新型量子点基分子印迹荧光传感器在快速检测中的应用

作为一种新型荧光纳米材料,量子点具有十分优异的光学特性,是分析化学、生物科学、医学等领域研究的热点标记材料.分子印迹聚合物是能够进行特异性识别和选择性吸附的"仿生"材料,它易于制备且具有较好的重现性和稳定性,因而分子印迹技术已成为具有广阔应用前景的识别技术.量子点基分子印迹荧光传感器结合了量子点和分子印迹技术的优势,由...     

Nanoparticles as scaffolds for FRET-based ratiometric detection of mercury ions in water with QDs as donors

The quenching of quantum dots' emission by some analytes (Hg(2+), Pb(2+), etc.) has long been hindering the fabrication of QD-based 'turn-on' or ratiometric fluorescent sensors for these analytes. In this study, we demonstrate a facile solution for constructing a robust FRET-based ratiometric sensor for Hg(2+) detection in water with CdTe QDs as the donor. By using the reverse microemulsion approach, CdTe QDs were first embedded into nanosized silica particles, forming the QDs/silica cores, a positively charged ultrathin spacer layer was then deposited on each QDs/silica core, followed by the coating of a mercury ion probe on the particle surfaces. The resultant multilayered QDs/silica composite nanoparticles are dispersible in HEPES buffered water; and in the presence of mercury ions, the QDs inside the nanoparticles will not be quenched by mercury ions due to the existence of the positively charged spacer layer, but can transfer their excited energy to the acceptors (probe/Hg(2+) complex), thus achieving the FRET-based ratiometric sensing for mercury ions in totally aqueous media. With its detection limit of 260 nM, this QD-based sensor exhibits high selectivity toward mercury ion and can be used in a wide pH range. This strategy may be used to construct QDs-based ratiometric assays for other ions which quench the emission of QDs.     

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.     

Simple and accurate quantification of quantum dots via single-particle counting

Quantification of quantum dots (QDs) is essential to the quality control of QD synthesis, development of QD-based LEDs and lasers, functionalizing of QDs with biomolecules, and engineering of QDs for biological applications. However, simple and accurate quantification of QD concentration in a variety of buffer solutions and in complex mixtures still remains a critical technological challenge. Here, we introduce a new methodology for quantification of QDs via single-particle counting, which is conceptually different from established UV-vis absorption and fluorescence spectrum techniques where large amounts of purified QDs are needed and specific absorption coefficient or quantum yield values are necessary for measurements. We demonstrate that single-particle counting allows us to nondiscriminately quantify different kinds of QDs by their distinct fluorescence burst counts in a variety of buffer solutions regardless of their composition, structure, and surface modifications, and without the necessity of absorption coefficient and quantum yield values. This single-particle counting can also unambiguously quantify individual QDs in a complex mixture, which is practically impossible for both UV-vis absorption and fluorescence spectrum measurements. Importantly, the application of this single-particle counting is not just limited to QDs but also can be extended to fluorescent microspheres, quantum dot-based microbeads, and fluorescent nano rods, some of which currently lack efficient quantification methods.     

Antioxidant activity assay based on the inhibition of oxidation and photobleaching of l-cysteine-capped CdTe quantum dots

Quantum dots (QDs) have recently been the focus of attention of many investigators for development of diagnostic tools in many research areas. In this work, we established a new QD-based assay to evaluate the antioxidant/polyphenolic activity. This assay is based on measurement of the inhibitory effect of the antioxidant/polyphenolic compounds on the UV-induced bleaching of CdTe QDs with l-cysteine capping. QDs exhibited excellent photostability without any UV exposure, while they bleached rapidly under UV irradiation. Generation of reactive oxygen species (ROS) under UV irradiation is probably the main cause of the photobleaching of QDs. By comparing the photostability of QDs in buffer solution in the absence and presence of sodium azide, as a known (1)O(2) quencher, the involvement of (1)O(2) in photobleaching of QDs was confirmed. The photobleaching effect induced by ROS could be reduced in the presence of antioxidant/polyphenolic compounds. We tested several antioxidant/polyphenolic compounds as well as known antioxidants such as trolox and 4 different types of tea. The results obtained by the QD-based assay revealed a very good correlation with the data acquired by Folin-Ciocalteu assay. Furthermore, a deeper understanding of the mechanism and the solution for photobleaching of QDs under UV irradiation might be very meaningful in promoting their clinical applications.     

Accurate detection of on-state quantum dot and biomolecules in a microfluidic flow with single-molecule two-color coincidence detection

Due to their unique optical and electronic properties, quantum dots (QDs) have been widely used in a variety of biosensors for sensitive detection of biomarkers and small molecules. However, single QD exhibits dynamic fluctuation of fluorescence intensity (i.e., blinking) with the transition between on and off states, which adversely influences the development of QD-based optical biosensors. Therefore, the methods for efficient evaluation of on-state QD are especially important and highly desirable. In this paper, a novel and unique approach based on single-molecule two-color coincidence detection is developed to simply and accurately evaluate the on-state QDs in a microfluidic flow. Our results demonstrate that improved QDs in the on state are detected in a microfluidic flow in comparison with that in the Brownian motion state, thus paving the way to the development of single QD-based biosensors for sensitive detection of low-abundance biomolecules. This single-molecule two-color coincidence detection has been applied for the homegeneous detection of nucleic acids in a microfluidic flow with the detection sensitivity of 5.0 fM.     

Fluorescent quantum dots for microbial imaging

QDs (Semiconductor QDs, CDs, SiQDs, and Pdots) are used in imaging microorganisms including viruses, bacteria, and fungi.     

Detection of DNA Hybridization via Fluorescence Intensity Variations of ZnSe-DNA Quantum Dot Biosensors

ZnSe quantum dots (QDs) that were capped with 11-mercaptoundecanoic acid (MUA) and conjugated to amino-modified ssDNA molecules exhibited variations in fluorescence emission intensity upon hybridization with complementary ssDNA in solution, a phenomenon that can be exploited for rapid detection of free ssDNA sequences. Conjugation of MUA-capped ZnSe QDs to amino-modified ssDNA molecules resulted in increased fluorescence emission intensity and stability at room temperature. Increasing the length of the ssDNA, that was conjugated to the QDs, resulted in increased fluorescence emission intensity up to a length of about 50 nucleotide bases, beyond which the peak emission intensity reached a plateau. Hybridization of QD-ssDNA conjugates with complementary ssDNA, either in free form or bound to QDs from the same population, resulted in additional fluorescence emission intensity amplification. A small red shift was observed when three-dimensional QD-dsDNA-QD structures were formed. The QD-ssDNA sensors with single ssDNA molecule per QD were developed and used for rapid quantitative detection of fully or partially complementary free ssDNA sequences in aqueous solution. Partial hybridization of the QD-ssDNA sensors with short ssDNA targets resulted in smaller QD emission intensity amplification, when compared to full hybridization. A QD-ssDNA sensor containing a sequence corresponding to the hemoglobin beta gene was used to detect and discriminate between free ssDNA targets consisting of a complementary ssDNA sequence and targets containing a single-base mutation that can cause sickle-cell anemia. Such QD-based biosensors can form the basis for rapid separation-free assays that can be used to detect target biomolecules in solution.     

A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots

Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs). We anticipate that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.     

Quantum Dots for Display Applications

This article offers a materials-chemistry perspective for colloidal quantum dots (QDs) in the field of display, including QD-enhanced liquid-crystal-display (QD-LCD) and QD-based light-emitting-diodes (QLEDs) display. The rapid successes of QDs for display in the past five years are not accidental but have a deep root in both maturity of their synthetic chemistry and their unique chemical, optical, and optoelectronic properties. This article intends to discuss the natural match of QD emitters for display and chemical means to eventually bring about their full potential.     

Bioconjugated quantum dots as fluorescent probes for bioanalytical applications

Quantum dots (QDs) are inorganic semiconductor nanocrystals that have unique optoelectronic properties responsible for bringing together multidisciplinary research to impel their potential bioanalytical applications. In recent years, the many remarkable optical properties of QDs have been combined with the ability to make them increasingly biocompatible and specific to the target. With this great development, QDs hold particular promise as the next generation of fluorescent probes. This review describes the developments in functionalizing QDs making use of different bioconjugation and capping approaches. The progress offered by QDs is evidenced by examples on QD-based biosensing, biolabeling, and delivery of therapeutic agents. In the near future, QD technology still faces some challenges towards the envisioned broad bioanalytical purposes.      

Quenching of photoluminescence in conjugates of quantum dots and single-walled carbon nanotube

Development of quantum dot (QD) based device components requires controlled integration of QDs into different photonic and electronic materials. In this regard, introduction of methods for regular arrangement of QDs and investigation of properties of QD-based assemblies are important. In the current work we report (1) controlled conjugation of CdSe-ZnS QDs to sidewall-functionalized single-walled carbon nanotube (SWCNT) templates (2) and the effect of conjugation of QDs to SWCNT on the photoluminescence (PL) properties of QDs. We identified that PL intensity and lifetime of QDs are considerably reduced after conjugation to SWCNT. The origin of the quenching of the PL intensity and lifetime was discussed in terms of F?rster resonance energy transfer (FRET). FRET involves nonradiative transfer of energy from a photoexcited QD (energy donor) to a nearby SWCNT (energy acceptor) in the ground state. This was examined by varying the density of QDs on SWCNT and conjugating smaller and bigger QDs to the same SWCNT. We estimated the FRET efficiency in QD-SWCNT conjugates from the quenching of the PL intensity and lifetime and identified that FRET is independent of the density and type of QDs on SWCNT but inherent to QD-SWCNT conjugates.     

量子点荧光探针检测抗坏血酸

以巯基丙酸（MPA）为稳定剂水相合成了高荧光CdTe量子点. 向量子点溶液中加入Mn2+,由于量子点表面状态发生改变而使其荧光淬灭,加入抗坏血酸后量子点荧光又得以恢复,且荧光恢复程度与抗坏血酸的浓度线性相关,从而建立了基于量子点的荧光“开关”探针检测抗坏血酸的新方法. 当CdTe量子点的浓度为1.67 uM（量子点的尺寸为1.91nm）,加入的Mn2+浓度为0.25 mM时,在优化的实验条件下,检测抗坏血酸的线性范围为0.25~16 uM,检出限为36 nM. 相对标准偏差为2.5%(10 uM, n=11). 该探针可用于维生素C药片和人血浆中抗坏血酸的快速、灵敏和选择性检测.     

In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots

A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications.     

Synthesis in aqueous solution and characterisation of a new cobalt-doped ZnS quantum dot as a hybrid ratiometric chemosensor

In this paper, cobalt (Co(2+))-doped (CoD) ZnS quantum dots (QDs) are synthesised in aqueous solution and characterised for the first time. L-Cysteine (L-Cys) ligands on the surface of CoD ZnS QDs can bind 2,4,6-trinitrotoluene (TNT) to form Meisenheimer complexes (MHCs) mainly through acid-base pairing interactions between TNT and L-Cys and the assistance of hydrogen bonding and electrostatic co-interactions among L-Cys intermolecules. The aggregation of inter-dots induced by MHCs greatly influenced the light scattering property of the QDs in aqueous solution, and Rayleigh scattering (RS) enhancement at the defect-related emission wavelengths as well as its left side was observed with the excitation of CoD ZnS QDs by violet light. RS enhancement, combining with the quenching of the orange transition emission induced by TNT anions, resulted in a change in the ratiometric visualisation of the system being investigated. A novel CoD ZnS QD-based hybrid ratiometric chemosensor has therefore been developed for simple and sensitive analysis of TNT in water. This ratiometric probe can assay down to 25 nM TNT in solution without interference from a matrix of real water sample and other nitroaromatic compounds. Because of the excellent electron-accepting ability and strong affinity of TNT to L-Cys on the surface of CoD ZnS QDs, the CoD photoluminescent nanomaterials reported here are well suited for detecting ultra-trace TNT and for distinguishing different nitro-compounds in aqueous solution.     

The application of amine-terminated silicon quantum dots on the imaging of human serum proteins after polyacrylamide gel electrophoresis (PAGE)

Novel amine-terminated silicon (Si) quantum dots (QDs) were synthesized and applied for the detection of human serum proteins on gels directly after polyacrylamide gel electrophoresis (PAGE). The diameter of these stable amine-terminated Si?QDs was in the range of 0.5-2.0 nm. In this study, the fluorescent imaging conditions, such as the buffer solution, pH value, buffer concentration and quantity of Si?QDs, were optimized and the possible mechanisms of Si?QDs-protein interaction were analyzed. The mode of Si?QDs and human serum albumin association was found to occur by hydrogen bond interactions; this was probably attributed to the interaction between the amino group of amine-terminated Si?QDs and the carboxyl group of proteins. Meanwhile, human serum proteins separated by native 1D and native 2D electrophoresis were detected by Si QD-based fluorescent imaging. Some proteins, such as isoform 1 of α-1-antitrypsin, complement C3 (Fragment) and hemopexin, which were identified by mass spectrometry (MS), were easily detected by using Si?QDs, but not with CBB-R250 staining. The Si?QDs-based fluorescent imaging technique with high resolution is a sensitive and dependable method for direct detection of human serum proteins, and has enormous potential in clinical diagnosis.     

Enhancement of the Modulation Response of Quantum-Dot-Based Down-Converted Light through Surface Plasmon Coupling

In this paper, we first elaborate on the effects of surface plasmon (SP) coupling on the modulation responses of the emission of a light-emitting diode (LED) and its down-converted lights through colloidal quantum dots (QDs). The results of our past efforts for this subject are briefly discussed. The discussions lay the foundation for the presentation of the new experimental data of such down-converted lights in this paper. In particular, the enhancement of the modulation bandwidth (MB) of a QD-based converted light through SP coupling is demonstrated. By linking green-emitting QDs (GQDs) and/or red-emitting QDs (RQDs) with synthesized Ag nano-plates via surface modifications and placing them on a blue-emitting LED, the MBs of the converted green and red emissions are significantly increased through the induced SP coupling of the Ag nano-plates. When both GQD and RQD exist and are closely spaced in a sample, the energy transfer processes of emission-reabsorption and Förster resonance energy transfer from GQD into RQD occur, leading to the increase (decrease) in the MB of green (red) light. With SP coupling, the MB of a mixed light is significantly enhanced.     

Reversible modulation of quantum dot photoluminescence using a protein- bound photochromic fluorescence resonance energy transfer acceptor

Multiple copies ( approximately 20) of Escherichia coli maltose binding protein (MBP) were coordinated to luminescent semiconductor quantum dots (QDs) via a C-terminal oligohistidine segment. The MBP was labeled with a sulfo-N-hydroxysuccinimide-activated photochromic BIPS molecule (1',3-dihydro-1'-(2-carboxyethyl)-3,3-dimethyl-6-nitrospiro[2H-1-benzopyran-2,2'-(2H)-indoline]) at two different dye-to-MBP ratios; D/P = 1 and 5. The ability of MBP-BIPS to modulate QD photoluminescence was tested by switching BIPS from the colorless spiropyran (SP) to the colored merocyanine (MC) using white light (>500 nm) or UV light ( approximately 365 nm), respectively. QDs surrounded by MBP-BIPS with D/P = 1 were quenched on average approximately 25% with consecutive repeated switches, while QDs surrounded by MBP-BIPS with D/P = 5 were quenched approximately 60%. This result suggests a possible use of BIPS-labeled proteins in QD-based nanostructures as part of a threshold switch or other biosensing device.     

pH-sensitive Photoluminescence of CdSe/ZnSe/ZnS Quantum Dots in Human Ovarian Cancer Cells

The photoluminescence of mercaptoacetic acid (MAA)-capped CdSe/ZnSe/ZnS semiconductor nanocrystal quantum dots (QDs) in SKOV-3 human ovarian cancer cells is pH-dependent, suggesting applications in which QDs serve as intracellular pH sensors. In both fixed and living cells the fluorescence intensity of intracellular MAA-capped QDs (MAA QDs) increases monotonically with increasing pH. The electrophoretic mobility of MAA QDs also increases with pH, indicating an association between surface charging and fluorescence emission. MAA dissociates from the ZnS outer shell at low pH, resulting in aggregation and loss of solubility, and this may also contribute to the MAA QD fluorescence changes observed in the intracellular environment.     

Quantum-dot-based homogeneous time-resolved fluoroimmunoassay of alpha-fetoprotein

Quantum dots (QDs) with novel photoproperties are not widely used in clinic diagnosis, and homogeneous time-resolved fluorescence assays possess many advantages over current methods for alpha-fetoprotein (AFP) detection. A novel QD-based homogeneous time-resolved fluorescence assay was developed and used for detection of AFP, a primary marker for many cancers and diseases. QD-doped carboxyl-modified polystyrene microparticles (QPs) were prepared by doping oil-soluble QDs possessing a 605 nm emission peak. The antibody conjugates (QPs-E014) were prepared from QPs and an anti-AFP monoclonal antibody, and luminescent terbium chelates (LTCs) were prepared and conjugated to a second anti-AFP monoclonal antibody (LTCs-E010). In a double-antibodies sandwich structure, QPs-E014 and LTCs-E010 were used for detection of AFP, serving as energy acceptor and donor, respectively, with an AFP bridge. The results demonstrated that the luminescence lifetime of these QPs was sufficiently long for use in a time-resolved fluoroassay, with the efficiency of time-resolved Förster resonance transfer (TR-FRET) at 67.3% and the spatial distance of the donor to acceptor calculated to be 66.1 Å. Signals from TR-FRET were found to be proportional to AFP concentrations. The resulting standard curve was log Y = 3.65786 + 0.43863·log X (R = 0.996) with Y the QPs fluorescence intensity and X the AFP concentration; the calculated sensitivity was 0.4 ng mL⁻¹. By assaying test samples against the standard curve, the coefficient of variations was <5%, indicating that QDs were suitable for this homogenous time-resolved fluoroimmunoassay. This work extended the potential applications of QDs in future homogeneous analytical bioassays. In the coming research, hepatitis B surface antigen, another primary marker for hepatocellular carcinoma, will be studied for practical detection using a QD-based homogenous multiplex fluoroimmunoassay.