Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging

Cell status changes are typically accompanied by the simultaneous changes of multiple microRNA (miRNA) levels. Thus, simultaneous and ultrasensitive detection of multiple miRNA biomarkers shows great promise in early cancer diagnosis. Herein, a facile single-molecule fluorescence imaging assay was proposed for the simultaneous and ultrasensitive detection of multiple miRNAs using only one capture anti-DNA/RNA antibody (S9.6 antibody). Two complementary DNAs (cDNAs) designed to hybridize with miRNA-21 and miRNA-122 were labelled with Cy3 (cDNA1) and Cy5 (cDNA2) dyes at their 5′-ends, respectively. After hybridization, both miRNA-21/cDNA1 and miRNA-122/cDNA2 complexes were captured by S9.6 antibodies pre-modified on a coverslip surface. Subsequently, the Cy3 and Cy5 dyes on the coverslip surface were imaged by the single-molecule fluorescence setup. The amount of miRNA-21 and miRNA-122 was quantified by counting the image spots from the Cy3 and Cy5 dye molecules in the green and red channels, respectively. The proposed assay displayed high specificity and sensitivity for singlet miRNA detection both with a detection limit of 5 fM and for multiple miRNA detection both with a detection limit of 20 fM. Moreover, it was also demonstrated that the assay could be used to detect multiple miRNAs simultaneously in human hepatocellular cancer cells (HepG2 cells). The proposed assay provides a novel biosensing platform for the ultrasensitive and simple detection of multiple miRNA expressions and shows great prospects for early cancer diagnosis.

A single-molecule assay for multiple microRNA detection.     

S-nitrosothiol chemistry at the single-molecule level

SNO patrol: S-Nitrosothiols (RSNO) are important molecules involved in cell signaling, which control physiological processes such as vasodilation and bronchodilation. By using the protein pore α-hemolysin as a nanoreactor, the biological chemistry of RSNO has been investigated at the single-molecule level.     

Mass action at the single-molecule level

We developed a system to reversibly encapsulate small numbers of molecules in an array of nanofabricated "dimples". This system enables highly parallel, long-term, and attachment-free studies of molecular dynamics via single-molecule fluorescence. In studies of bimolecular reactions of small numbers of confined molecules, we see phenomena that, while expected from basic statistical mechanics, are not observed in bulk chemistry. Statistical fluctuations in the occupancy of sealed reaction chambers lead to steady-state fluctuations in reaction equilibria and rates. These phenomena are likely to be important whenever reactions happen in confined geometries.     

Digital photoswitching of fluorescence based on the photochromism of diarylethene derivatives at a single-molecule level

Photochromic reactions of diarylethene derivatives were detected at a single-molecule level by using a fluorescence technique. Fluorescent photoswitching molecules in which photochromic diarylethene and fluorescent bis(phenylethynyl)anthracene units are linked through an adamantyl spacer were synthesized, and switching of fluorescence upon irradiation with UV and visible light was followed in solution as well as on polymer films at the single-molecule level. Although in solution the fluorescence intensity gradually changed upon irradiation with UV and visible light, digital on/off switching between two discrete states was observed at the single-molecule level. The "on"- and "off"-times were dependent on the power of UV and visible light. When the power of UV and visible light was increased, the average on- and off-times became short in proportion to the reciprocal power of the light. The response-times were found to show distribution. The distribution of the on- and off-times is considered to reflect the difference in the micro-environment as well as conformation of the molecules.     

Electrochemical proton relay at the single-molecule level

A scheme for the experimental study of single-proton transfer events, based on proton-coupled two-electron transfer between a proton donor and a proton acceptor molecule confined in the tunneling gap between two metal leads in electrolyte solution is suggested. Expressions for the electric current are derived and compared with formalism for electron tunneling through redox molecules. The scheme allows studying the kinetics of proton and hydrogen atom transfer as well as kinetic isotope effects at the single-molecule level under electrochemical potential control.     

Probing the charge-transfer dynamics in DNA at the single-molecule level

Photoinduced charge-transfer fluorescence quenching of a fluorescent dye produces the nonemissive charge-separated state, and subsequent charge recombination makes the reaction reversible. While the information available from the photoinduced charge-transfer process provides the basis for monitoring the microenvironment around the fluorescent dyes and such monitoring is particularly important in live-cell imaging and DNA diagnosis, the information obtainable from the charge recombination process is usually overlooked. When looking at fluorescence emitted from each single fluorescent dye, photoinduced charge-transfer, charge-migration, and charge recombination cause a "blinking" of the fluorescence, in which the charge-recombination rate or the lifetime of the charge-separated state (τ) is supposed to be reflected in the duration of the off time during the single-molecule-level fluorescence measurement. Herein, based on our recently developed method for the direct observation of charge migration in DNA, we utilized DNA as a platform for spectroscopic investigations of charge-recombination dynamics for several fluorescent dyes: TAMRA, ATTO 655, and Alexa 532, which are used in single-molecule fluorescence measurements. Charge recombination dynamics were observed by transient absorption measurements, demonstrating that these fluorescent dyes can be used to monitor the charge-separation and charge-recombination events. Fluorescence correlation spectroscopy (FCS) of ATTO 655 modified DNA allowed the successful measurement of the charge-recombination dynamics in DNA at the single-molecule level. Utilizing the injected charge just like a pulse of sound, such as a "ping" in active sonar systems, information about the DNA sequence surrounding the fluorescent dye was read out by measuring the time it takes for the charge to return.     

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.     

Measurement of submicrosecond intramolecular contact formation in peptides at the single-molecule level

We describe a single-molecule-sensitive method to determine the rate of contact formation and dissociation between tryptophan and an oxazine derivative (MR121) on the basis of measurements of the photon distance distribution. Two short peptides (15 and 20 amino acids) derived from the transactivation domain of the human oncoprotein p53 were investigated. With the fluorophore attached at the N-terminal end of the flexible peptides, fluorescence of the dye is efficiently quenched upon contact formation with a tryptophan residue. The mechanism responsible for the efficient fluorescence quenching observed in the complexes is assumed to be a photoinduced electron-transfer reaction occurring predominantly at van der Waals contact. Fluorescence fluctuations caused by intramolecular contact formation and dissociation were recorded using confocal fluorescence microscopy with two avalanche photodiodes and the time-correlated single-photon-counting technique, enabling a temporal resolution of 1.2 ns. Peptides containing a tryptophan residue at positions 9 and 8, respectively, show contact formation with rate constants of 1/120 and 1/152 ns(-1), respectively. Whereas the rate constants of contact formation most likely directly report on biopolymer chain mobility, the dissociation rate constants of 1/267 and 1/742 ns(-1), respectively, are significantly smaller and reflect strong hydrophobic interactions between the dye and tryptophan. Fluorescence experiments on point-mutated peptides where tryptophan is exchanged by phenylalanine show no fluorescence quenching.     

Electron transfer at the single-molecule level in a triphenylamine-perylene imide molecule.

Photoinduced electron transfer (ET) processes in a donor-acceptor system based on triphenylamine and perylene imide have been studied at the single-molecule (SM) and ensemble levels. The system exists as two isomers, one of which undergoes forward and reverse ET in toluene with decay constants of 3.0 and 2.2x10(9) s(-1), respectively, resulting in the dual emission of quenched and delayed fluorescence while the other isomer remains ET-inactive. The fluorescence of both isomers is heavily quenched in the more polar solvent, diethyl ether, by ET. A broad range of ET dynamics is seen at the SM level in polystryene with the two isomers nonresolvable indicating that the local nanoenvironment of the SMs varies considerably throughout the polymer matrix. Both the electronic coupling and the driving force for ET are shown to influence the ET dynamics. Many fluorescence trajectories of SMs show long periods (tens of milliseconds to seconds) where the count rate is attenuated either partly (a "dim" state) or to the background level (an "off-time"). During these periods, the reduction or interruption of emission is attributed to cycles of rapid charge separation followed by charge recombination to the ground state reducing the fluorescence quantum yield of the SM.     

Probing the polymer anomalous dynamics at solid/liquid interfaces at the single-molecule level

A goal across multiple scientific fields (e.g. separations, polymer processing, and biomaterials) is to understand polymer dynamics at solid/liquid interfaces. In the last two decades, rapid developments in single-molecule techniques have revolutionized our ability to directly observe molecular behaviors with ultra-high spatial/temporal resolution and to decouple the elementary processes that were often veiled in ensemble experiments. This review provided an overview of principle and realization of two single-molecule fluorescence techniques that were often used to study the interfacial dynamics. In addition, this review updated recent progress in the discovery and understanding of dynamical anomalies of polymers at solid/liquid interfaces using these single-molecule techniques, emphasizing important elementary processes of diffusion, adsorption, and desorption.     

DNA molecule manipulation by motor proteins for analysis at the single-molecule level

Massively parallel and individual DNA manipulation for analysis has been demonstrated by designing a fully self-assembled molecular system using motor proteins. DNA molecules were immobilized by trapping in a polyacrylamide gel replica, and were digested by a restriction enzyme, XhoI, for DNA analysis. One end of the λDNA was modified with biotin and the other end was modified with digoxin molecules by fragment labeling and ligation methods. The digoxin-functionalized end was immobilized on a glass surface coated with anti-digoxigenin antibody. The biotinylated end was freely suspended and experienced Brownian motion in a buffer solution. The free end was attached to a biotinylated microtubule via avidin–biotin biding and the DNA was stretched by a kinesin-based gliding assay. A stretched DNA molecule was fixed between the gel and coverslip to observe the cleavage of the DNA by the enzyme, which was supplied through the gel network structure. This simple process flow from DNA manipulation to analysis offers a new method of performing molecular surgery at the single-molecule scale. Figure DNA molecule manipulation by motor proteins for analysis at the single-molecule level     

Multicolor single-molecule spectroscopy with alternating laser excitation for the investigation of interactions and dynamics

We have developed confocal multicolor single-molecule spectroscopy with optimized detection sensitivity on three spectrally distinct channels for the study of biomolecular interactions and FRET between more than two molecules. Using programmable acousto-optical devices as beamsplitter and excitation filter, we overcome some of the limitations of conventional multichroic beamsplitters and implement rapid alternation between three laser lines. This enables to visualize the synthesis of DNA three-way junctions on a single-molecule basis and to resolve seven stoichiometric subpopulations as well as to quantify FRET in the presence of competing energy transfer pathways. Furthermore, the ability to study correlated molecular movements by monitoring several distances within a biomolecular complex simultaneously is demonstrated.     

Extremely strong self-assembly of a bimetallic salen complex visualized at the single-molecule level

A bis-Zn(salphen) structure shows extremely strong self-assembly both in solution as well as at the solid-liquid interface as evidenced by scanning tunneling microscopy, competitive UV-vis and fluorescence titrations, dynamic light scattering, and transmission electron microscopy. Density functional theory analysis on the Zn(2) complex rationalizes the very high stability of the self-assembled structures provoked by unusual oligomeric (Zn-O)(n) coordination motifs within the assembly. This coordination mode is strikingly different when compared with mononuclear Zn(salphen) analogues that form dimeric structures having a typical Zn(2)O(2) central unit. The high stability of the multinuclear structure therefore holds great promise for the development of stable self-assembled monolayers with potential for new opto-electronic materials.