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 Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells

Herein, a Janus three-dimensional (3D) DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual microRNAs (miRNAs) in cancer cells, which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells. In this system, the Janus nanoparticles were synthesized as the carrier for immobilizing two different oligonucleotides on two different functionalized hemispheres of the nanoparticles to form a Janus 3D DNA nanostructure, which could convert trace amounts of miRNA-21 and miRNA-155 targets into massive FAM and Cy5-labeled duplexes to induce two remarkable fluorescence emissions by the catalytic hairpin assembly (CHA) and 3D DNA walker cascade nucleic acid amplification strategy, realizing sensitive detection and imaging of miRNA targets in cancer cells. Impressively, in comparison with current miRNA imaging methods based on nanoparticle assemblies, the proposed strategy could efficiently eliminate “false positive” results obtained in single type miRNA detection and distinctly increase the immobilization concentration of two different signal probes using Janus nanoparticles as the carrier to further enhance fluorescence intensity, resulting in accurate co-location in the same position of living cells. Meanwhile, the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers, which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization (FISH) approach. Therefore, it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles, resulting in the further validation of their function in tumor proliferation and differentiation. This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.

A Janus three-dimensional DNA nanomachine was constructed for the simultaneous and sensitive fluorescent detection and imaging of dual microRNAs in the cancer cells.     

Rational Engineering of a Dynamic,Entropy‐Driven DNA Nanomachine for Intracellular MicroRNA Imaging

We rationally engineered an elegant entropy‐driven DNA nanomachine with three‐dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy‐driven catalytic reaction of intramolecular toehold‐mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye‐labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction‐based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity‐induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.     

Entropy-driven reactions in living cells for assay let-7a microRNA

Imaging of microRNA (miRNA) in living cells could facilitate the monitoring of the expression and distribution of miRNA and research on miRNA-related diseases. Given the low expression levels and even down-regulation of cellular miRNA that is associated with some diseases, enzyme-free amplification strategies are imperative for intracellular miRNA assay. In this work, we report an entropy-driven reaction for amplification assay miRNA with a detection limit of 0.27 pM. The resulting signal amplification provides excellent recognition and signal enhancement of specific miRNAs in living cells. This method supplies accurate information regarding cellular miRNA-related biological events and provides a new tool for highly sensitive and simultaneous imaging of multiple low-level biomarkers, thereby improving the accuracy of early disease diagnosis.     

基于双色荧光传感器的癌细胞成像及microRNA定量检测

癌细胞中microRNA(miRNA)的灵敏成像对于疾病的诊断治疗具有重要意义,其中miRNA-21通常在多种癌细胞中异常表达.本文将DNA功能化的金纳米颗粒与发射波长分离的荧光染料FAM和Cy5. 5修饰的DNA通过含有光控基团PC-linker的DNA4作为桥梁进行自组装,构建了纳米传感器GDC.将302 nm紫外光作为启动开关,用其照射该体系时,Cy5. 5修饰的DNA3被释放,其荧光强度可作为内参比信号,用于标定进入细胞的组装体含量;细胞中miRNA-21作为催化分子,与外加燃料Fuel DNA共同作用下可实现催化放大,FAM修饰的DNA2被释放且被猝灭的荧光信号得以恢复,并作为检测信号.通过2种荧光信号强度(FL)的检测及FL_FAM/FL_{Cy5. 5}比值的计算,达到定量分析细胞中miRNA含量的目的.该体系可扣除因细胞内组装体含量不同造成的背景信号误差,不仅能显著提高检测准确度,还因存在催化循环而大大降低了检出限,比传统方法至少降低了3个数量级.该传感器的检出限为23. 1 pmol/L,通过定量计算得出He La细胞中miRNA的...     

Time-dependent photoluminescence blue shift of the quantum dots in living cells: effect of oxidation by singlet oxygen

Time-dependent photoluminescence (PL) enhancement, blue shift, and photobleach were observed from the thiol-capped CdTe quantum dots (QDs) ingested in mouse myoblast cells and human primary liver cancer cells. It was revealed that the PL blue shift resulted from the photooxidation of the QD core by singlet oxygen molecules formed on the QD core surface.     

Tuning Optical Properties of Si Quantum Dots by π‐Conjugated Capping Molecules

The absorption and photoluminescence (PL) properties of silicon quantum dots (QDs) are greatly influenced by their size and surface chemistry. Herein, we examined the optical properties of three Si QDs with increasing σ–π conjugation length: octyl‐, (trimethylsilyl)vinyl‐, and 2‐phenylvinyl‐capped Si QDs. The PL photon energy obtained from as‐prepared samples decreased by 0.1–0.3 eV, while the PL excitation (PLE) extended from 360 nm (octyl‐capped Si QDs) to 400 nm (2‐phenylvinyl‐capped Si QDs). A vibrational PL feature was observed in all samples with an energy separation of about 0.192±0.013 eV, which was explained based on electron–phonon coupling. After soft oxidization through drying, all samples showed blue PL with maxima at approximately 410 nm. A similar high‐energy peak was observed with the bare Si QD sample. The changes in the optical properties of Si QDs were mainly explained by the formation of additional states arising from the strong σ–π conjugation and QD oxidation.     

A microfluidic surface-enhanced Raman scattering (SERS) sensor for microRNA in extracellular vesicles with nucleic acid-tyramine cascade amplification

Exosomal microRNA (miRNA) is an ideal candidate of noninvasive biomarker for the early diagnosis of cancer. Sensitive and accurate sensing of abnormal exosomal miRNA plays essential role for clinical promotion due to its close correlation with tumor proliferation and progression. Herein, a microfluidic surface-enhanced Raman scattering (SERS) sensor was proposed for an on-line detection of exosomal miRNA based on rolling circle amplification (RCA) and tyramine signal amplification (TSA) strategy. The microfluidic chip consists of a magnetic enrichment chamber, a serpentine fluidic mixer and a plasmonic SERS substrate functionalized with capture probes. The released miRNA activates the capture probe, triggers RCA reaction, and generates a large number of single-stranded DNA products to drive the catalysis of nanotags deposition via TSA, producing numerous “hot spots” to enhance the SERS signals. In merit of the microfluidics chip and nucleic acid-tyramine cascade amplification, the developed SERS sensor significantly improves the sensitivity for the exosomal miRNA assay, resulting in a limit of detection (LOD) as low as 1 pmol/L and can be successfully applied in the analysis of exosomes secreted from breast tumor cells, which demonstrates the potential utility in practical applications.     

Imaging pancreatic cancer using surface-functionalized quantum dots

In this study, CdSe/CdS/ZnS quantum dots (QDs) were used as optical contrast agent for imaging pancreatic cancer cells in vitro using transferrin and anti-Claudin-4 as targeting ligands. CdSe/CdS/ZnS was chosen because the CdSe/CdS/ZnS QDs have better photoluminescence (PL) efficiency and stability than those of CdSe/ZnS. The transferrin-mediated targeting is demonstrated in both a cell-free coprecipitation assay as well as using in vitro confocal microscopy. Pancreatic cancer specific uptake is also demonstrated using the monoclonal antibody anti-Claudin-4. This targeted QD platform will be further modified for the purpose of developing as an early detection imaging tool for pancreatic cancer.     

A NIR Programmable In Vivo miRNA Magnifier for NIR-II Imaging of Early Stage Cancer

Aberrant expressions of biomolecules occur much earlier than tumor visualized size and morphology change, but their common measurement strategies such as biopsy suffer from invasive sampling process. In vivo imaging of slight biomolecule expression difference is urgently needed for early cancer detection. Fluorescence of rare earth nanoparticles (RENPs) in second near-infrared (NIR-II) region makes them appropriate tool for in vivo imaging. However, the incapacity to couple with signal amplification strategies, especially programmable signal amplification strategies, limited their application in lowly expressed biomarkers imaging. Here we develop a 980/808 nm NIR programmed in vivo microRNAs (miRNAs) magnifier by conjugating activatable DNAzyme walker set to RENPs, which achieves more effective NIR-II imaging of early stage tumor than size monitoring imaging technique. Dye FD1080 (FD1080) modified substrate DNA quenches NIR-II downconversion emission of RENPs under 808 nm excitation. The miRNA recognition region in DNAzyme walker is sealed by a photo-cleavable strand to avoid “false positive” signal in systemic circulation. Upconversion emission of RENPs under 980 nm irradiation activates DNAzyme walker for miRNA recognition and amplifies NIR-II fluorescence recovery of RENPs via DNAzyme catalytic reaction to achieve in vivo miRNA imaging. This strategy demonstrates good application potential in the field of early cancer detection.     

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.     

Au:CdHgTe quantum dots for in vivo tumor-targeted multispectral fluorescence imaging

Near-infrared gold-doped CdHgTe quantum dots (QDs) with improved photoluminescence and biocompatibility were developed using an aqueous solution route with l-glutathione and l-cysteine as stabilizers. As-prepared Au:CdHgTe QDs were covalently linked to arginine–glycine–aspartic acid (RGD) peptide, anti-epidermal growth factor receptor (EGFR) monoclonal antibody (MAb), and anti- carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) MAb separately. Three Au:CdHgTe QD bioconjugates (QD800-RGD, QD820-anti-CEACAM1, and QD840-anti-EGFR) were successfully used as probes for in vivo tumor-targeted multispectral fluorescence imaging of xenografts. Fluorescence signals from the QD bioconjugates used to detect three tumor markers were spectrally unmixed, and their co-localization was analyzed. The results indicate that multiple tumor markers could be simultaneously detected by multispectral fluorescence imaging in vivo using QD bioconjugates as probes. This approach has excellent potential as an imaging method for the noninvasive exploration and detection of multiple tumor markers in vivo, thereby substantially aiding the diagnosis of cancer.     

Rational Engineering of a Dynamic,Entropy-Driven DNA Nanomachine for Intracellular MicroRNA Imaging

We rationally engineered an elegant entropy-driven DNA nanomachine with three-dimensional track and applied it for intracellular miRNAs imaging. The proposed nanomachine is activated by target miRNA binding to drive a walking leg tethered to gold nanoparticle with a high density of DNA substrates. The autonomous and progressive walk on the DNA track via the entropy-driven catalytic reaction of intramolecular toehold-mediated strand migration leads to continuous disassembly of DNA substrates, accompanied by the recovery of fluorescence signal due to the specific release of a dye-labeled substrate from DNA track. Our nanomachine outperforms the conventional intermolecular reaction-based gold nanoparticle design in the context of an improved sensitivity and kinetics, attributed to the enhanced local effective concentrations of working DNA components from the proximity-induced intramolecular reaction. Moreover, the nanomachine was applied for miRNA imaging inside living cells.     

Temperature-sensitive photoluminescence of CdSe quantum dot clusters

The formation of narrow size dispersed and nanometer size aggregates (clusters) of cadmium selenide (CdSe) quantum dots (QDs) and their temperature-sensitive photoluminescence (PL) spectral properties close to room temperature (298 K) are discussed. CdSe QDs formed stable clusters with an average diameter of approximately 27 nm in the absence of coordinating solvents. Using transmission electron microscopy (TEM) imaging, we identified the association of individual QDs with 2-5 nm diameters into clusters of uniform size. A suspension of these clusters in different solvents exhibited reversible PL intensity changes and PL spectral shifts which were correlated with temperature. Although the PL intensity of CdSe QDs encapsulated in host matrixes and the solid state showed a response to temperature under cryogenic conditions, the current work identified for the first time QD clusters showing temperature-sensitive PL intensity variations and spectral shifts at moderate temperatures above room temperature. Temperature-sensitive reversible PL changes of clusters are discussed with respect to reversible thermal trapping of electrons at inter-QD interfaces and dipole-dipole interactions in clusters. Reversible luminescence intensity variations and spectral shifts of QD clusters show the potential for developing sensors based on QD nanoscale assemblies.     

Forming highly fluorescent near-infrared emitting PbS quantum dots in water using glutathione as surface-modifying molecule

We present a new facile procedure for transferring oil-soluble oleic acid-capped NIR-emitting PbS quantum dots (QDs) into water, using hydrophilic thiol ligands as the surface-modifying agents of the primary capping molecules (oleic acid). The influence of exchange of the primary capping molecules with five different types of thiol molecules is investigated. The results show that highly fluorescent water-soluble PbS QDs are obtained using glutathione as a surface-modifying agent (photoluminescence quantum yield (PL QY), >30%); significantly less fluorescent water-soluble QDs were obtained using l-cysteine (PL QY, ~5%); with other three thiol molecules, PbS QDs lose almost completely their fluorescence in aqueous solution. This striking difference among the five thiol molecules may be attributed to the difference in the molecular structure. Next, we explored systematically the conditions of QD water solubilization, storage stability, photostability and cytotoxicity and tested further the resulting water-soluble PbS QDs for the imaging of living animals. The preliminary results from these studies illustrate that our synthesis procedure is very facile and that the as-prepared water-soluble PbS QDs are stable and low-cytotoxic and will be an important potential probe in the imaging of living animals due to free carboxyl and amino groups on the external surface of the QDs.     

Subsecond luminescence intensity fluctuations of single CdSe quantum dots

Photoluminescence (PL) intermittency characteristics are examined for single quantum dots (QDs) in a CdSe QD sample synthesized at a slow rate at 75 degrees C. Although the PL quantum efficiency was relatively low ( approximately 0.25), we noticed that the PL intensity of single CdSe QDs fluctuated on a subsecond time scale with short-lived "on" and "off" states. The subsecond PL intensity fluctuations of CdSe QDs are different from "on" and "off" PL blinking generally observed for QDs fluctuating on a millisecond to minute time scale. We characterized single QDs by identifying polarized excitations, topographic imaging using atomic force microscopy (AFM), and transmission electron microscopy (TEM). From analysis of the PL intensity trajectories from >100 single CdSe QDs, the average intermittency time was 213 ms. From the PL quantum efficiency, slow growth of QDs, intensity trajectory analyses, and previous reports relating surface trap states and PL properties of QDs, we attribute the subsecond PL intensity fluctuations of single CdSe QDs and short-lived "on" and "off" states to a high-density distribution of homogeneous surface trap states.     

Auger Ionization Beats Photo‐Oxidation of Semiconductor Quantum Dots: Extended Stability of Single‐Molecule Photoluminescence

Despite the bright and tuneable photoluminescence (PL) of semiconductor quantum dots (QDs), the PL instability induced by Auger recombination and oxidation poses a major challenge in single‐molecule applications of QDs. The incomplete information about Auger recombination and oxidation is an obstacle in the resolution of this challenge. Here, we report for the first time that Auger‐ionized QDs beat self‐sensitized oxidation and the non‐digitized PL intensity loss. Although high‐intensity photoactivation insistently induces PL blinking, the transient escape of QDs into the ultrafast Auger recombination cycle prevents generation of singlet oxygen (¹O₂) and preserves the PL intensity. By the detection of the NIR phosphorescence of ¹O₂ and evaluation of the photostability of single QDs in aerobic, anaerobic, and ¹O₂ scavenger‐enriched environments, we disclose relations of Auger ionization and ¹O₂‐mediated oxidation to the PL stability of single QDs, which will be useful during the formulation of QD‐based single‐molecule imaging tools and single‐photon devices.     

Facile synthesis of fluorescent quantum dot‐polymer nanocomposites via frontal polymerization

We report an available approach for quickly fabricating CdS QD‐polymer nanocomposites via frontal polymerization (FP). First, we synthesized (3‐mercaptopropyl)‐1‐trimethoxysilane (MPS)‐capped CdS quantum dots (QDs). With these MPS‐capped CdS QDs containing mercapto groups, MPS‐capped CdS QDs can be easily incorporated into a poly(N‐methylolacrylamide) (PNMA) matrix via FP. A variety of features for preparing QD‐polymer nanocomposites, such as initiator concentration and CdS concentration, were thoroughly investigated. The fluorescence properties of QD‐polymer nanocomposites prepared via FP are comparatively investigated on the basis of ultraviolet–visible (UV–vis) spectra and photoluminescence (PL) spectra. Results show that the PL intensity of QD‐polymer nanocomposites prepared via the FP method is superior to that obtained by the traditional batch polymerization (BP) method. In addition, by measuring the changes of PL intensity of the samples immersed in different concentrations of copper acetate solution, we found the QD‐polymer nanocomposites can be ultrasensitive to copper ions. This FP process can be exploited as a facile and rapid way for synthesis QD‐polymer nanocomposites on a large scale, avoiding the fluorescence quenching of nanocrystals during incorporation nanocrystals into polymer matrices. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2170–2177, 2010     

Target-triggered hairpin-free chain-branching growth of DNA dendrimers for contrast-enhanced imaging in living cells by avoiding signal dispersion

The development of amplification strategies is one of the central challenges for detection of lowabundance targets. One-to-many(1:M) amplification strategies in which one target lights many signal probes, has improved the detection sensitivity in bulk solution, but with discounted contrast in cell imaging, because the lighted probes are dissociative and dispersible. In this work, a one-to-large(1:L) signaling mechanism, in which the lighted probes were orderly connected to each other, was concep...     

The Influence of Surface Modification on the Photoluminescence of CdTe Quantum Dots: Realization of Bio‐Imaging via Cost‐Effective Polymer

To impart biocompatibility, stability, and specificity to quantum dots (QDs)—and to reduce their toxicity—it is essential to carry out surface modification. However, most surface‐modification processes are costly, complicated, and time‐consuming. In addition, the modified QDs often have a large size, which leads to easy aggregation in biological environments, making it difficult to excrete them from in vivo systems. To solve these problems, three kinds of conventional polymers, namely, polyvinyl alcohol (PVA, neutral), sodium polystyrene sulfonate (PSS, negative charged), and poly(diallyl dimethyl ammonium chloride) (PDDA, positive charged) were selected to modify the surface of QDs at low cost via a simple process in which the size of the QDs was kept small after modification. The effect of polymer modification on the photoluminescence (PL) properties of the QDs was systematically investigated. High quantum yields (QYs) of 65 % were reached, which is important for the realization of bio‐imaging. Then, the cytotoxicity of CdTe QD–polymer composites was systematically investigated via MTT assay using the Cal27 and HeLa cell lines, especially for high concentrations of QD–polymer composites in vitro. The experimental results showed that the cytotoxicity decreased in the order CdTe‐PDDA>CdTe>CdTe‐PSS>CdTe‐PVA, indicating that PSS and PVA can reduce the toxicity of the QDs. An obvious cytotoxicity of CdTe‐PVA and CdTe‐PSS was present until 120 h for the Cal27 cell line and until 168 h for the HeLa cell line. At last, the Cal27 cell line was selected to realize bio‐imaging using CdTe‐PSS and CdTe‐PVA composites with different emission colors under one excitation wavelength.