Solution-phase single quantum dot fluorescence resonance energy transfer

We present a single particle fluorescence resonance energy transfer (spFRET) study of freely diffusing self-assembled quantum dot (QD) bioconjugate sensors, composed of CdSe-ZnS core-shell QD donors surrounded by dye-labeled protein acceptors. We first show that there is direct correlation between single particle and ensemble FRET measurements in terms of derived FRET efficiencies and donor-acceptor separation distances. We also find that, in addition to increased sensitivity, spFRET provides information about FRET efficiency distributions which can be used to resolve distinct sensor subpopulations. We use this capacity to gain information about the distribution in the valence of self-assembled QD-protein conjugates and show that this distribution follows Poisson statistics. We then apply spFRET to characterize heterogeneity in single sensor interactions with the substrate/target and show that such heterogeneity varies with the target concentration. The binding constant derived from spFRET is consistent with ensemble measurements.     

Determination of the size of quantum dots by fluorescence spectroscopy

There has been a lack of quick, simple and reliable methods for determination of nanoparticle size. An investigation of the size of hydrophobic (CdSe) and hydrophilic (CdSe/ZnS) quantum dots was performed by using the maximum position of the corresponding fluorescence spectrum. It has been found that fluorescence spectroscopy is a simple and reliable methodology to estimate the size of both quantum dot types. For a given solution, the homogeneity of the size of quantum dots is correlated to the relationship between the fluorescence maximum position (FMP) and the quantum dot size. This methodology can be extended to the other fluorescent nanoparticles. The employment of evolving factor analysis and multivariate curve resolution-alternating least squares for decomposition of the series of quantum dots fluorescence spectra recorded by a specific measuring procedure reveals the number of quantum dot fractions having different diameters. The size of the quantum dots in a particular group is defined by the FMP of the corresponding component in the decomposed spectrum. These results show that a combination of the fluorescence and appropriate statistical method for decomposition of the emission spectra of nanoparticles may be a quick and trusted method for the screening of the inhomogeneity of their solution.     

Sizes of water-soluble luminescent quantum dots measured by fluorescence correlation spectroscopy

In this paper, fluorescence correlation spectroscopy (FCS) was applied to measure the size of water-soluble quantum dots (QDs). The measurements were performed on a home-built FCS system based on the Stokes-Einstein equation. The obtained results showed that for bare CdTe QDs the sizes from FCS were larger than the ones from transmission electron microscopy (TEM). The brightness of QDs was also evaluated using FCS technique. It was found that the stability of the surface chemistry of QDs would be significantly improved by capping it with hard-core shell. Our data demonstrated that FCS is a simple, fast, and effective method for characterizing the fluorescent quantum dots, and is especially suitable for determining the fluorescent nanoparticles less than 10 nm in water solution.     

Genotyping using single nucleotide polymorphism, fluorescence spectroscopy and pattern recognition

This paper describes a method for genetic screening using single nucleotide polymorphism. Fluorescence spectra with an excitation frequency of 488 nm are recorded over a range of 550 to 660 nm of fragments of human DNA together with two fluorescent probe dyes attached to specific primers, one for each type of allele and a background dye, prepared using the Taqman reaction. The fluorescence spectra are monitored and principal components analysis used to separate spectra into three groups, which are visually identified as allele 1 (wild type), allele 2 (mutant) and mixed allele by comparison to reference samples. Malahanobis distance using 4 principal components are used to correctly classify samples into groups.     

Studies on bioconjugation of quantum dots using capillary electrophoresis and fluorescence correlation spectroscopy

In this paper, we systematically investigated the conjugation of quantum dots (QDs) with certain biomolecules using capillary electrophoresis (CE) and fluorescence correlation spectroscopy (FCS) methods. Commercial QDs and aqueous-synthesized QDs in our lab were used as labeling probes, certain bio-macromolecules, such as proteins, antibodies, and enzymes, were used as mode samples, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfo-succinimide (Sulfo-NHS) were used as linking reagents. We studied the effects of certain factors such as the isoelectric points (pIs) of bio-macromolecules and buffer pH on the bioconjugation of QDs, and found that the pIs of bio-macromolecules played an important role in the conjugation reaction. By the optimization of the buffer pH some proteins with different pIs were efficiently conjugated with QDs using EDC and Sulfo-NHS as linking agents. Furthermore, we on-line investigated the kinetic process of QDs-bioconjugation by FCS and found that the conjugation reaction of QDs with protein was rapid and the reaction process almost completed within 10 min. We also observed that QDs conjugated with proteins were stable for at least 5 days in phosphate buffer. Our work described here will be very helpful for the improvement of the QDs conjugation efficiency in bioapplications.     

Inhibition assay of biomolecules based on fluorescence resonance energy transfer (FRET) between quantum dots and gold nanoparticles

An inhibition assay method was developed based on the modulation in the FRET efficiency between quantum dots (QDs) and gold nanoparticles (AuNPs) in the presence of the molecules which inhibit the interactions between QD- and AuNP-conjugated biomolecules. For the functionalization, AuNPs were first stabilized by chemisorption of n-alkanethiols and then capped with the first generation polyamidoamine (G1 PAMAM) dendrimers. By employing a streptavidin-biotin couple as a model system, avidin was quantitatively analyzed as an inhibitor by sensing the change in photoluminescence (PL) quenching of SA-QDs by biotin-AuNPs. The detection limit for avidin was about 10 nM. It is anticipated that the PL quenching-based sensing system can be used for the quantitative analysis and high throughput screening of molecules which inhibit the specific biomolecular interactions.     

Semiconductor quantum dot-inorganic nanotube hybrids

A synthetic route for preparation of inorganic WS(2) nanotube (INT)-colloidal semiconductor quantum dot (QD) hybrid structures is developed, and transient carrier dynamics on these hybrids are studied via transient photoluminescence spectroscopy utilizing several different types of QDs. Measurements reveal efficient resonant energy transfer from the QDs to the INT upon photoexcitation, provided that the QD emission is at a higher energy than the INT direct gap. Charge transfer in the hybrid system, characterized using QDs with band gaps below the INT direct gap, is found to be absent. This is attributed to the presence of an organic barrier layer due to the relatively long-chain organic ligands of the QDs under study. This system, analogous to carbon nanotube-QD hybrids, holds potential for a variety of applications, including photovoltaics, luminescence tagging and optoelectronics.     

A quantum dot-intercalating dye dual-donor FRET based biosensor

We report herein the development of a novel quantum dot-intercalating dye dual-donor FRET system that can be used for sensitive detection of pM level DNA and protein targets.     

Distribution analysis for single molecule FRET measurement

A new numerical analysis method for experimental single-pair fluorescence resonance energy transfer (sp-FRET) data is proposed. In this method, every single data point was plotted in a style of a cumulative distribution function and dedicated to curve-fitting analysis, so that the analysis does not depend on bin size. A series of numerical simulations showed that this analysis has a more efficient and accurate resolvability of components than a fitting method based on Gaussian functions to a histogram plot. A simulated data based on experimental FRET distributions were also used to discuss the fitting errors of this method. The proposed method was applied to sp-FRET experiments of doubly dye-labeled double-strand DNA with a short sequence. Mixtures of up to three species were analyzed, and the contributions up to four subpopulations were successfully resolved.     

Mechanically controlled FRET to achieve high-contrast fluorescence switching

Organic luminescent materials with the ability to reversibly switch the luminescence when subjected to external stimuli have attracted considerable interest in recent years. However, luminescent materials with mechanochromic and photochromic dual-responsive properties are rarely reported. Hererin, we designed and synthesized a molecule P1 with dipeptide as a spacer to link rhodamine B and spiropyran moieties. P1 exhibited efficient photochromic properties both in solution and solid state. High-contrast independent fluorescence switch was also realized under the stimulus of external force. Moreover, two-step ring opening reaction and subsequent fluorescence resonance energy transfer process between the donor-acceptor pairs within one single molecule achieved successive color switch by mechanical control. Therefore, this behavior of P1 made it a promising candidate for high-contrast and sensitive optical recording and mechanical sensing system.     

Study of fluorescence quenching and dialysis process of CdTe quantum dots, using ensemble techniques and fluorescence correlation spectroscopy

Luminescence properties of quantum dots (QDs) are closely related to their surface structure and chemical properties. In this work some ensemble techniques and fluorescence correlation spectroscopy (FCS) were used to study the fluorescence quenching and dialysis process of CdTe QDs. It is found that when some heavy metal ions, such as silver ions (Ag+), quench QDs, the free Ag+ ions bind with bare Te atoms and form the AgTe structure on the surface. The FCS experimental results show that the quenching process is not the gradual reduction of fluorescence intensity of single QDs, but the decrease in the number of bright QDs with the addition of Ag+ ions. In other words, the bright QDs turn into dark directly in the quenching process. It is observed that some dark QDs converse into the bright QDs in the dialysis experiments and the dialysis process can improve the brightness per QDs. Furthermore, the results of FCS and fluorescence spectroscopy illustrate that the increase of the fluorescence quantum yield (QY) is mainly attributed to the removal of excess unreacted Cd-MPA complex and the possible chemical change of the QDs surface in the dialysis process. These new results can help us to further understand the complex surface structure of water-soluble QDs, improve their surface chemical features, and expand their applications in some fields.     

Efficient assembly of multi-walled carbon nanotube-CdSe/ZnS quantum dot hybrids with high biocompatibility and fluorescence property

CdSe/ZnS core-shell quantum dots (QDs) were efficiently tethered onto polyamidoamine dendrimer-modified multi-walled carbon nanotubes (MWCNTs) by covalent linkage and mercapto-mediated assembly. The obtained MWCNT-QD hybrids were both photophysically and morphologically characterized. The QDs are well-distributed on single nanotube surface in high density and the assembly of QDs onto MWCNTs does not change the fluorescence emission wavelength of QDs but significantly decreases the emission density. Cytotoxicity of MWCNT-QD hybrids to HeLa cells and their fluorescence property in living cell system were evaluated in detail. The hybrids show a little effect on cell viability even at very high concentration (100 μg mL(-1)). Moreover, they possess intense red fluorescence signal under optical fluorescence microscopy and good fluorescence stability over 72-h exposure in living cell system.     

Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy

The interaction between the fluorescently labeled redox protein, azurin, and a thin gold film is characterized using single-molecule fluorescence intensity and lifetime measurements. Fluorescence quenching starts at distances below 2.3 nm from the gold surface. At shorter distances the quantum yield may decrease down to fourfold for direct attachment of the protein to bare gold. Outside of the quenching range, up to fivefold enhancement of the fluorescence is observed on average with increasing roughness of the gold layer. Fluorescence-detected redox activity of individual azurin molecules, with a lifetime switching ratio of 0.4, is demonstrated for the first time close to a gold surface.     

Langmuir-Blodgett thin films of quantum dots: synthesis, surface modification, and fluorescence resonance energy transfer (FRET) studies

We describe herein studies on as-prepared hydrophobic ZnS-CdSe quantum dots (QDs) at the air-water interface. Surface pressure-area (pi-A) isotherms have been used to study the monolayer behavior. Uniform, lamellar multilayer thin films of QDs were deposited by the Langmuir-Blodgett (LB) technique. The role of two different surfactant systems commonly employed in the synthesis of these QDs (trioctylphosphine oxide-octadecylamine (TOPO-ODA) system and trioctylphosphine oxide-tetradecylphosphonic acid (TOPO-TDPA) system) on the monolayer behavior and the quality of thin films produced has been investigated. The thin films were characterized by quartz crystal microgravimetry (QCM), contact angle measurements, fluorescence spectroscopy, and transmission electron microscopy (TEM). These QD films were further modified by an amphiphilic polymer, poly(maleic anhydride-alt-1-tetradecene) (PMA). The hydrophobic interaction between the polymers and the surfactants attached to the QDs drove the self-assembly process. The carboxylic acid functional groups in the polymer were also used to immobilize avidin. We have demonstrated a proof of concept for the biosensing strategy wherein the avidin-coated QD films attracted biotinylated gold nanoparticles, resulting in fluorescence resonance energy transfer (FRET) quenching of the thin films.     

The residence probability: single molecule fluorescence correlation spectroscopy and reversible geminate recombination

The residence probability of a freely diffusing particle within an open d-dimensional ball is calculated as a function of time, for an initial distribution that is either a spherical delta function or uniform within the sphere. The latter is equivalent to the autocorrelation function (ACF) of fluorescence correlation spectroscopy (FCS) when utilizing near-field scanning optical microscopy (NSOM) probes. Starting from the general equation for the Laplace transform of the residence probability, we solve it in Laplace space for any dimensionality, inverting it into the time domain in one- and three-dimensions. The short- and long-time asymptotic behaviors of the residence probability are derived and compared with the exact results. Approximations for the two-dimensional ACF are discussed, and a new approximation is derived for the NSOM-FCS ACF. Also of interest is an analytic expression for a three-dimensional ACF, which could be useful for two-photon FCS. Analogy with the binding probability for reversible geminate recombination suggests that more information could be extracted from the long-time tails in FCS experiments.     

A versatile standard for bathochromic fluorescence based on intramolecular FRET

A perylene and a terrylene tetracarboxylic bisimide dyad was prepared in which an efficient energy transfer from the former to the latter is observed. The absorption spectrum of this compound covers a broad range. Bathochromic fluorescence with a high quantum yield was obtained independent of excitation wavelengths (λ < 655 nm). The dyad can be recommended for the use of calibrating fluorescence spectrometers, as well as a fluorescence standard in the bathochromic region.     

Evidence for resonance optical trapping of individual fluorophore-labeled antibodies using single molecule fluorescence spectroscopy

We report single molecule fluorescence studies of the diffusion of individual multiple fluorophore-labeled antibodies in solution, which show that a trapping potential of about 3.6 k(B)T can be obtained at laser powers below 1 mW with resonant excitation. Individual antibodies can be trapped for up to 140 ms, and bound antibodies can also be used to trap a single virion for up to 1 s. Selective resonance trapping to sort and manipulate fluorophore-labeled biomolecules and complexes may be possible.     

Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe

The detection of single molecules in single cells has enabled biochemical analyses to be conducted with high sensitivity and high temporal resolution. In this work, detection of apoptosis was studied by single molecule fluorescence correlation spectroscopy (FCS) in single living cells. Caspase activity was assayed using a new red fluorogenic probe that avoids the spectral overlap of green fluorescent probes and cell autofluorescence. This new probe, 2SBPO-Casp, was synthesized by coupling a water-soluble Nile Blue derivative (2SBPO) to an aspartic acid residue. Upon apoptosis induction and caspase activation, free 2SBPO dye is shown to accumulate inside the cell after probe cleavage. In previous work in our lab, single molecule fluorescence in single apoptotic cells was detected 45 min after induction using a rhodamine 110-based probe. However, significant statistical analysis was needed to exclude false positives. The use of 2SBPO-Casp overcomes the autofluorescence problem and offers a steady fluorescence signal. In our single molecule FCS measurements, Ramos cells were determined apoptotic on the basis of their correlation coefficient value (R(2)). Cells that contain an R(2) ≥ 0.65 were identified as highly correlated and therefore determined to be apoptotic. Single apoptotic cells identified in this manner were found as early as 30 min after induction and the number of apoptotic cells reached a peak value at the 3rd hour, which is consistent with other techniques. Using single molecule techniques and a new apoptosis probe, the temporal dynamics were elucidated with better sensitivity and resolution than in previous studies.     

Determination of photophysical parameters from photon arrival time trajectories in single molecule fluorescence spectroscopy

Triplet blinking is a phenomenon observed commonly in single molecule fluorescence spectroscopy. At high to moderate excitation intensities one can distinguish bright (on) and dark (off) periods in the fluorescence intensity trajectory caused by sojourns into the nonemissive triplet state. In this work, we focus on triplet blinking of an immobilized molecule in the low excitation regime, where a threshold between on and off intensity levels cannot be set, and, therefore, a standard on/off analysis of fluorescence intensity trajectories is not possible. In the low excitation regime triplet blinking parameters can be recovered from photon arrival time trajectories, i.e., records of individual photon arrival time. We use computer-generated data to compare the recovery of the triplet blinking parameters from the intensity correlation function (ICF) and the histogram of interarrival time. We have found that the ICF offers a better statistics for the recovery of the triplet blinking parameters.