DNA Nanoflowers for Multiplexed Cellular Imaging and Traceable Targeted Drug Delivery

We present a facile approach to make aptamer‐conjugated FRET (fluorescent resonance energy transfer) nanoflowers (NFs) through rolling circle replication for multiplexed cellular imaging and traceable targeted drug delivery. The NFs can exhibit multi‐fluorescence emissions by a single‐wavelength excitation as a result of the DNA matrix covalently incorporated with three dye molecules able to perform FRET. Compared with the conventional DNA nanostructure assembly, NF assembly is independent of template sequences, avoiding the otherwise complicated design of DNA building blocks assembled into nanostructures by base‐pairing. The NFs were uniform and exhibited high fluorescence intensity and excellent photostability. Combined with the ability of traceable targeted drug delivery, these colorful DNA NFs provide a novel system for applications in multiplex fluorescent cellular imaging, effective screening of drugs, and therapeutic protocol development.     

A Brief Overview of Some Physical Studies on the Relaxation Dynamics and Förster Resonance Energy Transfer of Semiconductor Quantum Dots

This article highlights some physical studies on the relaxation dynamics and Förster resonance energy transfer (FRET) of semiconductor quantum dots (QDs) and the way these phenomena change with size, shape, and composition of the QDs. The understanding of the excited‐state dynamics of photoexcited QDs is essential for technological applications such as efficient solar energy conversion, light‐emitting diodes, and photovoltaic cells. Here, our emphasis is directed at describing the influence of size, shape, and composition of the QDs on their different relaxation processes, that is, radiative relaxation rate, nonradiative relaxation rate, and number of trap states. A stochastic model of carrier relaxation dynamics in semiconductor QDs was proposed to correlate with the experimental results. Many recent studies reveal that the energy transfer between the QDs and a dye is a FRET process, as established from 1/d⁶ distance dependence. QD‐based energy‐transfer processes have been used in applications such as luminescence tagging, imaging, sensors, and light harvesting. Thus, the understanding of the interaction between the excited state of the QD and the dye molecule and quantitative estimation of the number of dye molecules attached to the surface of the QD by using a kinetic model is important. Here, we highlight the influence of size, shape, and composition of QDs on the kinetics of energy transfer. Interesting findings reveal that QD‐based energy‐transfer processes offer exciting opportunities for future applications. Finally, a tentative outlook on future developments in this research field is given.     

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.     

Single‐Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots

Fluorescence barcoding based on nanoparticles provides many advantages for multiparameter imaging. However, creating different concentration‐independent codes without mixing various nanoparticles and by using single‐wavelength excitation and emission for multiplexed cellular imaging is extremely challenging. Herein, we report the development of quantum dots (QDs) with two different SiO₂ shell thicknesses (6 and 12 nm) that are coated with two different lanthanide complexes (Tb and Eu). FRET from the Tb or Eu donors to the QD acceptors resulted in four distinct photoluminescence (PL) decays, which were encoded by simple time‐gated (TG) PL intensity detection in three individual temporal detection windows. The well‐defined single‐nanoparticle codes were used for live cell imaging and a one‐measurement distinction of four different cells in a single field of view. This single‐color barcoding strategy opens new opportunities for multiplexed labeling and tracking of cells.     

Ion‐Selective Formation of a Guanine Quadruplex on DNA Origami Structures

DNA origami nanostructures are a versatile tool that can be used to arrange functionalities with high local control to study molecular processes at a single‐molecule level. Here, we demonstrate that DNA origami substrates can be used to suppress the formation of specific guanine (G) quadruplex structures from telomeric DNA. The folding of telomeres into G‐quadruplex structures in the presence of monovalent cations (e.g. Na⁺ and K⁺) is currently used for the detection of K⁺ ions, however, with insufficient selectivity towards Na⁺. By means of FRET between two suitable dyes attached to the 3′‐ and 5′‐ends of telomeric DNA we demonstrate that the formation of G‐quadruplexes on DNA origami templates in the presence of sodium ions is suppressed due to steric hindrance. Hence, telomeric DNA attached to DNA origami structures represents a highly sensitive and selective detection tool for potassium ions even in the presence of high concentrations of sodium ions.     

High‐Performance Förster Resonance Energy Transfer (FRET)‐Based Dye‐Sensitized Solar Cells: Rational Design of Quantum Dots for Wide Solar‐Spectrum Utilization

High‐performance Förster resonance energy transfer (FRET)‐based dye‐sensitized solar cells (DSSCs) have been successfully fabricated through the optimized design of a CdSe/CdS quantum‐dot (QD) donor and a dye acceptor. This simple approach enables quantum dots and dyes to simultaneously utilize the wide solar spectrum, thereby resulting in high conversion efficiency over a wide wavelength range. In addition, major parameters that affect the FRET interaction between donor and acceptor have been investigated including the fluorescent emission spectrum of QD, and the content of deposited QDs into the TiO₂ matrix. By judicious control of these parameters, the FRET interaction can be readily optimized for high photovoltaic performance. In addition, the as‐synthesized water‐soluble quantum dots were highly dispersed in a nanoporous TiO₂ matrix, thereby resulting in excellent contact between donors and acceptors. Importantly, high‐performance FRET‐based DSSCs can be prepared without any infrared (IR) dye synthetic procedures. This novel strategy offers great potential for applications of dye‐sensitized solar cells.     

Light‐Harvesting Nanoparticle Probes for FRET‐Based Detection of Oligonucleotides with Single‐Molecule Sensitivity

Controlling the emission of bright luminescent nanoparticles by a single molecular recognition event remains a challenge in the design of ultrasensitive probes for biomolecules. Herein, we developed 20‐nm light‐harvesting nanoantenna particles, built of a tailor‐made hydrophobic charged polymer poly(ethyl methacrylate‐co‐methacrylic acid), encapsulating circa 1000 strongly coupled and highly emissive rhodamine dyes with their bulky counterion. Being 87‐fold brighter than quantum dots QDots 605 in single‐particle microscopy (with 550‐nm excitation), these DNA‐functionalized nanoparticles exhibit over 50 % total FRET efficiency to a single hybridized FRET acceptor, a highly photostable dye (ATTO665), leading to circa 250‐fold signal amplification. The obtained FRET nanoprobes enable single‐molecule detection of short DNA and RNA sequences, encoding a cancer marker (survivin), and imaging single hybridization events by an epi‐fluorescence microscope with ultralow excitation irradiance close to that of ambient sunlight.     

Graphdiyne:Structure of Fluorescent Quantum Dots

Graphdiyne (GDY) as an emerging two‐dimensional carbon allotrope exhibits excellent performance in energy chemistry, catalytic chemistry, optoelectronics, electronics, etc. because of the unique structure combining an sp‐ and sp²‐hybrid carbon network. However, the poor solubility of pristine GDY is a major obstacle to its applications in many fields. Proposed here is a facile strategy to control the preparation of GDY quantum dots (GDY‐Py QDs), in which pyrene groups are covalently linked to GDY by using a Sonogashira cross‐coupling reaction. The as‐prepared GDY‐Py QDs, with an average diameter of about 3±0.1 nm, show superior dispersibility in many organic solvents and water. The GDY‐Py QDs display not only bright fluorescent with a high relative quantum yield (QY) of 42.82 %, but they are also well‐behaved as contrast agents in cell imaging. The GDY‐Py QDs are bestowed with high stability and non‐cytotoxicity, and exhibit long fluorescent times, and have potential for optical imaging and biomedical applications.     

Gold nanoparticle-quantum dot-polystyrene microspheres as fluorescence resonance energy transfer probes for bioassays

The paper describes the development of highly sensitive particle-based fluorescence resonance energy transfer (FRET) probes that do not use molecular fluorophores as donors and acceptors. In these probes, CdSe/ZnS luminescent quantum dots (QDs) were capped with multiple histidine-containing peptides to increase their aqueous solubility while maintaining their high emission quantum yield and spectral properties. The peptide-modified QDs (QD-His) were covalently attached to carboxyl-modified polystyrene (PS) microspheres to form highly emitting PS microspheres (QD-PS). Gold nanoparticles (AuNPs) were then covalently attached to the QD-PS surface to form AuNP-QD-PS composite microspheres that were used as FRET probes. Attachment of AuNPs to QD-PS completely quenched the QD emission through FRET interactions. The emission of QD-PS was restored when the AuNPs were removed from the surface by thiol ligand displacement. The new AuNP-QD-PS FRET platform is simple to prepare and highly stable, and it opens many new possibilities for carrying out FRET assays on microparticle-based platforms and in microarrays. The versatility of these assays could be greatly increased by replacing the linkers between the QDs and AuNPs with ones that selectively respond to specific cleaving agents or enzymes.     

Detection of influenza A virus based on fluorescence resonance energy transfer from quantum dots to carbon nanotubes

In this paper, a simple and sensitive approach for H5N1 DNA detection was described based on the fluorescence resonance energy transfer (FRET) from quantum dots (QDs) to carbon nanotubes (CNTs) in a QDs-ssDNA/oxCNTs system, in which the QDs (CdTe) modified with ssDNA were used as donors. In the initial stage, with the strong interaction between ssDNA and oxCNTs, QDs fluorescence was effectively quenched. Upon the recognition of the target, the effective competitive bindings of it to QDs-ssDNA occurred, which decreased the interactions between the QDs-ssDNA and oxCNTs, leading to the recovery of the QDs fluorescence. The recovered fluorescence of QDs was linearly proportional to the concentration of the target in the range of 0.01–20 μM with a detection limit of 9.39 nM. Moreover, even a single-base mismatched target with the same concentration of target DNA can only recover a limited low fluorescence of QDs, illustrating the good anti-interference performance of this QDs-ssDNA/oxCNTs system. This FRET platform in the QDs-ssDNA/oxCNTs system was facilitated to the simple, sensitive and quantitative detection of virus nucleic acids and could have a wide range of applications in molecular diagnosis.     

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.     

Transfer of Two‐Dimensional Oligonucleotide Patterns onto Stereocontrolled Plasmonic Nanostructures through DNA‐Origami‐Based Nanoimprinting Lithography

The precise functionalization of self‐assembled nanostructures with spatial and stereocontrol is a major objective of nanotechnology and holds great promise for many applications. Herein, the nanoscale addressability of DNA origami was exploited to develop a precise copy‐machine‐like platform that can transfer two‐dimensional oligonucleotide patterns onto the surface of gold nanoparticles (AuNPs) through a deliberately designed toehold‐initiated DNA displacement reaction. This strategy of DNA‐origami‐based nanoimprinting lithography (DONIL) demonstrates high precision in controlling the valence and valence angles of AuNPs. These DNA‐decorated AuNPs act as precursors in the construction of discrete AuNP clusters with desired chirality.     

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein–protein interaction and even protein modifications in living cells. Here, we analyze the E⁰GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro . The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R ₀ = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E⁰GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo . Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo ) reveals that this new pair can be used for very effective quantitative FRET imaging.     

Precisely Tailored DNA Nanostructures and their Theranostic Applications

A critical challenge in nanotechnology is the limited precision and controllability of the structural parameters, which brings about concerns in uniformity, reproducibility and performance. Self‐assembled DNA nanostructures, as a newly emerged type of nano‐biomaterials, possess low‐nanometer precision, excellent programmability and addressability. They can precisely arrange various molecules and materials to form spatially ordered complex, resulting in unambiguous physical or chemical properties. Because of these, DNA nanostructures have shown great promise in numerous biomedical theranostic applications. In this account, we briefly review the history and advances on construction of DNA nanoarchitectures and superstructures with accurate structural parameters. We focus on recent progress in exploiting these DNA nanostructures as platforms for quantitative biosensing, intracellular diagnosis, imaging, and smart drug delivery. We also discuss key challenges in practical applications.     

Reversible Reconfiguration of DNA Origami Nanochambers Monitored by Single‐Molecule FRET

Today, DNA nanotechnology is one of the methods of choice to achieve spatiotemporal control of matter at the nanoscale. By combining the peculiar spatial addressability of DNA origami structures with the switchable mechanical movement of small DNA motifs, we constructed reconfigurable DNA nanochambers as dynamic compartmentalization systems. The reversible extension and contraction of the inner cavity of the structures was used to control the distance‐dependent energy transfer between two preloaded fluorophores. Interestingly, single‐molecule FRET studies revealed that the kinetics of the process are strongly affected by the choice of the switchable motifs and/or actuator sequences, thus offering a valid method for fine‐tuning the dynamic properties of large DNA nanostructures. We envisage that the proposed DNA nanochambers may function as model structures for artificial biomimetic compartments and transport systems.     

Gold nanoparticle-based homogeneous fluorescent aptasensor for multiplex detection

We developed a homogeneous fluorescence assay for multiplex detection based on the target induced conformational change of DNA aptamers. DNA aptamers were immobilized on quantum dots (QDs), and QDs conjugated ssDNA was adsorbed on the surface of gold nanoparticles (AuNPs) by electrostatic interaction between uncoiled ssDNA and the AuNPs. Subsequently the fluorescence of QDs was effectively quenched by the AuNPs due to fluorescence resonance energy transfer (FRET) of QDs to AuNPs. In the presence of targets, the QDs conjugated aptamers were detached from AuNPs by target induced conformational change of aptamers, consequently the fluorescence of the QDs was recovered proportional to the target concentration. In this study, three different QD/aptamer conjugates were used for multiplex detection of mercury ions, adenosine and potassium ions. In a control experiment, all of the three targets were simultaneously detected with high selectivity.     

Photophysical Properties of Au‐CdTe Hybrid Nanostructures of Varying Sizes and Shapes

We design well‐defined metal‐semiconductor nanostructures using thiol‐functionalized CdTe quantum dots (QDs)/quantum rods (QRs) with bovine serum albumin (BSA) protein‐conjugated Au nanoparticles (NPs)/nanorods (NRs) in aqueous solution. The main focus of this article is to address the impacts of size and shape on the photophysical properties, including radiative and nonradiative decay processes and energy transfers, of Au‐CdTe hybrid nanostructures. The red shifting of the plasmonic band and the strong photoluminescence (PL) quenching reveal a strong interaction between plasmons and excitons in these Au‐CdTe hybrid nanostructures. The PL quenching of CdTe QDs varies from 40 to 86 % by changing the size and shape of the Au NPs. The radiative as well as the nonradiative decay rates of the CdTe QDs/QRs are found to be affected in the presence of both Au NPs and NRs. A significant change in the nonradiative decay rate from 4.72×10⁶ to 3.92×10¹⁰ s^?1 is obtained for Au NR‐conjugated CdTe QDs. It is seen that the sizes and shapes of the Au NPs have a pronounced effect on the distance‐dependent energy transfer. Such metal‐semiconductor hybrid nanostructures should have great potentials for nonlinear optical properties, photovoltaic devices, and chemical sensors.     

Preparation of liposomes loaded with quantum dots, fluorescence resonance energy transfer studies, and near-infrared in-vivo imaging of mouse tissue

We report on a simple, fast and convenient method to engineer lipid vesicles loaded with quantum dots (QDs) by incorporating QDs into a vesicle-type of lipid bilayer using a phase transfer reagent. Hydrophilic CdTe QDs and near-infrared (NIR) QDs of type CdHgTe were incorporated into liposomes by transferring the QDs from an aqueous solution into chloroform by addition of a surfactant. The QD-loaded liposomes display bright fluorescence, and the incorporation of the QDs into the lipid bilayer leads to enhanced storage stability and reduced sensitivity to UV irradiation. The liposomes containing the QD were applied to label living cells and to image mouse tissue in-vivo using a confocal laser scanning microscope, while NIR images of mouse tissue were acquired with an NIR fluorescence imaging system. We also report on the fluorescence resonance energy transfer (FRET) that occurs between the CdTe QDs (the donor) and the CdHgTe QDs (the acceptor), both contained in liposomes. Based on these data, this NIR FRET system shows promise as a tool that may be used to study the release of drug-loaded liposomes and their in vivo distribution.

Use of quantum dots in the development of assays for cancer biomarkers

Biomarker assays may be useful for screening and diagnosis of cancer if a set of molecular markers can be quantified and statistically differentiated between cancerous cells and healthy cells. Markers of disease are often present at very low concentrations, so methods capable of low detection limits are required. Quantum dots (QDs) are nanoparticles that are emerging as promising probes for ultrasensitive detection of cancer biomarkers. QDs attached to antibodies, aptamers, oligonucleotides, or peptides can be used to target cancer markers. Their fluorescent properties have enabled QDs to be used as labels for in-vitro assays to quantify biomarkers, and they have been investigated as in-vivo imaging agents. QDs can be used as donors in assays involving fluorescence resonance energy transfer (FRET), or as acceptors in bioluminescence resonance energy transfer (BRET). The nanoparticles are also capable of electrochemical detection and are potentially useful for “lab-on-a-chip” applications. Recent developments in silicon QDs, non-blinking QDs, and QDs with reduced-size and controlled-valence further make these QDs bioanalytically attractive because of their low toxicity, biocompatibility, high quantum yields, and diverse surface modification flexibility. The potential of multiplexed sensing using QDs with different wavelengths of emission is promising for simultaneous detection of multiple biomarkers of disease.

Detection of FRET efficiency in imaging systems by photo-bleaching acceptors

Fluorescence resonance energy transfer (FRET) is widely used to obtain the distance between a donor and an acceptor in biological research. However, the detection of FRET efficiencies with fluorescence microscopy imaging systems remains a great challenge due to the difficulties of transferring gray scales of the images into fluorescence intensities, and the absence of exact quantum yields of donors and acceptors. Herein, we presented a new method to detect the FRET efficiency in imaging systems by analyzing the photo-bleaching-induced changes in fluorescent intensities of quantum dots (QDs, donors) and Cy5 dyes (acceptors). Our method is different from the previous acceptor-photo-bleaching studies in imaging systems by theoretically analyzing the bleaching process, and bringing forward a new parameter which is universal for samples of the same kind. It is convenient for calculating FRET efficiencies. There is hardly any spectral crosstalk between 605QD and Cy5, thus the FRET result is more accurate than that of many other common FRET pairs. The lengths of single-stranded and double-stranded DNA fragments in solution were determined via the analysis of FRET efficiency values. This technique provides a reliable approach to study biomacromolecules in living cells through fluorescent imaging and in situ measurements.