroscopy, photoluminescence, and TEM. It is anticipated that studies such as these will elucidate the dynamics of QD surface ligand modifications for use in sensors.      

Homogenous rapid detection of nucleic acids using two-color quantum dots

We report a homogenous method for rapid and sensitive detection of nucleic acids using two-color quantum dots (QDs) based on single-molecule coincidence detection. The streptavidin-coated quantum dots functioned as both a nano-scaffold and as a fluorescence pair for coincidence detection. Two biotinylated oligonucleotide probes were used to recognize and detect specific complementary target DNA through a sandwich hybridization reaction. The DNA hybrids were first caught and assembled on the surface of 605 nm-emitting QDs (605QDs) through specific streptavidin-biotin binding. The 525 nm-emitting QDs (525QDs) were then added to bind the other end of DNA hybrids. The coincidence signals were observed only when the presence of target DNA led to the formation of 605QD/DNA hybrid/525QD complexes. In comparison with a conventional QD-based assay, this assay provided high detection efficiency and short analysis time due to its high hybridization efficiency resulting from the high diffusion coefficient and no limitation of temperature treatment. This QD-based single-molecule coincidence detection offers a simple, rapid and ultra sensitive method for genomic DNA analysis in a homogenous format.     

Characterization and separation of semiconductor quantum dots and their conjugates by capillary electrophoresis

Semiconductor quantum dots (QDs) are very important luminescent nanomaterials with a wide range of potential applications. Currently, QDs as labeling probes are broadly used in bioassays, including immunoassay, DNA hybridization, and bioimaging, due to their excellent physical and chemical properties, such as broad excitation spectra, narrow and size‐dependent emission profiles, long fluorescence life time, and good photostability. The characterization of QDs and their conjugates is crucial for their wide bioapplications. CE has become a powerful tool for the separation and characterization of QDs and their conjugates. In this review, some CE separation models of QDs are first introduced, mainly including CZE, CGE, MEKC, and ITP. And then, some key applications, such as the measurements of size, surface charge, and concentration of QDs and the characterization of QDs conjugates (e.g. QD–protein, QD–DNA, QD–small molecule), are also described. Finally, future perspectives are discussed.     

Aptamer based lysozyme assay using fluorescent CuInS<Subscript>2</Subscript> quantum dots and graphene oxide,and its application to inhibitor screening

We report on a widely applicable approach for protein detection by using triple-helix DNA mediated CuInS₂ quantum dot (QD) and graphene oxide (GO) nanocomposite. The CuInS₂ QDs were coated with mercaptopropionic acid and then covalently linked to a hairpin aptamer against lysozyme (HLA). Single-stranded DNA (triple helix-forming oligonucleotide; THFO) readily absorbs on the surface of GO via π-stacking interaction, and this results in the formation of THFO-GO. If HLA-CuInS₂ QDs are added to the THFO-GO system, the fluorescence of HLA-CuInS₂ QDs (at excitation/emission wavelengths of 590/665 nm) is quenched. Lysozyme has a higher affinity for HLA than THFO. Therefore, in the presence of lysozyme, it will bind to the HLA-CuInS₂ QD and displace the THFO-GO. This results in the restoration of fluorescence that is related to the concentration of lysozyme. The fluorescence of the QDs is turned on. The calibration plot is linear in the 0.01 to 1.8 ng·mL￣¹ concentration range, with a 3 pg·mL￣¹ detection limit (at a signal-to-noise ratio of 3). The method was also applied to study the inhibition of lysozyme by Ivy _Ec . In our perception, this method has a wide scope in that it may become applicable to any protein for which an appropriate aptamer is available.

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Graphical abstract A novel convenient and universal fluorescence nanoprobe for sensitive and selective detection of lysozyme and inhibitor screening was established using triple-helix DNA mediated CuInS₂ QDs and GO nanocomposites

     

Compatibility of quantum dots with immunobuffers, and its effect on signal/background of quantum dot-based immunoassay

In this work, the compatibility of quantum dots (QDs) with immunobuffers was studied by investigating the fluorescence stability of QDs in immunobuffers (in this research immunobuffers were defined as buffers for immunoaffinity binding or separation). Experimentally, the fluorescence signals of QDs with different surface chemistries (amine-terminated, streptavidin-coated, or antibody-conjugated) in commonly used immunobuffers were monitored versus time. The effect of some buffer composition on the compatibility of QDs with these buffers was also explored. Based on experimental data, the QD compatibility with these buffers is summarized, and it is found that a trace amount of bovine serum albumin added to most of these buffers helps QDs to achieve compatibility with them. Moreover, with QD as fluorescence label and C-reactive protein as a model analyte, a magnetic bead-based assay was performed using compatible and incompatible QD–immunobuffer systems. It is shown that compatible QD–immunobuffer systems can be used to achieve a higher assay signal/background ratio.      

Bio serves nano: biological light-harvesting complex as energy donor for semiconductor quantum dots

Light-harvesting complex (LHCII) of the photosynthetic apparatus in plants is attached to type-II core-shell CdTe/CdSe/ZnS nanocrystals (quantum dots, QD) exhibiting an absorption band at 710 nm and carrying a dihydrolipoic acid coating for water solubility. LHCII stays functional upon binding to the QD surface and enhances the light utilization of the QDs significantly, similar to its light-harvesting function in photosynthesis. Electronic excitation energy transfer of about 50% efficiency is shown by donor (LHCII) fluorescence quenching as well as sensitized acceptor (QD) emission and corroborated by time-resolved fluorescence measurements. The energy transfer efficiency is commensurable with the expected efficiency calculated according to F?rster theory on the basis of the estimated donor-acceptor separation. Light harvesting is particularly efficient in the red spectral domain where QD absorption is relatively low. Excitation over the entire visible spectrum is further improved by complementing the biological pigments in LHCII with a dye attached to the apoprotein; the dye has been chosen to absorb in the "green gap" of the LHCII absorption spectrum and transfers its excitation energy ultimately to QD. This is the first report of a biological light-harvesting complex serving an inorganic semiconductor nanocrystal. Due to the charge separation between the core and the shell in type-II QDs the presented LHCII-QD hybrid complexes are potentially interesting for sensitized charge-transfer and photovoltaic applications.     

Nuclease tolerant FRET probe based on DNA-quantum dot conjugation.

We have developed a fluorescence resonance energy transfer (FRET) probe based on the conjugation of a quantum dot (QD) with dye (YOYO-3) intercalated DNA. The FRET-inducing electrostatic coupling of DNA and the QD made structural changes to the QD-DNA conjugates, which significantly prevented an enzymatic reaction between the DNA and a conventional restriction endonuclease (EcoRI).     

Surface effects on quantum dot-based energy transfer

CdSe quantum dot (QD)-phthalocyanine (Pc) conjugates were prepared as energy transfer donor-acceptor pairs, and the efficiency of the energy transfer process in this system was investigated as a function of QD size and under different surface chemistry conditions. The kinetics and efficiency of the energy transfer process were studied by femtosecond time-resolved laser spectroscopy. We observed that the energy transfer efficiency does not follow a linear dependence on spectral overlap integrals as predicted by the F?rster theory for molecules. This observation is found to be due to the involvement of QD surface states in the energy transfer process from the photoexcited QDs to the molecular energy acceptor.     

CsPbBr3 QD/AlOx Inorganic Nanocomposites with Exceptional Stability in Water,Light, and Heat

Herein, the assembly of CsPbBr₃ QD/AlO_x inorganic nanocomposites, by using atomic layer deposition (ALD) for the growth of the amorphous alumina matrix (AlO_x ), is described as a novel protection scheme for such QDs. The nucleation and growth of AlO_x on the QD surface was thoroughly investigated by miscellaneous techniques, which highlighted the importance of the interaction between the ALD precursors and the QD surface to uniformly coat the QDs while preserving the optoelectronic properties. These nanocomposites show exceptional stability towards exposure to air (for at least 45 days), irradiation under simulated solar spectrum conditions (for at least 8 h), and heat (up to 200 °C in air), and finally upon immersion in water. This method was extended to the assembly of CsPbBr_x I_3−x QD/AlO_x and CsPbI₃ QD/AlO_x nanocomposites, which were more stable than the pristine QD films.     

Colloidally Stable CdS Quantum Dots in Water with Electrostatically Stabilized Weak-Binding,Sulfur-Free Ligands

Colloidal quantum dot (QD) photocatalysts have the electrochemical and optical properties to be highly effective for a range of redox reactions. QDs are proven photo-redox catalysts for a variety of reactions in organic solvents but are less prominent for aqueous reactions. Aqueous QD photocatalysts require hydrophilic ligand shells that provide long-term colloidal stability but are not so tight-binding as to prevent catalytic substrates from accessing the QD surface. Common thiolate ligands, which also poison many co-catalysts and undergo photo-oxidative desorption, are therefore often not an option. This paper describes a framework for the design of water-solubilizing ligands that are in dynamic exchange on and off the QD surface, but still provide long-term colloidal stability to CdS QDs. The binding affinity and inter-ligand electrostatic interactions of a bifunctional ligand, aminoethyl phosphonic acid (AEP), are tuned with the pH of the dispersion. The key to colloidal stability is electrostatic stabilization of the monolayer. This work demonstrates a means of mimicking the stabilizing power of a thiolate-bound ligand with a zwitterionic tail group, but without the thiolate binding group.     

CsPbBr3 QD/AlOx Inorganic Nanocomposites with Exceptional Stability in Water,Light, and Heat

Herein, the assembly of CsPbBr₃ QD/AlO_x inorganic nanocomposites, by using atomic layer deposition (ALD) for the growth of the amorphous alumina matrix (AlO_x ), is described as a novel protection scheme for such QDs. The nucleation and growth of AlO_x on the QD surface was thoroughly investigated by miscellaneous techniques, which highlighted the importance of the interaction between the ALD precursors and the QD surface to uniformly coat the QDs while preserving the optoelectronic properties. These nanocomposites show exceptional stability towards exposure to air (for at least 45 days), irradiation under simulated solar spectrum conditions (for at least 8 h), and heat (up to 200 °C in air), and finally upon immersion in water. This method was extended to the assembly of CsPbBr_x I_3−x QD/AlO_x and CsPbI₃ QD/AlO_x nanocomposites, which were more stable than the pristine QD films.     

Optical and dynamic properties of water-soluble highly luminescent CdTe quantum dots

CdTe quantum dots (QDs) were synthesized in aqueous solution using thioglycolic acid (HS-CH2COOH, TGA) as a stabilizer. The phenomenon of "on" and "off" luminescence intermittency (blinking) of CdTe QDs in PVA and trehalose was investigated by single-molecule optical microscopy, and we identified that the intermittencies of single QDs were correlated with the interaction of water molecules absorbed on the QD surface. The "off" times, the interval between adjacent "on" states, remained essentially unaffected with an increase in excitation intensity. Every QD showed a similar power law behavior for the "off" time distribution regardless of the excitation intensity and aqueous environment of the QDs. In the case of "on" time distribution, power law behavior with an exponential cutoff tail is observed at longer time scales. The time traces indicated that the "on" time was inversely proportional to the excitation intensity; the duration of "on" time became shorter with increasing excitation intensity. An increase in the duration of "on" time was observed in trehalose with respect to that in PVA. We obtained a clear decrease in the power law exponent when PVA was replaced with trehalose. These observations indicate that the luminescence blinking statistics of water-soluble single CdTe QDs is significantly dependent on the aqueous environment, which is interpreted in terms of passivation of the surface trap states of QDs.     

Role of Emissive and Non‐Emissive Complex Formations in Photoinduced Electron Transfer Reaction of CdTe Quantum Dots

Bimolecular photoinduced electron transfer (PET) from excited state CdTe quantum dot (QD*) to an electron deficient molecule 2,4‐dinitrotoluene (DNT) is studied in toluene. We observed two types of QD‐DNT complex formations; (i) non‐emissive complex, in which DNT is embedded deep inside the surface polymer layer of QD and (ii) emissive complex, in which DNT molecules are attached to QDs but approach to the QD core is shielded by polymer layer. Because of its non‐emissive nature, the lifetime of QD is not affected by dark complex formation, though the steady‐state emission is greatly quenched. However, emissive complex formation causes both, lifetime and steady‐state emission quenching. In our fitting model, consideration of Poisson distribution of the attached quencher (DNT) molecules at QD surface enables a comprehensive fitting to our time resolved data. QD‐DNT complex formation was confirmed by an isothermal titration calorimetry (ITC) study. Fitting to the time resolved data using a stochastic kinetic model shows moderate increase (0.05 ns^?1 to 0.072 ns^?1) of intrinsic quenching rate with increasing the QD particle size (from ≈3.2 nm to ≈5.2 nm). Our fitting also reveals that the number of DNT molecules attached to a single QD increases from ≈0.1–0.2 to ≈1.2–1.7, as the DNT concentration is increased from ≈1 mm to 17.5 mm . Complex formation at higher quencher concentration assures that the observed PET kinetics is a thermodynamically controlled process where solvent diffusion has no role on it.     

Assembly kinetics of nanocrystals via peptide hybridization

The assembly kinetics of colloidal semiconductor quantum dots (QDs) on solid inorganic surfaces is of fundamental importance for implementation of their solid-state devices. Herein an inorganic binding peptide, silica binding QBP1, was utilized for the self-assembly of nanocrystal quantum dots on silica surface as a smart molecular linker. The QD binding kinetics was studied comparatively in three different cases: first, QD adsorption with no functionalization of substrate or QD surface; second, QD adsorption on QBP1-modified surface; and, finally, adsorption of QBP1-functionalized QD on silica surface. The surface modification of QDs with QBP1 enabled 79.3-fold enhancement in QD binding affinity, while modification of a silica surface with QBP1 led to only 3.3-fold enhancement. The fluorescence microscopy images also supported a coherent assembly with correspondingly increased binding affinity. Decoration of QDs with inorganic peptides was shown to increase the amount of surface-bound QDs dramatically compared to the conventional methods. These results offer new opportunities for the assembly of QDs on solid surfaces for future device applications.     

l-Cysteine-induced photoluminescence enhancement of CdSe/ZnSe quantum dots in aqueous solution

l-Cysteine molecules dramatically enhance the photoluminescence of colloidal CdSe/ZnSe quantum dots (i.e., ^CTAB/TOPOQD). Based on our spectroscopic studies of temporal variations in QD quantum yields as well as the in situ infrared spectral features of QDs, we propose that adsorption and rearrangement of l-cysteine molecules at the QD–water interface induces the observed unusual enhancement of the photoluminescence quantum yield. Upon addition of l-cysteine to the ^CTAB/TOPOQD solution, the adsorption of l-cysteine to the ^CTAB/TOPOQD colloidal particles is driven by the formation of a kinetically favorable intermediate species, which is formed by the coordination of thiol groups to the QD surface Cd atoms. The above species then reacts further to form a thermodynamically stable QD species, which probably involves coordination of both the amine and thiol groups of l-cysteine on the QD surface. Additional comparison studies using ^MPAQD and other small ligands (i.e., l-alanine, l-serine, and MPA) confirmed our proposed mechanism of l-cysteine adsorption at the ^CTAB/TOPOQD–water interfaces. In addition to these adsorption structures, we also propose that the dramatic enhancement of QY observed in this study is probably induced by the rearrangement and structural organization of l-cysteine and CTAB molecules at the QD–water interface, which improves the homogeneity and self-organization of the interfacial molecules.     

Single domain antibody–quantum dot conjugates for ricin detection by both fluoroimmunoassay and surface plasmon resonance

The combination of stable biorecognition elements and robust quantum dots (QDs) has the potential to yield highly effective reporters for bioanalyses. Llama-derived single domain antibodies (sdAb) provide small thermostable recognition elements that can be easily manipulated using standard DNA methods. The sdAb was self-assembled on dihydrolipoic acid (DHLA) ligand-capped CdSe–ZnS core–shell QDs made in our laboratory through the polyhistidine tail of the protein, which coordinated to zinc ions on the QD surface. The sdAb–QD bioconjugates were then applied in both fluorometric and surface plasmon resonance (SPR) immunoassays for the detection of ricin, a potential biothreat agent. The sdAb–QD conjugates functioned in fluoroimmunoassays for the detection of ricin, providing equivalent limits of detection when compared to the same anti-ricin sdAb labeled with a conventional fluorophore. In addition, the DHLA-QD–sdAb conjugates were very effective reporter elements in SPR sandwich assays, providing more sensitive detection with a signal enhancement of ∼10-fold over sdAb reporters and 2–4 fold over full sized antibody reporters. Commercially prepared streptavidin-modified polymer-coated QDs also amplified the SPR signal for the detection of ricin when applied to locations where biotinylated anti-ricin sdAb was bound to target; however, we observed a 4-fold greater amplification when using the DHLA-QD–sdAb conjugates in this format.     

High-intensity fluorescence imaging and sensitive electrochemical detection of cancer cells by using an extracellular supramolecular reticular DNA-quantum dot sheath

Acting as a cage-type cellular probe, an extracellular supramolecular reticular DNA-quantum dot (QD) sheath has been developed for high-intensity fluorescence microscopy imaging and the sensitive electrochemical detection of Ramos cells. The extracellular supramolecular reticular DNA-QD sheath is constructed from layer-by-layer self-assembly of DNA-CdTe QD probes and DNA nanowire frameworks functionalized with a Ramos cell-binding aptamer. The DNA-QD sheath forms specifically and quickly on the surface of Ramos cells at physiological temperature, and the assembly of large numbers of DNA-CdTe QD probes on the surface of Ramos cells produces exceedingly high fluorescence intensity. Using the extracellular supramolecular reticular DNA-QD sheath as the cellular probe, Ramos cells can be clearly observed and easily distinguished from a mixture of multiple cancer cells by fluorescence microscopy imaging. Using the new cage-type cellular probe, a sensitive sandwich-type electrochemical strategy has also been developed to achieve accurate quantitative analysis of Ramos cells. Under the optimized conditions, Ramos cells can be detected quantitatively in a range from 10 to 1000 cells with a detection limit of 10 cells. This strategy presents a promising platform for highly sensitive and convenient evaluation of cancer cell levels.