A compact functional quantum Dot-DNA conjugate: preparation, hybridization, and specific label-free DNA detection

In this letter, we report the preparation of a compact, functional quantum dot (QD)-DNA conjugate, where the capturing target DNA is directly and covalently coupled to the QD surface. This enables control of the separation distance between the QD donor and dye acceptor to within the range of the F?rster radius. Moreover, a tri(ethylene glycol) linker is introduced to the QD surface coating to effectively eliminate the strong, nonspecific adsorption of DNA on the QD surface. As a result, this QD-DNA conjugate hybridizes specifically to its complementary DNA with a hybridization rate constant comparable to that of free DNAs in solution. We show this system is capable of specific detection of nanomolar unlabeled complimentary DNA at low DNA probe/QD copy numbers via a "signal-on" fluorescence resonance energy transfer (FRET) response.     

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.     

DNA‐Programmed Dynamic Assembly of Quantum Dots for Molecular Computation

Despite the widespread use of quantum dots (QDs) for biosensing and bioimaging, QD‐based bio‐interfaceable and reconfigurable molecular computing systems have not yet been realized. DNA‐programmed dynamic assembly of multi‐color QDs is presented for the construction of a new class of fluorescence resonance energy transfer (FRET)‐based QD computing systems. A complete set of seven elementary logic gates (OR, AND, NOR, NAND, INH, XOR, XNOR) are realized using a series of binary and ternary QD complexes operated by strand displacement reactions. The integration of different logic gates into a half‐adder circuit for molecular computation is also demonstrated. This strategy is quite versatile and straightforward for logical operations and would pave the way for QD‐biocomputing‐based intelligent molecular diagnostics.     

Discrete nanostructures of quantum dots/Au with DNA

Nanostructures of colloidal CdSe/ZnS core/shell quantum dots (QDs) surrounded by a discrete number of Au nanoparticles were generated via DNA hybridization and purified by gel electrophoresis. Statistics from TEM analysis showed a high yield of designed structures. The distance between Au particles and QD, the number of Au around the central QD, and the size of Au and QD can be adjusted. Rationally designed structures such as these hold great promise for researching the physical interactions between semiconductor and Au nanoparticles and for developing more efficient nanoprobes.     

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.     

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.     

Self-assembled donor comprising quantum dots and fluorescent proteins for long-range fluorescence resonance energy transfer

We report on the development of a self-assembled donor for long-range fluorescence resonance energy transfer (FRET). To this end, a three-chromophore FRET (3Ch-FRET) system was constructed, which consists of a luminescent quantum dot (QD), enhanced yellow fluorescent proteins (EYFP), and Atto647-dye-modified oligonucleotides. The system was assembled by electrostatic binding of covalent EYFP-ssDNA conjugate to the QD and subsequent hybridization with complementary oligonucleotides labeled with Atto647-dye. The final conjugates comprise three different two-chromophore FRET (2Ch-FRET) subsystems, QD/EYFP, QD/Atto647, and EYFP/Atto647, respectively, which were studied in detail by steady-state and time-resolved photoluminescence measurements. The helicity of DNA allowed us to control donor/acceptor separations and thus enabled the detailed analysis of the various FRET processes. We found that the 2Ch-FRET and the 3Ch-FRET (QD/EYFP/Atto647) systems revealed FRET efficiencies and transfer rates that were affected by the availability of distinct FRET pathways. The derived energy-transfer efficiencies and F?rster radii indicated that within the 3Ch-FRET system, the 2Ch-FRET subsystem QD/EYFP showed highest FRET efficiencies ranging from 64 to 72%. Thus, it can be used as a powerful donor system that combines the intrinsic advantages of QDs (large and spectrally broad absorption cross section) and EYFP (high quantum yield) and enables long-distance FRET processes for donor-acceptor distances of up to 13 nm.     

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.     

A Fluorescent Polymeric Quantum Dot/Aptamer Superstructure and Its Application for Imaging of Cancer Cells

In this work, a novel polymeric quantum dot/aptamer superstructure with a highly intense fluorescence was fabricated by a molecular engineering strategy and successfully applied to fluorescence imaging of cancer cells. The polymeric superstructure, which is composed of both multiple cell‐based aptamers and a high ratio of quantum dot (QD)‐labeled DNA, exploits the target recognition capability of the aptamer, an enhanced cell internalization through multivalent effects, and cellular disruption by the polymeric conjugate. Importantly, the polymeric superstructure exhibits an increasingly enhanced fluorescence with recording time and is thus suitable for long‐term fluorescent cellular imaging. The unique and excellent fluorescence property of the QD superstructure paves the way for developing polymeric QD superstructures that hold promise for applications such as in vivo imaging.     

Fluorescence resonance energy transfer between a quantum dot donor and a dye acceptor attached to DNA

We show that direct coupling of a dye-labelled DNA (acceptor) to a quantum dot (QD) donor significantly reduces the donor-acceptor distance and improves the FRET efficiency: a highly efficient FRET (approximately 88%) at a low acceptor-to-donor ratio of 2 has been achieved at the single-molecule level.     

Catalytic Molecular Imaging of MicroRNA in Living Cells by DNA‐Programmed Nanoparticle Disassembly

Molecular imaging is an essential tool for disease diagnostics and treatment. Direct imaging of low‐abundance nucleic acids in living cells remains challenging because of the relatively low sensitivity and insufficient signal‐to‐background ratio of conventional molecular imaging probes. Herein, we report a class of DNA‐templated gold nanoparticle (GNP)–quantum dot (QD) assembly‐based probes for catalytic imaging of cancer‐related microRNAs (miRNA) in living cells with signal amplification capacity. We show that a single miRNA molecule could catalyze the disassembly of multiple QDs with the GNP through a DNA‐programmed thermodynamically driven entropy gain process, yielding significantly amplified QD photoluminescence (PL) for miRNA imaging. By combining the robust PL of QDs with the catalytic amplification strategy, three orders of magnitude improvement in detection sensitivity is achieved in comparison with non‐catalytic imaging probe, which enables facile and accurate differentiation between cancer cells and normal cells by miRNA imaging in living cells.     

A two-dimensional molecular beacon for mRNA-activated intelligent cancer theranostics

Ideal theranostics should possess directly correlated imaging and therapy modalities that could be simultaneously activated in the disease site to generate high imaging contrast and therapeutic efficacy with minimal side effects. However, so far it still remains challenging to engineer all these characteristics into a single theranostic probe. Herein, we report a new type of photosensitizer (PS)-derived “two-dimensional” molecular beacon (TMB) that could be specifically activated within tumor cells to exhibit both high imaging contrast and therapeutic efficacy that outperforms conventional photosensitizers for cancer theranostics. The TMB is constructed by integrating a photosensitizer (chlorin e6 (Ce6)), a quantum dot (QD), and a dark quencher (BHQ3) into a hairpin DNA molecule to generate multiple synergistic FRET modes. The imaging modality and therapy modality, which are mediated by FRET between the QD and BHQ3 and FRET between the QD and Ce6 respectively, are interconnected within the TMB and could be simultaneously activated by tumor mRNA molecules. We show that highly effective cancer imaging and therapy could be achieved for cancer cell lines and xenografted tumor models. The reported TMB represents an unprecedented theranostic platform for intelligent cancer theranostics.     

Controlled stoichiometric synthesis of DNA-quantum dot conjugates using Ni-mediated coordination chemistry

An oligonucleotide modified with Ni-nitrilotriacetate (NTA) was successfully synthesized and used for the stoichiometric functionalization of QDs. This synthetic approach allowed for the facile preparation of DNA-QD conjugates with a defined DNA/QD ratio using well-known Ni-histidine coordination chemistry. A FRET based DNA-QD nanoprobe was prepared using this method highlighting the great potential of this synthetic strategy.     

Self-assembled quantum dot-sensitized multivalent DNA photonic wires

Combining the inherent scaffolding provided by DNA structure with spatial control over fluorophore positioning allows the creation of DNA-based photonic wires with the capacity to transfer excitation energy over distances greater than 150 ?. We demonstrate hybrid multifluorophore DNA-photonic wires that both self-assemble around semiconductor quantum dots (QDs) and exploit their unique photophysical properties. In this architecture, the QDs function as both central nanoscaffolds and ultraviolet energy harvesting donors that drive Fo?rster resonance energy transfer (FRET) cascades through the DNA wires with emissions that approach the near-infrared. To assemble the wires, DNA fragments labeled with a series of increasingly red-shifted acceptor-dyes were hybridized in a predetermined linear arrangement to a complementary DNA template that was chemoselectively modified with a hexahistidine-appended peptide. The peptide portion facilitated metal-affinity coordination of multiple hybridized DNA-dye structures to a central QD completing the final nanocrystal-DNA photonic wire structure. We assembled several such hybrid structures where labeled-acceptor dyes were excited by the QDs and arranged to interact with each other via consecutive FRET processes. The inherently facile reconfiguration properties of this design allowed testing of alternate formats including the addition of an intercalating dye located in the template DNA or placement of multiple identical dye acceptors that engaged in homoFRET. Lastly, a photonic structure linking the central QD with multiple copies of DNA hybridized with 4-sequentially arranged acceptor dyes and demonstrating 4-consecutive energy transfer steps was examined. Step-by-step monitoring of energy transfer with both steady-state and time-resolved spectroscopy allowed efficiencies to be tracked through the structures and suggested that acceptor dye quantum yields are the predominant limiting factor. Integrating such DNA-based photonic structures with QDs can help create a new generation of biophotonic wire assemblies with widespread potential in nanotechnology.     

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.     

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.     

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.     

Single nanoparticle imaging and characterization of different phospholipid-encapsulated quantum dot micelles

Phospholipid quantum dot (QD) micelles have been extensively used as fluorescent tags in single nanoparticle imaging for biomedical imaging. In this work, the microscopic structures and photophysical properties of the phospholipid QD micelles were studied at the single nanoparticle level. Two commonly used types of phospholipid QD micelles were prepared and tested both on a solid-phase surface and in liquid phase, including 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-encapsulated QD micelles (DSPE-QDMs) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-encapsulated QD micelles (PEG-DSPE-QDMs). Their fluorescence intensities and diffusion trajectories were determined by a total internal reflection fluorescence-based single nanoparticle imaging platform and comparatively analyzed carefully. It was demonstrated that DSPE-QDMs possessed a comparably wider intensity distribution and lower diffusion coefficient than that of PEG-DSPE-QDMs. PEG-DSPE-QDMs exhibited an obvious fluorescent intermittence. The results suggested that for most of the DSPE-QDMs, more than one QD were encapsulated in a single micelle. On the other hand, only one QD was embedded in a single micelle of PEG-DSPE-QDMs for most of the cases. Such variances suggested that phospholipids play a key role in the fabrication of the QD micelles. This work provides a useful foundation for their further biomedical applications.     

TCNQ Interlayers for Colloidal Quantum Dot Light‐Emitting Diodes

CdSe/CdS/ZnS quantum dot light‐emitting diodes (QD‐LEDs) show increased brightness (from ca. 18 000 to 27 000 cd m^?2) with 7,7,8,8‐tetracyanoquinodimethane (TCNQ) between the QD and electron‐transfer layers of ZnO nanoparticles. As QD/ZnO layers are known to have interface defects, our finding leads to the importance of interface engineering for QD‐LEDs. Although the photoluminescent intensity and decay lifetime of ZnO/TCNQ/QD layers are similar to those of ZnO/QD layers, cyclic voltammetry suggests improved charge transfer of TCNQ/ZnO layers compared to that of pure ZnO layers. This helps us to understand the mechanism of electrically driven QD‐LED behavior, which differs from that of conventional solid‐state LEDs, and enables the rational design of QD‐based optoelectronic devices.     

Development and characterization of a magnetic bead-quantum dot nanoparticles based assay capable of Escherichia coli O157:H7 quantification

The development and characterization of a magnetic bead (MB)-quantum dot (QD) nanoparticles based assay capable of quantifying pathogenic bacteria is presented here. The MB-QD assay operates by having a capturing probe DNA selectively linked to the signaling probe DNA via the target genomic DNA (gDNA) during DNA hybridization. The signaling probe DNA is labeled with fluorescent QD₅₆₅ which serves as a reporter. The capturing probe DNA is conjugated simultaneously to a MB and another QD₆₅₅, which serve as a carrier and an internal standard, respectively. Successfully captured target gDNA is separated using a magnetic field and is quantified via a spectrofluorometer. The use of QDs (i.e., QD₅₆₅/QD₆₅₅) as both a fluorescence label and an internal standard increased the sensitivity of the assay. The passivation effect and the molar ratio between QD and DNA were optimized. The MB-QD assay demonstrated a detection limit of 890 zeptomolar (i.e., 10⁻²¹ mol L⁻¹) concentration for the linear single stranded DNA (ssDNA). It also demonstrated a detection limit of 87 gene copies for double stranded DNA (dsDNA) eaeA gene extracted from pure Escherichia coli (E. coli) O157:H7 culture. Its corresponding dynamic range, sensitivity, and selectivity were also presented. Finally, the bacterial gDNA of E. coli O157:H7 was used to highlight the MB-QD assay's ability to detect below the minimum infective dose (i.e., 100 organisms) of E. coli O157:H7 in water environment.