A new method for the detection of ATP using a quantum-dot-tagged aptamer

Fluorescence resonance energy transfer (FRET) between a quantum dot as donor and an organic fluorophore as acceptor has been widely used for detection of nucleic acids and proteins. In this paper, we developed a new method, characterized by 605-nm quantum dot (605QD) fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease, to detect adenosine triphosphate (ATP). The new method involved the use of three different oligonucleotides: 3′-biotin-modified DNA that binds to streptavidin-conjugated 605QD; 3′-Cy5-labelled DNA; and a capture DNA consisting of an ATP aptamer and a sequence which could hybridize with both 3′-biotin-modified DNA and 3′-Cy5-labelled DNA. In the absence of the target ATP, the capture DNA binds to 3′-biotin-modified DNA and 3′-Cy5-labelled DNA, bringing quantum dot and Cy5 into close proximity for greater FRET efficiency. When ATP is introduced, the release of the 3′-Cy5-labelled DNA from the hybridization complex took place, triggering 605QD fluorescence intensity increase and corresponding Cy5 fluorescence intensity decrease. Taken together, the virtue of FRET pair 605QD/Cy5 and the property of aptamer-specific conformation change caused by aptamer–ATP interaction, combined with the fluorescence intensity change of both 605QD and Cy5, provide prerequisites for simple and convenient ATP detection. Zhang Chen and Guang Li contributed equally to this work.     

Long time scale blinking kinetics of cyanine fluorophores conjugated to DNA and its effect on Förster resonance energy transfer

The blinking kinetics of individual Cy5 fluorophores conjugated to DNA are directly measured using single-molecule spectroscopy. Under deoxygenated aqueous conditions, Cy5 fluorescence exhibits spontaneous and reversible on/off fluctuations with a period lasting seconds. This blinking is observed when directly exciting Cy5 with 640 nm light and by Forster resonance energy transfer (FRET). We find that Cy5 blinking is influenced by the proximity of the donor, the structure of the donor, the presence of 514 nm excitation, and FRET. In the context of single-molecule FRET, blinking of the acceptor produces anticorrelated donor-acceptor intensity fluctuations, which can be difficult to discern from variations in the interdye distance. Slow blinking is, in particular, problematic because it overlaps with biologically relevant time scales. By employing an alternating 514640 nm laser excitation scheme, we show that the dark states can be readily resolved and discriminated from FRET distance fluctuations.     

Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics

Single-molecule F?rster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.     

Quantum dot-conjugated hybridization probes for preliminary screening of siRNA sequences

In the present study, we describe the design and fabrication of quantum dot-conjugated hybridization probes and their application to the development of a comparatively simple and rapid procedure for the selection of highly effective small-interfering RNA (siRNA) sequences for RNA interference (RNAi) in mammalian cells, for example, siRNAs with high accessibility and affinity to the respective mRNA target. A single-stranded siRNA was conjugated with a quantum dot and used as a hybridization probe. The target mRNA was amplified in the presence of Cy5-labeled nucleotides, and Cy5-mRNA served as a hybridization sample. The formation of siRNA/mRNA duplexes during a comparatively short hybridization time (1 h) was used as a criterion for the selection of highly effective, target-specific siRNA sequences. The accessibility and affinity of the siRNA sequence for the target mRNA site were determined by fluorescence resonance energy transfer (FRET) between a quantum dot (donor) and a fluorescent dye molecule (Cy5, acceptor) localized at an appropriate distance from each other when hybridization occurred. The FRET signal was observed only when there was high accessibility between an antisense siRNA and a sense mRNA and did not appear in the case of mismatch siRNAs. Moreover, the amplitude of the FRET signal significantly correlated with the specific effect of siRNA on the expression of the target mRNA and protein, determined in native cells by RT-PCR and immunoblot analysis, respectively.     

Fluorescence resonance energy transfer (FRET) for DNA biosensors: FRET pairs and Förster distances for various dye-DNA conjugates

Fluorescence resonance energy transfer (FRET) between the extrinsic dye labels Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black Fluorescence Quencher (IabFQ), and Iowa Black RQ (IabRQ) has been studied. The F?rster distances for these FRET-pairs in single- and double-stranded DNA conjugates have been determined. In particular, it should be noted that the quantum yield of the donors Cy3 and TAMRA varies between single- and double-stranded DNA. While this alters the F?rster distance for a donor-acceptor pair, this also allows for detection of thermal denaturation events with a single non-intercalating fluorophore. The utility of FRET in the development of nucleic acid biosensor technology is illustrated by using TAMRA and IabRQ as a FRET pair in selectivity experiments. The differential quenching of TAMRA fluorescence by IabRQ in solution has been used to discriminate between 0 and 3 base pair mismatches at 60 degrees C for a 19 base sequence. At room temperature, the quenching of TAMRA fluorescence was not an effective indicator of the degree of base pair mismatch. There appears to be a threshold of duplex stability at room temperature which occurs beyond two base pair mismatches and reverses the observed trend in TAMRA fluorescence prior to that degree of mismatch. When this experimental system is transferred to a glass surface through covalent coupling and organosilane chemistry, the observed trend in TAMRA fluorescence at room temperature is similar to that obtained in bulk solution, but without a threshold of duplex stability. In addition to quenching of fluorescence by FRET, it is believed that several other quenching mechanisms are occurring at the surface.     

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

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

Autophagy Caught in the Act: A Supramolecular FRET Pair Based on an Ultrastable Synthetic Host–Guest Complex Visualizes Autophagosome–Lysosome Fusion

A supramolecular FRET pair based on the ultrahigh binding affinity between cyanine 3 conjugated cucurbit[7]uril (CB[7]‐Cy3) and cyanine 5 conjugated adamantylamine (AdA‐Cy5) was exploited as a new synthetic tool for imaging cellular processes in live cells. Confocal laser scanning microscopy revealed that CB[7]‐Cy3 and AdA‐Cy5 were intracellularly translocated and accumulated in lysosomes and mitochondria, respectively. CB[7]‐Cy3 and AdA‐Cy5 then formed a host–guest complex, reported by a FRET signal, as a result of the fusion of lysosomes and mitochondria. This observation not only indicated that CB[7] forms a stable complex with AdA in a live cell, but also suggested that this FRET pair can visualize dynamic organelle fusion processes, such as those involved in the degradation of mitochondria through autophagy (mitophagy), by virtue of its small size, chemical stability, and ease of use.     

Subunit exchange of MjHsp16.5 studied by single-molecule imaging and fluorescence resonance energy transfer

MjHsp16.5 was separately labeled by fluorescent dye Cy3 and Cy5.5. The dissociation event of a single 24-mer MjHsp16.5 molecule was captured by single-molecule imaging (SMI). Temperature-regulated subunit exchange was revealed by the real-time fluorescence resonance energy transfer (FRET). The combination of single-molecular statistics and kinetic parameters from FRET experiments leads to the conclusion that below 75 degrees C the rate-determining step of the subunit exchange was the dissociation of the dye-labeled 24-mer in which the dimer was intact, whereas above 75 degrees C, smaller units emerged in the exchange and the rate-determining step had the character of a bimolecular reaction.     

Accurate single molecule FRET efficiency determination for surface immobilized DNA using maximum likelihood calculated lifetimes

Single molecule fluorescent lifetime trajectories of surface immobilized double-stranded DNA coupled with a tetramethylrhodmaine and Cy5 FRET pair were directly measured using time-tagged single-photon counting and scanning confocal microscopy. A modified maximum likelihood estimator (MLE) was developed to compensate for localized background fluorescence and instrument response. With this algorithm, we were able to robustly extract fluorescent lifetimes from their respective decays with as few as 20 photons. Fluorescent lifetimes extracted using an MLE were found to be highly dependent on background fluorescence. We show that appropriate factors are required to extract true lifetime trajectories from single fluorophores.     

Capillary electrophoretic studies on displacement and proteolytic cleavage of surface bound oligohistidine peptide on quantum dots

Subtle changes in the chemical structure or the composition of surface bound ligands on quantum dots (QDs) remain difficult to detect. Here we describe a facile setup for fluorescence detection coupled capillary electrophoresis (CE-FL) and its application in monitoring ligand displacement on QDs through metal-affinity driven assembly. We also describe the use of CE-FL to monitor amide bond cleavage by a specific protease, based on Förster resonance energy transfer (FRET) between Cy5 and QDs spaced by a hexahistidine peptide (H6–Cy5). CE-FL allowed separation of unbound QDs and ligand bound QDs and also revealed an ordered assembly of H6–Cy5 on QDs. In a ligand displacement experiment, unlabeled hexahistidine peptide gradually displaced surface bound H6–Cy5 until finally reaching equilibrium. The displacement intermediates were clearly separated on CE-FL. Proteolytic cleavage of surface bound H6–Cy5 by thrombin was monitored by CE-FL through mobility shift, peak broadening, and FRET changes. Enzymatic parameters thus obtained were comparable with those measured by fluorescence spectroscopy.     

The oxidation state of a protein observed molecule-by-molecule.

We report the observation of the redox state of the blue copper protein azurin on the single-molecule level. The fluorescence of a small fluorophore attached to the protein is modulated by the change in absorption of the copper center via fluorescence resonance energy transfer (FRET). In our model system, the fluorescence label Cy5 was coupled to azurin from Pseudomonas aeruginosa via cysteine K27C. The Cy5 fluorescence was partially quenched by the absorption of the copper center of azurin in its oxidized state. In the reduced state, absorption is negligible, and thus no quenching occurs. We report on single-molecule measurements, both in solution by using fluorescence correlation spectroscopy (FCS) combined with fluorescence intensity distribution analysis (FIDA), and on surfaces by using wide-field fluorescence microscopy.     

Separation‐free single‐base extension assay with fluorescence resonance energy transfer for rapid and convenient determination of DNA methylation status at specific cytosine and guanine dinucleotide sites

A separation‐free single‐base extension (SBE) assay utilizing fluorescence resonance energy transfer (FRET) was developed for rapid and convenient interrogation of DNA methylation status at specific cytosine and guanine dinucleotide sites. In this assay, the SBE was performed in a tube using an allele‐specific oligonucleotide primer (i.e., extension primer) labeled with Cy3 as a FRET donor fluorophore at the 5′‐end, a nucleotide terminator (dideoxynucleotide triphosphate) labeled with Cy5 as a FRET acceptor, a PCR amplicon derived from bisulfite‐converted genomic DNA, and a DNA polymerase. A single base‐extended primer (i.e., SBE product) that was 5′‐Cy3‐ and 3′‐Cy5‐tagged was formed by incorporation of the Cy5‐labeled terminator into the 3′‐end of the extension primer, but only if the terminator added was complementary to the target nucleotide. The resulting SBE product brought the Cy3 donor and the Cy5 acceptor into close proximity. Illumination of the Cy3 donor resulted in successful FRET and excitation of the Cy5 acceptor, generating fluorescence emission from the acceptor. The capacity of the developed assay to discriminate as low as 10% methylation from a mixture of methylated and unmethylated DNA was demonstrated at multiple cytosine and guanine dinucleotide sites.     

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.     

Joint statistical analysis of multichannel time series from single quantum dot-(Cy5)n constructs

One of the major challenges in single-molecule studies is how to extract reliable information from the inevitably noisy data. Here, we demonstrate the unique capabilities of multichannel joint statistical analysis of multispectral time series using F?ster resonance energy transfer (FRET) in single quantum dot (QD)-organic dye hybrids as a model system. The multispectral photon-by-photon registration allows model-free determination of intensity change points of the donor and acceptor channels independently. The subsequent joint analysis of these change points gives high-confidence assignments of acceptor photobleaching events despite the interference from background noise and from intermittent blinking of the QD donors and acceptors themselves. Finally, the excited-state lifetimes of donors and acceptors are calculated using the joint maximum likelihood estimation (MLE) method on the donor and acceptor decay profiles, guided by a four-state kinetics model.     

FRET-based modified graphene quantum dots for direct trypsin quantification in urine

A versatile nanoprobe was developed for trypsin quantification with fluorescence resonance energy transfer (FRET). Here, fluorescence graphene quantum dot is utilized as a donor while a well-designed coumarin derivative, CMR2, as an acceptor. Moreover, bovine serum albumin (BSA), as a protein model, is not only served as a linker for the FRET pair, but also a fluorescence enhancer of the quantum dots and CMR2. In the presence of trypsin, the FRET system would be destroyed when the BSA is digested by trypsin. Thus, the emission peak of the donor is regenerated and the ratio of emission peak of donor/emission peak of acceptor increased. By the ratiometric measurement of these two emission peaks, trypsin content could be determined. The detection limit of trypsin was found to be 0.7 μg/mL, which is 0.008-fold of the average trypsin level in acute pancreatitis patient's urine suggesting a high potential for fast and low cost clinical screening.     

A multi-ion particle sensor

The first sub-micron polyacrylic sensor containing two independent ion-sensing systems is shown, that uses a single excitation wavelength and separates signals by using quantum dot donors to form FRET pairs with other fluorophores.     

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.     

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)–probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism.