Protease-activated quantum dot probes

We have developed a novel nanoparticulate luminescent probe with inherent signal amplification upon interaction with a targeted proteolytic enzyme. This construct may be useful for imaging in cancer detection and diagnosis. In this system, quantum dots (QDs) are bound to gold nanoparticles (AuNPs) via a proteolytically degradable peptide sequence to non-radiatively suppress luminescence. A 71% reduction in luminescence was achieved with conjugation of AuNPs to QDs. Release of AuNPs by peptide cleavage restores radiative QD photoluminescence. Initial studies observed a 52% rise in luminescence over 47 h of exposure to 0.2 mg/mL collagenase. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker.     

Fluorescence resonance energy transfer in single enzyme molecules with a quantum dot as donor

H(+)-ATPsynthases couple a transmembrane proton transport with ATP synthesis and ATP hydrolysis. Previously, the relative subunit movement during this process has been measured by fluorescence resonance energy transfer (FRET) between two organic fluorophores covalently bound to different subunits. To improve the photophysical stability, a luminescent CdSe/ZnS nanocrystal (quantum dot) was bound to the enzyme and an organic fluorophore, Alexa568, was used as fluorescence acceptor. Single-molecule spectroscopy with the membrane integrated labeled H(+)-ATPsynthase was carried out. Single-pair FRET indicates three different conformations of the enzyme. During ATP hydrolysis relative intramolecular subunit movements are observed in real time.     

Near infrared sensing based on fluorescence resonance energy transfer between Mn:CdTe quantum dots and Au nanorods

A novel sensing system based on the near infrared (NIR) fluorescence resonance energy transfer (FRET) between Mn:CdTe quantum dots (Qdots) and Au nanorods (AuNRs) was established for the detection of human IgG. The NIR-emitting Qdots linked with goat anti-human IgG (Mn:CdTe-Ab1) and AuNRs linked with rabbit anti-human IgG (AuNRs-Ab2) acted as fluorescence donors and acceptors, respectively. FRET occurred by human IgG with the specific antigen–antibody interaction. And human IgG was detected based on the modulation in FRET efficiency. The calibration graph was linear over the range of 0.05–2.5 μM of human IgG under optimal conditions. The proposed sensing system can decrease the interference of biomolecules in NIR region and increase FRET efficiency in optimizing the spectral overlap of AuNRs with Mn:CdTe Qdots. This method has great potential for multiplex assay with different donor–acceptor pairs.     

Modeling bidirectional transport of quantum dot nanoparticles in membrane nanotubes

This paper develops a model of transport of quantum dot (QD) nanoparticles in membrane nanotubes (MNTs). It is assumed that QDs are transported inside intracellular organelles (called here nanoparticle-loaded vesicles, NLVs) that are propelled by either kinesin or dynein molecular motors while moving on microtubules (MTs). A vesicle may have both types of motors attached to it, but the motors are assumed to work in a cooperative fashion, meaning that at a given time the vesicle is moved by either kinesin or dynein motors. The motors are assumed not to work against each other, when one type of motors is pulling the vesicle, the other type is inactive. From time to time the motors may switch their roles: passive motors can become active motors and vice versa, resulting in the change of the vesicle’s direction of motion. It is further assumed that QDs can escape NLVs and become free QDs, which are then transported by diffusion. Free QDs can be internalized by NLVs. The effects of two possible types of MT orientation in MNTs are investigated: when all MTs have a uniform polarity orientation, with their plus-ends directed toward one of the cells connected by an MNT, and when MTs have a mixed polarity orientation, with half of MTs having their plus-ends directed toward one of the cells and the other half having their plus-ends directed toward the other cell. Computational results are presented for three cases. The first case is when organelles are as likely to be transported by kinesin motors as by dynein motors. The second case is when organelles are more likely to be transported by kinesin motors than by dynein motors, and the third case is when NLVs do not associate with dynein motors at all.     

Molecular beacons: an optimal multifunctional biological probe

Molecular beacon technology is set up based on fluorescence resonance energy transfer (FRET) and the complementary pairing principles. These fluorescent molecular probes, which are very highly specific and sensitive, have now become one important tool in medical and biological researches. This review introduces the molecular beacons structure, principle, the main impact factors, the labeling of the molecular beacons, and research progress on molecular beacons fluorescent-label in the polymerase chain reaction (PCR), DNA sequence analysis, gene dynamic detection in living cells, protein (enzyme)-nucleic acid interactions and applications in clinical medicine.     

Influence of the nature of quantum dot surface cations on interactions with DNA

Quantum dots are semiconductor nanoparticles that are approximately 1-10nm in diameter, similar to small proteins, and their photoluminescence is sensitive to the presence and nature of adsorbates. We have deployed these nanomaterials as luminescent probes of DNA structure. Sequence dependent conformational flexibility of DNA is of great interest due to its implications for drug-DNA and DNA-protein interactions. The counterion atmosphere surrounding DNA plays an important role in its structure, dynamics, and packaging. In this paper, we investigate the effect that various monovalent and divalent cations have on the binding of 4.5 nm CdS quantum dots to oligonucleotides that have sequence-directed intrinsic structure.     

Synthesis and photophysical studies of CdTe quantum dot-monosubstituted zinc phthalocyanine conjugates

The linkage of unsymmetrically monosubstituted 4-aminophenoxy zinc phthalocyanine (ZnAPPc, 5) to CdTe quantum dots capped with mercaptopropionic acid (MPA), l-cysteine (l-cys) or thioglycolic acid (TGA) has been achieved using the coupling agents ethyl-N(3-dimethylaminopropyl) carbodiimide and N-hydroxy succinimide, which facilitate formation of an amide bond to form the QD-ZnAPPc-linked conjugate. The formation of the amide bond was confirmed using Raman and IR spectroscopies. Atomic force microscopy (AFM) and UV-Vis spectroscopy were used further to characterise the conjugate. Förster resonance energy transfer (FRET) resulted in stimulated emission of ZnAPPc in both the linked (QD-ZnAPPc-linked) and mixed (QD:ZnAPPc-mixed) conjugates. The linked l-cys and TGA QDs conjugates (QD-ZnAPPc-linked) gave the largest FRET efficiencies hence showing the advantages of covalent linking. Fluorescence quantum yields of QDs were decreased in QD:ZnAPPc-mixed and QD:ZnAPPc-linked.     

Establishment of a fluorescence resonance energy transfer-based bioassay for detecting dioxin-like compounds

Dioxins comprise a group of compounds which contain a double aromatic ring-like structure. They are among the most prevalent and toxic environmental pollutants. Accumulation of dioxins in human tissues poses a potential threat to human health. Currently, analytical chemical procedures dominate dioxin-detection protocols. In this study, we established a fluorescence resonance energy transfer (FRET)-based dioxin-detection bioassay. Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) fused-cyan fluorescent protein (CFP) and -yellow fluorescent protein (YFP) constructed were transiently co-transfected into rat hepatoma cell line, H4IIEC3 cells. Our results showed that no FRET signals were detected in AHR-CFP- and ARNT-YFP-transfected H4IIEC3 cells. However, dioxin treatments upregulated FRET signals in these transfected cells in a dose-dependent manner. This work highlighted the potential of FRET technique in the detection of dioxin-like compounds.     

Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties

Novel -COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding -COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time (t1/2) of the coated beads increases to 537 s--seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 microg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.     

复合探针荧光定量PCR方法的建立

为了对特定基因进行实时检测,根据荧光能量转移(FRET)原理,设计及合成了一种新的FRET复合探针,该探针由一条长的荧光杂交探针和短的淬灭探针构成,其中荧光探针5′端接一荧光素分子,3′端接一延伸阻断分子磷酸,淬灭探针3′端连接一个淬灭分子对甲基红,淬灭探针与荧光探针5′端互补,无模板时,该探针杂交形成复合探针。无荧光产生,当有模板时,荧光探针与模板杂交,荧光不能被淬灭,产生的荧光与模板量成正比。根据复合探针的反应原理,研究了该探针的FRET性质及影响因素包括淬灭探针及扩增片段长度、荧光探针与淬灭探针的合适比例及镁离子浓度。实验结果显示淬灭探针及扩增片段长度对复合探针的作用有明显的影响,本实验采用淬灭探针长21个核苷酸,扩增片段长127bp,荧光探针与淬灭探针的合适比例为1：1,镁离子浓度为3mmo1／L,可获得最佳的反应体系;该复合探针合成简单,淬灭彻底,具有良好的准确性与特异性,敏感性达10^2拷贝,并具有较宽的动力学定量范围,可对10^2—10^9拷贝范围内的待检样品进行准确的定量。复合探针技术可应用于病毒感染水平、转基因拷贝数及单核苷酸多态性等检测。     

A PMMA microcapillary quantum dot linked immunosorbent assay (QLISA)

The development of a simple and inexpensive quantum dot based immunoassay for detecting myeloperoxidase (MPO) in stool samples is reported (QLISA). The method developed utilizes readily available polymethylmethacrylate (PMMA) microcapillaries as substrates for performing the sandwich assay. High power (80 mW) and low power (10 mW) UV-LEDs were tested for their efficiency in maximizing detection sensitivity in a waveguide illumination or a side illumination mode. The results obtained indicate that both waveguide and side illumination modes can be employed for detecting MPO down to 15 ng/mL, however the high power LED in a side illumination mode improves sensitivity and simplifies the data acquisition process. The protocol and sensor robustness was evaluated with animal stool samples spiked with MPO and the results indicate that the sensitivity of detection is not compromised when used in stool samples. The effect of the ionic strength of the environment on the fluorescence stability of quantum dots was evaluated and found to affect the assay only if long imaging times are employed. Replacing the buffer with glycerol during imaging increased the fluorescence intensity of quantum dots while significantly minimized the loss in intensity even after 2 h.     

Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.     

Comparison of quantum dot-binding protein tags: Affinity determination by ultracentrifugation and FRET

Background

Hybrid complexes of proteins and colloidal semiconductor nanocrystals (quantum dots, QDs) are of increasing interest in various fields of biochemistry and biomedicine, for instance for biolabeling or drug transport. The usefulness of protein–QD complexes for such applications is dependent on the binding specificity and strength of the components. Often the binding properties of these components are difficult and time consuming to assess.

Methods

In this work we characterized the interaction between recombinant light harvesting chlorophyll a/b complex (LHCII) and CdTe/CdSe/ZnS QDs by using ultracentrifugation and fluorescence resonance energy transfer (FRET) assay experiments. Ultracentrifugation was employed as a fast method to compare the binding strength between different protein tags and the QDs. Furthermore the LHCII:QD stoichiometry was determined by separating the protein–QD hybrid complexes from unbound LHCII via ultracentrifugation through a sucrose cushion.

Results

One trimeric LHCII was found to be bound per QD. Binding constants were evaluated by FRET assays of protein derivatives carrying different affinity tags. A new tetra-cysteine motif interacted more strongly (K_a = 4.9 ± 1.9 nM^− 1) with the nanoparticles as compared to a hexahistidine tag (His₆ tag) (K_a ~ 1 nM^− 1).

Conclusion

Relative binding affinities and binding stoichiometries of hybrid complexes from LHCII and quantum dots were identified via fast ultracentrifugation, and binding constants were determined via FRET assays.

General significance

The combination of rapid centrifugation and fluorescence-based titration will be useful to assess the binding strength between different types of nanoparticles and a broad range of proteins.     

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of interlaboratory data as well as quality control in clinical flow cytometry. In this article, we describe a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, which is tunable on the basis of dot size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication, we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, namely commercial fluorescein calibration beads. The utility of the calibration beads is also extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dot-labeled virus particles to cells.     

Sulfur signaling: is the agent sulfide or sulfane?

Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction.     

A sensitive and quantitative autolysosome probe for detecting autophagic activity in live and prestained fixed cells

Autophagy is a complex, multi-step and biologically important pathway mediated by autophagosomes and autolysosomes. Accurately dissecting and detecting different stages of autophagy is important to elucidate its molecular mechanism and thereby facilitate the discovery of pharmaceutical molecules. We herein reported a small-molecule synthetic probe, Zn-G₄, which is only fluorescent upon starvation- or chemical agent-induced autophagy within the autolysosome or possible the late endosome/lysosome networks. The probe can be detected by one-photon microscopy, which gives a high signal-to-noise ratio readout of autophagic activity. The pH gradient-independent fluorescence can be detected both in live and prestained fixed cells. Moreover, the fluorescent recording can be used to quantify autophagic activity at a single point without transfection or false positive signals due to protein aggregation. Furthermore, autophagy-induced fluorescence in autolysosomes can also be detected by two-photon microscopy, suggesting potential applications in deep tissue and in vivo. In conclusion, we have developed a sensitive and specific autolysosomal probe that can be used for monitoring autophagy during later stages along with quantitative assays together with widely used early markers or microtubule-associated protein 1 light chain 3 (LC3)-based probes.     

A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes

Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-terminus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported "signaling endosome" model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibrium (tau(1/2) < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF.     

Nano-bio-chips for high performance multiplexed protein detection: Determinations of cancer biomarkers in serum and saliva using quantum dot bioconjugate labels

The integration of semiconductor nanoparticle quantum dots (QDs) into a modular, microfluidic biosensor for the multiplexed quantitation of three important cancer markers, carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and Her-2/Neu (C-erbB-2) was achieved. The functionality of the integrated sample processing, analyte capture and detection modalities was demonstrated using both serum and whole saliva specimens. Here, nano-bio-chips that employed a fluorescence transduction signal with QD-labeled detecting antibody were used in combination with antigen capture by a microporous agarose bead array supported within a microfluidics ensemble so as to complete the sandwich-type immunoassay. The utilization of QD probes in this miniaturized biosensor format resulted in signal amplification 30 times relative to that of standard molecular fluorophores as well as affording a reduction in observed limits of detection by nearly 2 orders of magnitude (0.02 ng/mL CEA; 0.11 pM CEA) relative to enzyme-linked immunosorbent assay (ELISA). Assay validation studies indicate that measurements by the nano-bio-chip system correlate to standard methods at R² = 0.94 and R² = 0.95 for saliva and serum, respectively. This integrated nano-bio-chip assay system, in tandem with next-generation fluorophores, promises to be a sensitive, multiplexed tool for important diagnostic and prognostic applications.     

Visualization of beta-secretase cleavage in living cells using a genetically encoded surface-displayed [corrected] FRET probe

The human beta-secretase, BACE, plays a key role in the generation of pathogenic amyloid beta-peptide (Abeta) in Alzheimer's disease and has been identified as an ideal target for therapy. Previous studies reported the monitoring of BACE activity in vitro utilizing chemical synthesized sensors. Here we describe the first genetically encoded FRET probe that can detect BACE activity in vivo. The FRET probe was constructed with the BACE substrate site (BSS) and two mutated green fluorescent proteins. In living cell, the FRET probe was directed to the secretory pathway and anchored on the cell surface to measure BACE enzymatic activity. The results show that the FRET probe can be cleaved by BACE effectively in vivo, suggesting that the probe can be used for real-time monitoring of BACE activity. This assay provides a novel platform for BACE inhibitor screening in vivo.     

CdTe quantum dots (QDs) improve the affinities of baicalein and genistein for human serum albumin in vitro

Baicalein and genistein were studied for the affinities for human serum albumin (HSA) in the presence and absence of three CdTe quantum dots (QDs) with different sizes. Three typical CdTe QDs with maximum emissions of 535 nm (green-emitting, G-QDs), 598 nm (yellow-emitting, Y-QDs), and 654 nm (red-emitting, R-QDs) were tested. The fluorescence intensities of HSA decreased remarkably with increasing concentration of QDs. Baicalein resulted in an obvious blue-shift of the λ_em of HSA from 340 to 334 nm. However, the extents of blue-shifts induced by baicalein and genistein in the presence of QDs were much bigger than that in the absence of QDs. The quenching process of baicalein for HSA was easily affected by the QDs size than that of genistein. QDs increased the quenching constant from 136.97% to 162.24% for baicalein. However, QDs only increased the quenching constants from 20.56% to 32.23% for genistein. G-QDs, Y-QDs, and R-QDs increased the affinities of baicalein for HSA about 3.02%, 6.38% and 9.40%. G-QDs, Y-QDs, and R-QDs increased the affinities of genistein for HSA about 2.56%, 13.46% and 19.44%. The binding affinities of baicalein and genistein for HSA increased with increasing QDs size.