Surfactant‐Dependent Charge Transfer between Polyoxometalates and Single‐Walled Carbon Nanotubes: A Fluorescence Spectroscopic Study

Hybridizations of redox‐active polyoxometalates (POMs) with single‐walled carbon nanotubes (SWNTs) have been widely investigated for their diverse applications. For the purpose of constructing high‐quality electronic devices, controlling charge transfer within POM/SWNT hybrids is an inevitable issue. As determined by means of fluorescence spectroscopy, electron transfer between SWNTs and a common POM dopant, phosphomolybdic acid (PMo₁₂), can be tuned simply by an alteration of nanotube surfactant type from anionic to nonionic. The mechanism is attributed to the influence of surfactant type on the stabilization of the electron donor–acceptor hybrid and effect of surfactant–nanotube interactions. These results will be important to control charge‐transport behavior in nanohybrids consisting of carbon nanotubes.     

Single-molecule fluorescence imaging of nanocatalytic processes

This tutorial review covers recent developments in using single-molecule fluorescence microscopy to study nanoscale catalysis. The single-molecule approach enables following catalytic and electrocatalytic reactions on nanocatalysts, including metal nanoparticles and carbon nanotubes, at single-reaction temporal resolution and nanometer spatial precision. Real-time, in situ, multiplexed measurements are readily achievable under ambient solution conditions. These studies provide unprecedented insights into catalytic mechanism, reactivity, selectivity, and dynamics in spite of the inhomogeneity and temporal variations of catalyst structures. Prospects, generality, and limitations of the single-molecule fluorescence approach for studying nanocatalysis are also discussed.     

Single-molecule fluorescence spectroelectrochemistry of cresyl violet

Here we report a new path to study single molecule electron transfer dynamics by coupling scanning fluorescence microscopy with a potentiostat via a conventional electrochemical cell to enable single-molecule fluorescence spectroelectrochemistry of cresyl violet in aqueous solution, demonstrating that the single-molecule fluorescence intensity of cresyl violet is modulated synchronously with the cyclic voltammetric potential scanning.     

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.     

应用全内反射单分子荧光成像研究蛋白复合物亚基组成

细胞的生化过程大都是由蛋白复合物完成的,研究蛋白复合物亚基的组成对于了解蛋白质的结构和生物学功能具有重要的意义,然而如何准确确定蛋白复合物中蛋白质亚基的数量（stoichiometry）仍然是一个挑战.近年来,活细胞体系单分子荧光成像技术的不断发展为原位实时动态地研究蛋白质的结构和性质提供了新的手段.本文主要介绍了应用活细胞全内反射单分子荧光成像技术表征细胞膜区蛋白复合物组成的3种方法,包括单分子漂白步数分析、荧光强度统计分布以及蛋白运动分析,并结合其基本原理介绍了这几种方法在活细胞体系膜蛋白研究中的应用.     

A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation.

We report on the dynamics of fluorescence from individual molecules of a mutant of the wild-type green fluorescent protein (GFP) from Aequorea victoria, super folder GFP (SFGFP). SFGFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. It shows increased thermal stability and is able to retain its fluorescence when fused to poorly folding proteins. We use a recently developed single-molecule technique which combines fluorescence-fluctuation spectroscopy and time-correlated single photon counting in order to characterize the photophysical properties of SFGFP under one- (OPE) and two- (TPE) photon excitation conditions. We use Rhodamine 110 as a model chromophore to validate the methodology and to explain the single-molecule results of SFGFP. Under OPE, single SFGFP molecules undergo fluorescence flickering on the time scale of micros and tens of micros due to triplet formation and ground-state protonation-deprotonation, respectively, as demonstrated by excitation intensity- and pH-dependent experiments. OPE single-molecule fluorescence lifetimes indicate heterogeneity in the population of SFGFP, indicating the presence of the deprotonated I and B forms of the SFGFP chromophore. TPE of single SFGFP molecules results in the photoconversion of the chromophore. TPE of single SFGFP molecules show fluorescence flickering on the time scale of micros due to triplet formation. A flicker connected with protonation-deprotonation of the SFGFP chromophore is detected only at low pH. Our results show that SFGFP is a promising fusion reporter for intracellular applications using OPE and TPE microscopy.     

细胞膜上信号转导蛋白的单分子成像与分析

结合本课题组的研究工作,介绍了单分子荧光成像原理、荧光标记方法及数据分析方法,并进一步综述了单分子荧光成像在几种重要的膜蛋白信号转导分子机制和相关药物研究中的进展.     

Fiddling the string of carbon nanotubes with amphiphiles

The field of carbon nanotube-amphiphile self assembly has been reviewed to address the ongoing debate regarding their binding. Based on our spectrophotometry and transmission electron microscopy studies, this report shows the binding of lysophospholipids onto carbon nanotubes is dependent on the charge and geometry of the lipids and the pH of the solvent, and independent of solvent temperature. From molecular dynamics simulations, the binding of lysophospholipids onto carbon nanotubes does not fully obey any of the models proposed in the literature. We also studied carbon nanotube diffusion using single-molecule fluorescence microscopy, and carbon nanotube-lipid binding and dissociation using the technique of fluorescence resonance energy transfer. The use of carbon nanotube-lipid assembly for enabling nanotoxicological studies is demonstrated by the uptake of the assembly in the living organism Daphnia magna.     

Intermittent single-molecule interfacial electron transfer dynamics

We report on single-molecule studies of photosensitized interfacial electron transfer (ET) processes in Coumarin 343 (C343)-TiO(2) nanoparticles (NP) and Cresyl Violet (CV(+))-TiO(2) NP systems, using time-correlated single-photon counting coupled with scanning confocal fluorescence microscopy. Fluorescence intensity trajectories of individual dye molecules adsorbed on a semiconductor NP surface showed fluorescence fluctuations and blinking, with time constants distributed from milliseconds to seconds. The fluorescence fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the interfacial ET reaction dynamics. We attribute fluorescence fluctuations to the interfacial ET reaction rate fluctuations, associating redox reactivity intermittency with the fluctuations of molecule-TiO(2) electronic and Franck-Condon coupling. Intermittent interfacial ET dynamics of individual molecules could be characteristic of a surface chemical reaction strongly involved with and regulated by molecule-surface interactions. The intermittent interfacial reaction dynamics that likely occur among single molecules in other interfacial and surface chemical processes can typically be observed by single-molecule studies but not by conventional ensemble-averaged experiments.     

Using single-molecule fluorescence spectroscopy to study electron transfer.

Techniques in single-molecule fluorescence spectroscopy now allow sophisticated studies of photophysical processes in single molecules. As interest grows in the possibilities of molecular electronics, researchers have begun to turn these techniques to the study of electron transfer. Electron-transfer reactions have now been detected and measured at the single-molecule level in a variety of systems and on a variety of timescales by adapting techniques from previous single-molecule fluorescence studies.     

A comparison of potential molecular wires as components for molecular electronics

The field of electronics using single-molecule components has recently received much attention as a possible new design concept for the continued miniturisation of electronics. Molecular wires are the conceptually simplest components of such electronic systems and several different compound types have been used to produce molecular wires. Examples of some of the most promising families of molecular wires are presented, namely conjugated hydrocarbons, carbon nanotubes, porphyrin oligomers and DNA. Discussion centres around their potential use in functioning electronic architectures in terms of their electronic properties, ease and controllability of synthesis and potential for self-assembly.     

Single Molecule Detection of Nitric Oxide Enabled by d(AT)(15) DNA Adsorbed to Near Infrared Fluorescent Single-Walled Carbon Nanotubes

We report the selective detection of single nitric oxide (NO) molecules using a specific DNA sequence of d(AT)(15) oligonucleotides, adsorbed to an array of near-infrared fluorescent semiconducting single-walled carbon nanotubes (AT(15)-SWNT). While SWNT suspended with eight other variant DNA sequences show fluorescence quenching or enhancement from analytes such as dopamine, NADH, l-ascorbic acid, and riboflavin, d(AT)(15) imparts SWNT with a distinct selectivity toward NO. In contrast, the electrostatically neutral polyvinyl alcohol enables no response to nitric oxide, but exhibits fluorescent enhancement to other molecules in the tested library. For AT(15)-SWNT, a stepwise fluorescence decrease is observed when the nanotubes are exposed to NO, reporting the dynamics of single-molecule NO adsorption via SWNT exciton quenching. We describe these quenching traces using a birth-and-death Markov model, and the maximum likelihood estimator of adsorption and desorption rates of NO is derived. Applying the method to simulated traces indicates that the resulting error in the estimated rate constants is less than 5% under our experimental conditions, allowing for calibration using a series of NO concentrations. As expected, the adsorption rate is found to be linearly proportional to NO concentration, and the intrinsic single-site NO adsorption rate constant is 0.001 s(-1) μM NO(-1). The ability to detect nitric oxide quantitatively at the single-molecule level may find applications in new cellular assays for the study of nitric oxide carcinogenesis and chemical signaling, as well as medical diagnostics for inflammation.     

Characterization of the chemical interaction between single-walled carbon nanotubes and titanium dioxide nanoparticles by thermogravimetric analyses and resonance Raman spectroscopy

Raman spectroscopy is a powerful technique that is used to characterize or observe alterations in the structure or properties of carbon nanotubes and its composites. This method can provide information about electronic changes or quantify them. We used Raman spectroscopy to study the chemical and electronic changes in a composite formed by titanium dioxide nanoparticles and single-walled carbon nanotubes. This composite was characterized by scanning electron microscopy to investigate the morphology and by thermogravimetric analyses to assess the thermal stability of the isolated carbon nanotubes as compared with the nanotubes by titanium dioxide nanoparticles. The Raman results showed that the modification of the nanotubes with the TiO₂ nanoparticles generates a new material with different structure of the nanotubes, resulting in a decrease in defects. The charge transfer from the TiO₂ nanoparticles to the nanotubes alters the electronic properties of both moieties in the hybrid material. The interaction between the nanotubes and nanoparticles decreases the CC bound order of the nanotubes and decreases their thermal stability.     

Temperature dependence of the Raman spectra of individual carbon nanotubes

Resonant Raman scattering (RRS) spectra of individual carbon nanotubes on a SiO2 substrate have been investigated first in the temperature range of 100-600 K (Phys. Rev. B 2002, 66, 115411). It was revealed by the intensity abnormality of the radial breathing mode (RBM) that the carbon nanotubes have a temperature-dependent density of electronic states. This means that the previously reported temperature coefficients of RBM of carbon nanotubes are smaller than their "real" ones for the bulk samples of single- or double-walled carbon nanotubes. Comparatively, the G line of individual nanotubes shows no observable difference relative to the bulk samples.     

Nanotubes from isomeric dibenzoylmethane molecules

Organic nanotubes of various diameters were fabricated from the isomeric molecule dibenzoylmethane (DBM) by using an immersing technique with ordered porous alumina membrane as the template. The ratio of the enol isomers of DBM increased as the diameters of the nanotubes decreased. In addition, although almost no fluorescence could be detected for the DBM monomer, a striking enhancement in the fluorescence emission intensity of the nanotubes was observed as the diameters decreased. This is due to the increased ratio of the enol isomers.     

Simultaneous High Throughput Single Molecule Electrophoresis and Single Molecule Spectroscopy

Single molecule detection (SMD) has developed rapidly in recent years, especially high-throughput single molecule detection. Such research facilitated several fundamental studies at the single molecule level. In the fixture, SMD may be successfully applied to biological, clinical and medical research for DNA sequencing and single-molecule scans for disease detection. Presently, single-molecule identification of DNA and proteins is performed using fluorescence intensity, mobility or hybridization with a selective probe. In some cases, such methods are insufficient for confident single-molecule identification. Therefore, we invented a high-throughput combination single-molecule spectroscopy/imaging technique for monitoring the spectroscopic differences of several different individual molecules while they migrate in solution. The technique can offer three-dimensional data for each molecule:mobility, fluorescence intensity and spectroscopy information. Two sample systems were selected as test cases. In the first case, λ DNA is labeled with YOYO-Ⅰ,POPO-Ⅲ and a combination of the two dyes. Many individual λ DNA molecules are simultaneously imaged and identified by their spectroscopic differences. In the second case, a biotinylated 2.1 kb PCR product (also labeled with YOYO-Ⅰ) was reacted with avidin-conjugated R-phycoerythrin. The individual reactants and products are also simultaneously imaged and identified by their spectroscopic differences. This technique can be used for high-throughput DNA screening, molecular identification and monitoring intermolecular interactions with a speed of over 2,000,000 molecules per second. The existing method is the highest and most powerful single-molecule screening method available to date. Such technology is expected to have a great impact on single-molecule diagnosis and monitoring molecular interaction at the single molecule level and will be beneficial to early detection and diagnosis of disease (e.g. cancer, HIV). Furthermore, this technique allows one to directly observe and evaluate the data without any complicated calculations.     

Monitoring molecular beacon DNA probe hybridization at the single-molecule level

We have monitored the reaction dynamics of the DNA hybridization process on a liquid/solid interface at the single-molecule level by using a hairpin-type molecular beacon DNA probe. Fluorescence images of single DNA probes were recorded by using total internal reflection fluorescence microscopy. The fluorescence signal of single DNA probes during the hybridization to individual complementary DNA probes was monitored over time. Among 400 molecular beacon DNA probes that we tracked, 349 molecular beacons (87.5 %) were hybridized quickly and showed an abrupt fluorescence increase, while 51 probes (12.5 %) reacted slowly, resulting in a gradual fluorescence increase. This ratio stayed about the same when varying the concentrations of cDNA in MB hybridization on the liquid/surface interface. Statistical data of the 51 single-molecule hybridization images showed that there was a multistep hybridization process. Our results also showed that photostability for the dye molecules associated with the double-stranded hybrids was better than that for those with the single-stranded molecular beacon DNA probes. Our results demonstrate the ability to obtain a better understanding of DNA hybridization processes using single-molecule techniques, which will improve biosensor and biochip development where surface-immobilized molecular beacon DNA probes provide unique advantages in signal transduction.     

Polyelectrolyte‐Assisted Noncovalent Functionalization of Carbon Nanotubes with Ordered Self‐Assemblies of a Water‐Soluble Porphyrin

The self‐assembly and induced supramolecular chirality of meso‐tetrakis(4‐sulfonatophenyl)porphyrin (TSPP) on both single‐wall (SWCNT) and multiwall carbon nanotubes (MWCNT) are investigated. Under mild pH conditions (pH 3), TSPP forms aggregates when CNTs are dispersed in an aqueous solution containing positively charged polyelectrolytes such as poly‐L ‐lysine (PLL) or poly(allylamine hydrochloride) (PAH). Evidence for the geometry of the porphyrin aggregates is obtained from absorption spectra, whereby the fingerprints of J‐ and H‐aggregates are clearly seen only in the presence of smaller‐diameter nanotubes. J‐aggregates are better stabilized with PLL, whereas in the presence of PAH mainly H‐aggregates prevail. Excited‐state interactions within these nanohybrids are studied by steady‐state and time‐resolved fluorescence. The porphyrin emission intensity in the nanohybrid solution is significantly quenched compared to that of TSPP alone, and this implies strong electronic interaction between CNTs and porphyrin molecules. Fluorescence lifetime imaging microscopy (FLIM) further supports that porphyrin arrays are associated with the MWCNT sidewalls wrapped in PLL. In the case of the SWCNT hybrid, spherical structures associated with longer fluorescence lifetime appeared after one week, indicative of H‐aggregates of TSPP. The latter are the result of π–π stacking of porphyrin units on neighboring nanotubes facilitated by the strong tendency of these nanotubes to interact with each other. These results highlight the importance of optimum dimensions and surface‐area architectures of CNTs in the control/stability of the porphyrin aggregates with promising properties for light harvesting.     

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.     

Bleaching-resistant,Near-continuous Single-molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA Binding

Photobleaching of fluorescent probes limits the observation span of typical single-molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near-continuous single-molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.