Markov processes in single molecule fluorescence

This article examines the current status of Markov processes in single molecule fluorescence. For molecular dynamics to be described by a Markov process, the Markov process must include all states involved in the dynamics and the first-passage time (FPT) distributions out of those states must be describable by a simple exponential law. The observation of non-exponential FPT distributions or other evidence of non-Markovian dynamics is common in single molecule studies and offers an opportunity to expand the Markov model to include new dynamics or states that improve understanding of the system.     

A glass nanopore electrode for single molecule detection

<正>We have developed a simple method for fabricating robust and low noise glass nanopore electrodes with pore size 10±5 nm to detect single molecules.β-Cyclodextrin was used as model compound for characterization.In 1.0 mol/L NaCl solution,the molecules generated current pulses of 2-5 pA with noise level less than 0.8 pA.A slide mode and a plug mode were suggested for the way ofβ-cyclodextrin single molecule moving into the glass nanopores.     

An improved synthesis of a fluorescent gabapentin-choline conjugate for single molecule detection

Voltage-gated calcium ion channels comprise pore-forming α₁ and auxiliary α₂δ, β, and γ subunits. They are important molecular devices involved in a variety of cell functions. Fluorescently labeled acylcholine analogues are important in studies such as ion channel regulation. Cy3-3-acetylcholine has recently been synthesized for single molecule detection studies; albeit in an extremely low overall yield (0.06%). In this work, an alternative route to that used in the previous Cy3-3-acetylcholine synthesis was developed with a 90% yield at a significantly lower material cost.     

A microfluidic protease activity assay based on the detection of fluorescence polarization

This article describes a fluorescence polarization (FP)-based protease assay on a microfluidic device that is compatible with fast and reproducible analyses of protease activities. The optical systems were arranged for simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes, and the binding of tetramethylrhodamine (TMR) labeled-biotin with streptavidin was utilized for optimizing FP detection in continuously flowing solutions within 74-μm wide, 12-μm deep microchannels of a glass chip. In developing off-chip FP-based assays for proteinase K, trypsin, papain and elastase, TMR conjugated-casein protein (TMR-α-casein) was employed as a universal substrate. After optimization of the hydrodynamic flow control to allow complete mixing of TMR-α-casein and short proteolysis time as possible, and of buffer composition to minimize protein sticking problems, the developed assay was transferred to the microfluidic chip by monitoring FP changes of TMR-α-casein in the main microchannel. The results indicate that the proposed device would serve as an integrated microfluidic platform with automated injection of reacting species, diffusion-controlled mixing, reaction and detection for protease activities without the need to separate the products.     

Velocity measurement of particulate flow in microfluidic channels using single point confocal fluorescence detection.

This article presents a non-invasive, optical technique for measuring particulate flow within microfluidic channels. Confocal fluorescence detection is used to probe single fluorescently labeled microspheres (0.93 microm diameter) passing through a focused laser beam at a variety of flow rates (50 nL min(-1)-8 microL min(-1)). Simple statistical methods are subsequently used to investigate the resulting fluorescence bursts and generate velocity data for the flowing particles. Fluid manipulation is achieved by hydrodynamically pumping fluid through microchannels (150 microm wide and 50 microm deep) structured in a polydimethylsiloxane (PDMS) substrate. The mean fluorescence burst frequency is shown to be directly proportional to flow speed. Furthermore, the Poisson recurrence time and width of recovered autocorrelation curves is demonstrated to be inversely proportional to flow speed. The component-based confocal fluorescence detection system is simple and can be applied to a diversity of planar chip systems. In addition, velocity measurement only involves interrogation of the fluidic system at a single point along the flow stream, as opposed to more normal multiple-point measurements.     

Nanosensors lost in space. A random walk study of single molecule detection with single-nanopore sensors

Nanopores by providing single molecule detection and manipulation are lately in the forefront of life science and nanotechnology research. While single nanopore sensors can detect the residence of even one molecule or nanoparticle within the nanopore, the analytical significance of this process is often misunderstood. A fundamental problem of nanosensors is that their sensing zone is generally infinitesimal with respect of the probed sample volume. Consequently, the probability to have in extremely diluted solutions target molecules or nanoparticles encountering the nanosensor is low. Thus, eventhough the sensor by itself has single molecule detection capability the average time frame in which this occurs is by far not irrelevant for the analysis. In this paper we report on random walk simulations to determine the average time (encounter time) needed by a single molecule to encounter a single nanopore sensor. By assigning the simulation environment with real space and time values a semi-empirical equation for expressing the average encounter time in purely diffusive systems is provided. We also show that random walk simulations can be adapted to evaluate the encounter time in the presence of an external force field acting on the target molecule. As practically relevant application the case of electrophoretically driving DNA strands towards the nanopore sensor is presented and a semi-empirical equation for the encounter time is provided.     

Determining under- and oversampling of individual particle distributions in microfluidic electrophoresis with orthogonal laser-induced fluorescence detection

This report investigates the effects of sample size on the separation and analysis of individual biological particles using microfluidic devices equipped with an orthogonal LIF detector. A detection limit of 17 +/- 1 molecules of fluorophore is obtained using this orthogonal LIF detector under a constant flow of fluorescein, which is a significant improvement over epifluorescence, the most common LIF detection scheme used with microfluidic devices. Mitochondria from rat liver tissue and cultured 143B osteosarcoma cells are used as model biological particles. Quantile-quantile (q-q) plots were used to investigate changes in the distributions. When the number of detected mitochondrial events became too large (>72 for rat liver and >98 for 143B mitochondria), oversampling occurs. Statistical overlap theory is used to suggest that the cause of oversampling is that separation power of the microfluidic device presented is not enough to adequately separate large numbers of individual mitochondrial events. Fortunately, q-q plots make it possible to identify and exclude these distributions from data analysis. Additionally, when the number of detected events became too small (<55 for rat liver and <81 for 143B mitochondria) there were not enough events to obtain a statistically relevant mobility distribution, but these distributions can be combined to obtain a statistically relevant electrophoretic mobility distribution.     

Evaluating cell migration in vitro by the method based on cell patterning within microfluidic channels

Cell migration is an early-stage and critical step for cancer metastasis. The most common approach to monitor this process is wound-healing assay. However, this traditional method has some unavoidable limitations. We observed that simply scratching the monolayer of cultured cells might cause local cell damage around the injury line. The cells along the scratched border seemed to be irritated and exhibited abnormal distribution of cytoskeleton reassembly with protruding "cell islands" and "pseudopodia" during wound healing, which might potentially affect the assessment of cell migration behavior. Herein, we applied a microfluidic device that mechanically constrained cells seeded in a designed pattern inside microchannels, and monitored cell movement in a way of mimicking the natural microenvironment of cancerous tissues. We illustrated the capacity of this simple method to probe cellular migration behaviors and to screen some biological active agents that reflected in their influence on cellular motility.     

Power-law blinking in the fluorescence of single organic molecules.

The blinking behavior of perylene diïmide molecules is investigated at the single‐molecule level. We observe long‐time scale blinking of individual multi‐chromophoric complexes embedded in a poly(methylmethacrylate) matrix, as well as for the monomeric dye absorbed on a glass substrate at ambient conditions. In both these different systems, the blinking of single molecules is found to obey analogous power‐law statistics for both the on and off periods. The observed range for single‐molecular power‐law blinking extends over the full experimental time window, covering four orders of magnitude in time and six orders of magnitude in probability density. From molecule to molecule, we observe a large spread in off‐time power‐law exponents. The distributions of off‐exponents in both systems are markedly different whereas both on‐exponent distributions appear similar. Our results are consistent with models that ascribe the power‐law behavior to charge separation and (environment‐dependent) recombination by electron tunneling to a dynamic distribution of charge acceptors. As a consequence of power‐law statistics, single molecule properties like the total number of emitted photons display non‐ergodicity.     

Investigation on the folding mode of a polymer chain in a spiral crystal by single molecule force spectroscopy

Investigation on the folding mode of a single polymer chain in its crystal is significant to the understanding of the mechanism of the fundamental crystallization as well as the engineering of new polymer crystal-based materials. Herein, we use the combined techniques of atomic force microscopy （AFM） imaging and force spectroscopy to pull a single polyethylene oxide （PEO） chain out of its spiral crystal in amyl acetate. From these data, the folding mode of polymer chains in the spiral crystal has been reconstructed. We find that the stems tilt in the typical flat area, leading to the decrease in the apparent lamellar height. While in the area of screw dislocation, the lamellar height gradually increases in the range of several nanometers. These results indicate that the combined techniques present a novel tool to directly unravel the chain folding mode of spiral crystals at single-molecule level.     

Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries

Laser-induced fluorescence detection of labeled amino acids in a flowing stream at femtomolar (10(-15)M) concentrations was achieved by using a fused-silica capillary flow-cell comprising a constricted thin-walled detection region with inner diameters (IDs) ranging from 2 to 8 microm. The diameter of the constricted region was made to match a diffraction-limited focus of a uniphase transverse electromagnetic mode (TEM(00)) laser beam. Optimization of capillary dimensions and geometries (i.e., curvature, wall thickness, and outer-inner diameter ratio were performed in order to minimize cylindrical lensing of the focused laser beam. The fluorescence was collected in a confocal optical setup using a 1.3 numerical aperture (NA), 100x oil-immersion objective and a single-photon-counting avalanche diode (SPAD). Under conditions of fluid flow, the constriction in the capillary forces all analytes to traverse across the laser probe volume, resulting in a high sampling efficiency. Fluorescein isothiocyanate-labeled glutamate (FITC-Glu) was electrophoretically separated and detected in capillaries having an ID of 2 microm at the constricted region with detection limits of 250 fM (signal-to-noise ratio (S/N) = 3) in the injected solution.     

Sensitive and simple detection of Escherichia coli strain based on time-resolved fluorescence DNA hybridization assay

A two-probe tandem DNA hybridization assay based on time-resolved fluorescence was employed to detect Escherichia coli strain. The amino modified capture probe was covalently immobilized on the common glass slide surface. The Eu(TTA)₃(5-NH₂-phen) with the characteristics of long lifetime and intense luminescence was labeled with reporter probe. The original extracted DNA samples without the purification and amplification process were directly used in the hybridization assay. The concentration of capture probe, hybridization temperature, hybridization and washing time were optimized. The detection limit is about 1.49 × 10³ CFU mL⁻¹E. coli cells, which is comparable to the value of most microbiology methods. The proposed method has the advantages of easy operation, satisfactory sensitivity and specificity, which can provide a promising technique for monitoring the microorganisms.     

Recent advances in microfluidic cell sorting techniques based on both physical and biochemical principles

Microfluidic technologies for isolating cells of interest from a heterogeneous sample have attracted great attentions, due to the advantages of less sample consumption, simple operating procedure, and high separation accuracy. According to the working principles, the microfluidic cell sorting techniques can be categorized into biochemical (labeled) and physical (label‐free) methods. However, the inherent drawbacks of each type of method may somehow influence the popularization of these cell sorting techniques. Using the multiple complementary isolation principles is a promising strategy to overcome this problem, therefore there appears to be a continuing trend to integrate two or more sorting methods together. In this review, we focus on the recent advances in microfluidic cell sorting techniques relied on both physical and biochemical principles, with emphasis on the mechanisms of cell separation. The biochemical cell sorting techniques enhanced by physical principles and the physical cell sorting techniques enhanced by biochemical principles, are first introduced. Then, we highlight on‐chip magnetic‐activated cell sorting, on‐chip fluorescence‐activated cell sorting, multi‐step cell sorting and multi‐principle cell sorting techniques, which are based on both physical and biochemical separation mechanisms. Finally, the challenges and future perspectives of the integrated microfluidics for cell sorting are discussed.     

Antigen detection based on background fluorescence quenching immunochromatographic assay

Gold immunochromatographic assay (GICA) has been around for quite a while, but it is qualitative in the vast majority of applications. A fast, simple and quantitative GICA is in call for better medicine. In the current study, we have established a novel, quantitative GICA based on fluorescence quenching and nitrocellulose membrane background signals, called background fluorescence quenching immunochromatographic assay (bFQICA). Using model analyte alpha-fetoprotein (AFP), the present study assessed the performance of bFQICA in numerous assay aspects. With serial dilutions of the international AFP standard, standard curves for the calculation of AFP concentration were successfully established. At 10 and 100 ng mL⁻¹ of the international AFP standard, the assay variability was defined with a coefficient of variance at 10.4% and 15.2%, respectively. For samples with extended range of AFP levels, bFQICA was able to detect AFP at as low as 1 ng mL⁻¹. Fluorescence in bFQICA strips stayed constant over months. A good correlation between the results from bFQICA and from a well-established Roche electrochemiluminescence immunoassay was observed in 27 serum samples (r = 0.98, p < 0.001). In conclusion, our study has demonstrated distinctive features of bFQICA over conventional GICA, including utilization of a unique fluorescence ratio between nitrocellulose membrane background and specific signals (F1/F2) to ensure accurate measurements, combined qualitative and quantitative capabilities, and exceptionally high sensitivity for detection of very low levels of antigens. All of these features could make bFQICA attractive as a model for antigen-antibody complex based GICA, and could promote bFQICA to a broad range of applications for investigation of a variety of diseases.     

Ratiometric fluorescence detection of mercuric ion based on the nanohybrid of fluorescence carbon dots and quantum dots

A novel nanohybrid ratiometric fluorescence probe comprised of carbon dots (C-dots) and hydrophilic CdSe@ZnS quantum dots (QDs) has been developed by simply mixing the blue-emission C-dots with red-emission carboxylmethyldithiocarbamate modified CdSe@ZnS QDs (GDTC-QDs). The nanohybrid ratiometric fluorescence probe exhibits dual emissions at 436 nm and 629 nm under a single excitation wavelength. Due to the strong chelating ability of GDTC on the surface of QDs to mercuric ion (Hg²⁺), the fluorescence of the GDTC-QDs in the nanohybrid system could be selectively quenched in the presence of Hg²⁺ while the fluorescence of the C-dots remained constant, resulting in an obviously distinguishable fluorescence color evolution (from red to blue) of the nanohybrid system. The detection limit of this method was found to be as low as 0.1 μM. Furthermore, the recovery result for Hg²⁺ in real samples including tap water and lake water by this method was satisfying, suggesting its potential application for Hg²⁺ sensing.     

Immunoassay of P-glycoprotein on single cell by capillary electrophoresis with laser induced fluorescence detection

The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5-10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future.     

Electrochemical electron transfer and its relation to charge transport in single molecule junctions

Ability to control charge transport at nanometer scale lies in the heart of design of fast reliable electronic devices. Molecular electronics thrive to use functional molecules for such transport. If the molecule contains redox center(s), a diode-like or transistor-like behavior can be easily explored by controlling not only the voltage difference between two metallic contacts of the molecular junction but also the potential of one of the contacting electrodes with respect to some reference. Thus, one needs to understand the relationship between electrochemical electron transfer and charge transport in metal–molecule–metal junctions. This review presents latest theoretical approaches toward understanding of such relationship and discusses pivotal experimental works to validate them. Tunneling and hopping pathways may operate in parallel (two-channel model), but experimental conditions dictate the channel preference.     

High-throughput determination of glutathione and reactive oxygen species in single cells based on fluorescence images in a microchannel

A novel high-throughput method is presented based on fluorescence images of cells in a microchannel for determination of glutathione (GSH) and reactive oxygen species (ROS) inside single cells. We first present a method to determine GSH and ROS separately, in which GSH in cells is derivatized by 2,3-naphthalenedicarboxaldehyde (NDA), and intracellular ROS is labeled using dihydrorhodamine 123. The cells with either fluorescent derivatized GSH or fluorescent labeled ROS are introduced into a microchannel and fluorescence images of every moving cell in the microchannel are taken continuously using a highly sensitive thermoelectrically cooled electron-multiplying CCD. The fluorescence intensities of the images correspond to the masses of GSH or ROS. An average detection rate of 80-120 cells/min is achieved. We then propose a method for simultaneously determining GSH and ROS, in which ROS is first labeled in the cells. The labeled cells are then introduced into the whole channel and allowed to immobilize onto the glass substrate. The fluorescence images of all the cells in the channel are taken. NDA is then introduced into the channel to derivatize the GSH in the immobilized cells, and fluorescence images of all cells are taken again. An average analysis rate of 20 cells/min is achieved. The masses of GSH and ROS in the single cells can be obtained from the fluorescence intensities of the images using their calibration curves. Since the cells are not lysed, there is no problem with adsorption of biological macromolecules and cellular debris on the channel wall, so that channel treatment, necessary in usual single-cell analysis techniques using CE and microchip electrophoresis, is no longer necessary. For single global cells, this method can also be used to determine the concentrations of ROS and GSH, which has not been reported previously. The concentrations of ROS and GSH in single global cells can be calculated from the determined masses and the cell volume (derived from the diameter of the round fluorescence image of the derivatized GSH). For gastric cancer cells, the concentrations of GSH and ROS are in the range 0.35x10(-3)-1.3x10(-3) mol/L and 0.77x10(-) (6)-1.5x10(-6) mol/L, respectively.