单分子流式检测仪的研制

采用激光诱导荧光和流体动力学聚焦技术成功地研制出单分子流式检测仪, 实现了对水溶液中单个藻红蛋白及单个DNA分子片段的检测, 检测速率可达到每秒几十次. 与单分子荧光显微术相比, 流式分析将固定的标本台改为流动的单分子悬液, 大大提高了检测速率和统计精确性, 更加适合生物样品的快速、超高灵敏分析.     

Single molecule blinking and photobleaching separated by wide-field fluorescence microscopy

Single molecule fluorescence detection of Atto590 in poly(vinyl alcohol) was achieved by using a wide-field epifluorescence microscope with CCD-camera detection. Image sequences are obtained from which the time traces of the detected molecules are built. We find a distinctive difference between the time evolution of the fluorescence originating from the molecules detected in the first image of the sequence compared to the time evolution of the fluorescence of the molecules detected in each image of the sequence. Atto590 shows very long blinking times and photobleaching and photoblinking that are both quadratically dependent on the irradiation power density. Our approach allows kinetic separation of photobleaching from blinking. The possibility of choosing different ensembles of molecules is demonstrated and taken advantage of for this aim. Initially dark molecules or low emitting ones that might be overlooked are important to describe the complete ensemble behavior.     

Optical Properties and Response Mechanism Analysis of Multi-branched Fluorescent Probes Based on Intramolecular Charge Transfer

In this work, the optical properties of fluorescent probes used for detection of biothiol were studied by employing time-dependent density functional theory. By calculating the single photon absorption and emission properties of probe Mol.1, Mol.2 and Mol.3 before and after reaction with cysteine and homocysteine, we have investigated the effect of carboncarbon triple bond and benzene ring on the properties of fluorescent probes. It is found that the oscillator strength of probe molecules increases gradually with the improvement of the structure of the electron donor triphenylamine and the addition of carbon-carbon triple bonds, and better properties of fluorescence probes have also been demonstrated. At the same time, the effect of different number of side branches on the molecular properties of the probe was also studied. The results showed that compared with single-branched molecule Z1 and tribranched probe Mol.3, two side probe molecules Z2 had higher oscillator strength andbetter detection effect. In addition, the new single-branched probe Mol.4 with the addition of carbon-carbon triple bonds and benzene rings has better probe properties and simpler structure than the tribranched probe Mol.3.     

Isolation and characterization of R-phycoerythrin subunits and enzymatic digests

Subunits and enzymatic digests of the highly fluorescent phycobiliprotein R-phycoerythrin (R-PE) were analyzed by several separation and detection techniques including HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), CE, and HPLC-electrospray ionization (ESI) MS. R-PE subunits were isolated by HPLC and detected as single molecules by total internal reflection fluorescence microscopy. The results show efficient absorption and fluorescence of the R-PE subunits and digest peptides, originating from the incorporation of phycoerythrobilin and phycourobilin chromophores in them. In addition, HPLC-ESI-MS and SDS-PAGE were optimized to determine the molecular masses of phycobiliprotein subunits and the chromophore-containing peptides, as well as the amino acid sequences of the latter. Favorable spectroscopic and structural properties of R-PE subunits and enzymatic digests, even under denaturing conditions, make these molecules suitable for use as fluorescence labels for biomolecules.     

Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.     

LOCAL ELECTRIC FIELD EFFECT ON THE CHLOROPHYLL FLUORESCENCE

Abstract. Polarized absorption and fluorescence of chlorophyll a and bacteriochlorophyll solutions in nematic liquid crystal mixtures have been studied in the presence of an external electric field.
The electric field caused a reorientation of the pigment molecules as concluded from changes in the polarized absorption. However, no correlation was found between the change in the polarized absorption and the change in the polarized fluorescence as a function of the field strength. The field influence was much stronger than expected only from the molecular orientation. The fluorescence polarized parallel to the direction of the liquid crystal was found to increase, whereas the similarly polarized absorption decreases. As a whole, the fluorescence yield significantly increased. It is suggested that the additional electric field is reinforced by a local field, most probably due to a protonation of the liquid crystal molecules.
Charged solvent molecules are reoriented in an external electric field which changes the local electric field acting on chlorophylls. Similar changes in CHI fluorescence yield due to local electric field can be created in vivo by the shift of charged molecules present in surrounding pigments. Such effects can be at least partially responsible for slow induction of fluorescence phenomena.     

Ion recognition by 1,2,3-triazole moieties synthesized via “click chemistry”

1,2,3-Triazole-based ligands obtained through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) have been exploited in vast array of research domains owing to the stitching of simpler molecules through a needle of Cu(I) catalyst. The numerous reports on ion(s) detection capabilities of synthesized 1,4-disubstituted 1,2,3-triazole ligands using absorption and fluorescence spectroscopy are accessible. This review enlists substituted 1,2,3-triazole-based sensor probes, since 2010, synthesized selectively by CuAAC, having the ability to sense either a single ion or multiple ions under specific set of conditions along with their detection limits. The review also apprehends the different techniques and sensing mechanisms involved in the detection of ions by chemosensor probes.     

An integrated optics microfluidic device for detecting single DNA molecules

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.     

A maximum likelihood estimator to distinguish single molecules by their fluorescence decays

We have developed a maximum likelihood estimator to distinguish between similar molecules at the single molecule level based upon fluorescence decay measurements. Time resolved fluorescence measurements for single Rhodamine 6G and tetramethylrhodamine isothiocyanate molecules in fluid flow are derived from time-correlated single photon counting. A maximum likelihood estimator is developed and applied to data from a mixture of molecules. Single molecules are identified and distinguished by their fluorescence time decays. Comparison is made between identification error rates and theoretical predictions. To our knowledge, this is the first reported example of single molecule identification by fluorescence decay in a mixture.     

激发态光物理的拓扑控制:对(N,N'-二正癸基)氨基苯甲腈的簇集与光诱导分子内电荷转移

在水-二氧六环混合介质中,对二正癸基氨基苯甲腈(DDABN)分子,在水的体积分数超过0.5后,发生簇集并可观察到双重荧光发射且长波长发射带位置与在纯二氧六环中相近。该发射带被证实系分子内扭转电荷转移(TICT)带。吸收光谱表明DDABN簇集体为J-型。     

Derivatization of peptides and small proteins for improved identification and detection in capillary zone electrophoresis (CZE)

Peptides and small proteins, of limited molecular weight (MW) can be derivatized with a 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (6-AQC) reagent, leading to a single capillary zone electrophoresis (CZE) peak, suggestive of a completely tagged product. The number of tags per molecule was demonstrated by matrix assisted, laser desorption, time-of-flight mass spectrometry (MALDI-TOFMS) studies. In CZE, these species have greatly improved plate count and peak shape, improved (lowered) detectabilities, and in general, improved identification properties in the CZE mode in high performance capillary electrophoresis (HPCE). The formation of what appears to be a single, homogeneously tagged product is a function of how the derivatizations are performed. Once these conditions are optimized, virtually all peptides and small proteins tested (limited MW) can form single, fully tagged products, with the desirable CZE properties. These derivatization approaches thus lead to products that perform and are detected much better in CZE than their precursors (native, untagged peptides). The determined plate counts for these tagged peptides were as high as 6 million plates/m, which was very reproducible, and 59–12,000 times higher than the untagged (native) molecules. The peak symmetry was also improved greatly. The limit of detection (LOD) of some tested 6-AQC tagged peptides were nine to 209 times improved (lower) with ultraviolet (UV) absorption detection, again as compared with that for the native species. The LOD could be further lowered via laser induced fluorescence (LIF) detection in CZE, especially when acetonitrile (ACN) containing buffers were used.     

Investigations of condensed heteroaromatic systems including a thiophene ring

The electronic absorption and fluorescence spectra of a number of condensed heteroaromatic compounds containing a thiophene ring were studied. In connection with the fact that the theoretical treatment of the closeness of the electronic structure of thiophene and benzene molecules is based, in particular, on the similarity between the electronic absorption spectra of thiophene-containing systems and their polycyclic aromatic analogs, the spectra of the investigated compounds were compared with the corresponding polycyclic aromatic hydrocarbons. It was demonstrated that the differences introduced by the thiophene ring depend on the length of the chain of condensed benzene rings in the molecules of the thiophene-containing systems. The results can be used to analyze mixtures of such compounds.See [1] for communication XX.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 7, pp. 925–932, July, 1972.     

Responsive Fluorescence Probe for Selective and Sensitive Detection of Hypochlorous Acid in Live Cells and Animals

The development of effective bioanalytical methods for rapid, sensitive and specific detection of HOCl in vitro and in vivo plays a key role for better understanding the roles of this molecule in normal and diseased conditions, but remains challenging due to the highly reactive nature of HOCl and the complicated biological conditions. In this work, a new fluorescence probe, PQI , was developed for monitoring of the HOCl level in biological samples. PQI was easily synthesized by a one‐step condensation reaction. Upon addition of HOCl, significant changes in the absorption spectra and the color of the solution were noticed, facilitating the “naked eye” detection of HOCl in PBS buffer. The fluorescence of PQI was found to be significantly increased within a few seconds, leading to “OFF‐ON” fluorescence response towards HOCl. The sensing mechanism, oxidation of thioether by HOCl, was confirmed by HRMS titration analysis. PQI features a large Stokes shift, high sensitivity and selectivity, and rapid fluorescence response towards HOCl. Quantitative detection of HOCl in single live cells was demonstrated through fluorescence imaging and flow cytometry analysis. PQI was then successfully used in visualisation of HOCl in live zebrafish and nude mice.     

Express and sensitive detection of multiple miRNAs via DNA cascade reactors functionalized photonic crystal array

Array based detection techniques with fluorescence signal reading is a powerful tool for multiple targets analysis. However,when applied fluorescence array for micro RNA detection, time-consuming multi-steps surface signal amplification is usually required due to the low abundance of micro RNA in total RNA expressions, which impairs detection efficiency and limits its application in point of care test(POCT) manner. Herein, DNA cascade reactors(DCRs) functionalized photonic crystal(PC)array was fabricated for express and sensitive detections of mi RNA-21 and mi RNA-155. DCRs were assembled by interval conjugation of self-quenched hairpin DNA probes to single strand DNA nanowire synthesized by rolling circle amplification,which generated cascade DNA hybridization reactions in response to target mi RNAwith instant fluorescence recovery signal. PC array patterns with multi-structure colors further amplified fluorescence with their respective photonic bandgaps(PBGs)matching with the emission peaks of fluorescence molecules labelled on DCRs. The as-prepared DCRs functionalized PC array demonstrated express and sensitive simultaneous detections of mi RNA-21 and mi RNA-155 with hundreds f M detection limits only in 15 min, and was successfully applied in fast quantifications of low abundance mi RNAs from cell lysates and spiked mi RNAs from human serum, which would hold great potential for disease diagnosis and therapeutic effect monitoring with a POCT manner.     

Luminescent energy transfer between cadmium telluride nanoparticle and lanthanide(III) chelate in competitive bioaffinity assays of biotin and estradiol

Fluorescence resonance energy transfer has been studied between lanthanide(III) chelates as donors and protein-coupled CdTe semiconductor nanoparticles as acceptors. Wide excitation spectra and large Stokes shift of semiconductor nanoparticles and timeresolved fluorescence detection were shown to provide a combination for successful energy transfer assay. Different intrinsically fluorescent europium(III) and terbium(III) chelates coupled to single biotin molecules were studied for optimal energy transfer with streptavidin labeled semiconductor nanoparticles. No significant differences between the studied chelates were observed. The strength of the methodology was demonstrated in a clinically relevant competitive and separation-free immunoassay of estradiol, where subnanomolar limit of detection was achieved with the coefficient of variation 2-11%. The data suggested that relatively short distance was needed to obtain adequate energy transfer. Therefore, biomolecules were coupled onto the semiconductor nanoparticles without any spacers.     

Fluorescence lifetime correlation spectroscopy combined with lifetime tuning: new perspectives in supported phospholipid bilayer research

A new concept based on fluorescence lifetime correlation spectroscopy (FLCS) is presented allowing the simultaneous determination of diffusion coefficients of identical molecules located in different environments. The difference in fluorescence lifetimes, which is the main prerequisite for FLCS, is reached by locating one population of the dye close to a light-absorbing surface. Since such surfaces quench fluorescence, the fluorescence lifetime of chromophores located close to these surfaces can be tuned in a specific manner. This approach has been demonstrated for a BODIPY-tail-labeled lipid in supported phospholipid bilayers (SPBs) as well as in phospholipid multilayers adsorbed onto solid supports. In particular, the effect of the solid support type on the fluorescence lifetime as well as its dependence on the BODIPY-support distance has been characterized and verified by theoretical considerations based on precise determination of refractive indices of the used supports. While the fluorescence lifetime of BODIPY dye is 5.6 ns in small unilamellar vesicles (SUVs) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), the lifetime is 1.8 ns in DOPC/DOPS SPBs adsorbed onto ITO-covered glass or 3.0 ns in a DOPC/DOPS monolayer adsorbed onto seven 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) layers on oxidized silicon. Using these particular systems, we demonstrated that FLCS enables one to characterize simultaneously two-dimensional lipid diffusion in the planar lipid layers and three-dimensional vesicle diffusion in bulk above the lipid layers using single dye labeling. The autocorrelation functions obtained by this new approach do agree with those obtained by standard FCS on isolated SPBs or vesicles. Possible applications of this virtual two-channel measurement using single dye labeling as well as one detection channel are discussed.     

Recognition of polycyclic aromatic hydrocarbons and their derivatives by the 1,3-dinaphthalimide conjugate of calix[4]arene: emission, absorption, crystal structures, and computational studies

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants as well as well-known carcinogens. Therefore, it is important to develop an effective receptor for the detection and quantification of such molecules in solution. In view of this, a 1,3-dinaphthalimide derivative of calix[4]arene (L) has been synthesized and characterized, and the structure has been established by single crystal XRD. In the crystal lattice, intermolecular arm-to-arm π···π overlap dominates and thus L becomes a promising receptor for providing interactions with the aromatic species in solution, which can be monitored by following the changes that occur in its fluorescence and absorption spectra. On the basis of the solution studies carried out with about 17 derivatives of the aromatic guest molecular systems, it may be concluded that the changes that occur in the fluorescence intensity seem to be proportional to the number of aromatic rings present and thus proportional to the extent of π···π interaction present between the naphthalimide moieties and the aromatic portion of the guest molecule. Though the nonaromatic portion of the guest species affects the fluorescence quenching, the trend is still based on the number of rings present in these. Four guest aldehydes are bound to L with K(ass) of 2000-6000 M(-1) and their minimum detection limit is in the range of 8-35 μM. The crystal structure of a naphthaldehyde complex, L.2b, exhibits intermolecular arm-to-arm as well as arm-to-naphthaldehyde π···π interactions. Molecular dynamics studies of L carried out in the presence of aromatic aldehydes under vacuum as well as in acetonitrile resulted in exhibiting interactions observed in the solid state and hence the changes observed in the fluorescence and absorption spectra are attributable for such interactions. Complex formation has also been delineated through ESI MS studies. Thus L is a promising receptor that can recognize PAHs by providing spectral changes proportional to the aromatic conjugation of the guest and the extent of aromatic π···π interactions present between L and the guest.     

Binary optical encoding strategy for multiplex assay

A binary optical encoding strategy is proposed to meet the increasing requirements of multiplex bioassays. As illustrated in fluorescence immunodetection of multiplex antigen molecules, photonic crystal beads (PCBs) and quantum dots (QDs) can be used as biomolecular microcarriers and fluorescence labels, respectively. The categories of antigens were deciphered by the binary combination of optical spectra of PCBs and QDs as independent encoding elements. The number of categories that could be detected was theoretically m × n, where m and n represent the number of encoding PCBs and QDs, respectively. In addition, the concentrations of the antigens were determined by the fluorescence signals of the QDs. Results of sensitivity analysis indicate that a low-level detection of 58 pg/mL was achieved. Because of the special nanostructures of these two encoding elements, the binary encoding strategy demonstrated its superiority and practicability when compared with single PCB or QD encoding. This supports potential application in multiplex bioassays.