Color-tuned FRET polystyrene microspheres by single wavelength excitation

Fluorescent monodisperse polystyrene microspheres were prepared by two-stage dispersion polymerization, which successfully covalently labeled microspheres with two dyes without disturbing the final particle size and size distribution. By varying the dye concentrations, microspheres show tuned colors with different fluorescent intensity under a single wavelength excitation. Fluorescence resonance energy transfer (FRET) between two labeled dyes was proved to contribute to the emission of the longer-wavelength dye at a shorter-wavelength excitation. There is no dye leakage for microspheres because of the covalent incorporation of dye molecules. The microsphere matrix provides good protection of dye molecules and blocks the influence of media outside on the fluorescence of microspheres. Single microsphere shows intense fluorescence due to a large number of encapsulated dye molecules. These uniform barcoding fluorescent microspheres have potential application in multiplexed bioanalysis.     

Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy

Capillary flow experiments are described with fluorescent molecules, bacteria, and microspheres using fluorescence correlation spectroscopy as an analytical tool. The flow velocity in the microcapillary is determined by fitting autocorrelation traces with a model containing parameters related to diffusion and flow. The flow profile of pressure-driven flow inside a microcapillary is determined by using the fluorescence fluctuations of a small dye molecule. It was found that bacteria and microspheres are retarded in their flow by optical forces produced by the laser beam.     

羧基功能化聚苯乙烯荧光微球的制备及表征

以苯乙烯、丙烯酸为单体,引入疏水性荧光染料罗丹明6G(Rh6G),采用微乳聚合法制备羧基聚苯乙烯荧光微球,并分析了表面活性剂、引发剂、丙烯酸用量对产物粒径分布的影响,考察了羧基聚苯乙烯荧光微球的浓度对荧光强度的影响。通过粒度分析仪、扫描电子显微镜、红外光谱仪、紫外吸收光谱以及荧光光谱仪对样品的纳米特性、形貌、结构和荧光性能进行了表征。结果表明,用微乳聚合法制备出50~250nm的羧基聚苯乙烯荧光微球,粒径均一且呈单分散性。紫外光谱图测试表明,在533nm左右有吸收峰。荧光光谱测试表明,羧基功能化的荧光聚苯乙烯微球浓度≤0.01%,其荧光最大激发峰为527nm,最大发射峰在555nm处;浓度高于0.01%时,荧光光谱出现红移,且荧光强度减弱。     

聚苯乙烯微球在污染物吸附领域的研究进展

目的 单分散聚苯乙烯微球是一种性能优异的功能材料,被广泛应用于医学、标准计量、分析化学和吸附等领域.方法 本文综述了聚苯乙烯(PS)微球的制备方法,讨论了单体、分散剂、引发剂、反应介质和温度等反应条件对PS微球粒径和粒径分布的影响.接着介绍了功能化聚苯乙烯的方法,如磺化、羧基化、氨基化等,接着探讨了功能化聚苯乙烯微球在染料分子、重金属、残留农药等环境污染物吸附领域的研究进展,最后对聚苯乙烯微球的应用前景进行了展望.结论 聚苯乙烯微球未来的研究重点将集中在功能化的制备以及吸附性能的提升上,具有很广阔的应用前景.     

Shah and sine convolution Fourier transform detection for microchannel electrophoresis with a charge coupled device

This paper describes an improved format for Shah convolution Fourier transform (SCOFT) detection that utilizes the spatial resolution of a charge-coupled device (CCD) rather than a fixed optical mask to perform a Shah or sine convolution over a fluorescence signal. The laser-induced fluorescence from a 9-mm section of microfabricated channel is collected with a CCD at 28 Hz. Each image frame is multiplied by a convolution function to modulate the collected signal through space. Each frame is then summed to generate an intensity-versus-time data set for Fourier analysis. The fluorescence signal oscillates at a frequency dependent upon both the convolution function multiplied across each data frame and the velocity of fluorescent microspheres or a plug of fluorescent dye flowing through the channel. This SCOFT technique affords more flexibility over formats that employ a physical mask and provides data that can be optimized for signal-to-noise (S/N) or resolution information. A 1,000-fold improvement in S/N is demonstrated for a plug of fluorescein dye. Detection of fluorescent beads exhibited frequency signals that were dependent upon the bead size distribution, the electric field, and the electrophoresis buffer concentration. Data are presented demonstrating the quantitation of fluorescent microspheres.     

Simultaneous topographic and fluorescence imaging of single DNA molecules for DNA analysis with a scanning near-field optical/atomic force microscope.

High-resolution fluorescence imaging of lambda-phage DNA molecules, intercalated with the dye YOYO-1, has been performed by a SNOM/AFM based on a bent-type optical fiber probe. A modified design of the optical probe has been made, and successful near-field optical resolution has been obtained for the strongly stretched lambda-phage DNA molecules. The best optical resolution was estimated at 45 nm for the dye-intercalated single lambda-DNA molecules by a mean width evaluation. In our comparison between the far-field fluorescence and high-resolution near-field fluorescence images for the DNA, it has been found that the near-field images much better defined the intercalation state of the dye. Finally, the relation between the DNA shapes and the dye distribution states, and the discrimination between the double-stranded and single-stranded DNA molecules, are discussed by comparing the topography and fluorescence images of the SNOM/AFM.     

表面可修饰的荧光复合微球的制备及表征

采用反相悬浮乳液法合成了丙烯酸(AA)含量不同的N-异丙基丙烯酰胺(NIPAM)共聚丙烯酸(AA)的聚合物模板P(NIPAM-co-AA), 以此作为微反应器, 分别用两种不同的方法制备了P(NIPAM-co-AA)-co-Alq3 (八羟基喹啉铝)荧光复合微球, 再用四乙氧基硅烷(TEOS)对其表面进行修饰, 获得了表面功能化的P(NIPAM-co-AA)-Alq3-SiO₂荧光复合微球。用SEM、 IR、 FM和XRD等手段对其结构和性能进行表征。结果表明: 所制备的荧光复合微球单分散性好、 荧光发光效率较高且性能稳定, 同时具有较大的比表面积; 功能化的荧光复合微球可用于高通量药物筛选和识别生物大分子等生物医学领域。      

Staining method for protein analysis by capillary gel electrophoresis

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS-protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS-protein-dye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS-protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli.     

Probes for detection of specific DNA sequences at the single-molecule level

A method has been developed for highly sensitive detection of specific DNA sequences in a homogeneous assay using labeled oligonucleotide molecules in combination with single-molecule photon burst counting and identification. The fluorescently labeled oligonucleotides are called smart probes because they report the presence of complementary target sequences by a strong increase in fluorescence intensity. The smart probes consist of a fluorescent dye attached at the terminus of a hairpin oligonucleotide. The presented technique takes advantage of the fact that the used oxazine dye JA242 is efficiently quenched by complementary guanosine residues. Upon specific hybridization to the target DNA, the smart probe undergoes a conformational change that forces the fluorescent dye and the guanosine residues apart, thereby increasing the fluorescence intensity about six fold in ensemble measurements. To increase the detection sensitivity below the nanomolar range, a confocal fluorescence microscope was used to observe the fluorescence bursts from individual smart probes in the presence and absence of target DNA as they passed through the focused laser beam. Smart probes were excited by a pulsed diode laser emitting at 635 nm with a repetition rate of 64 MHz. Each fluorescence burst was identified by three independent parameters: (a) the burst size, (b) the burst duration, and (c) the fluorescence lifetime. Through the use of this multiparameter analysis, higher discrimination accuracies between smart probes and hybridized probe-target duplexes were achieved. The presented multiparameter detection technique permits the identification of picomolar target DNA concentrations in a homogeneous assay, i.e., the detection of specific DNA sequences in a 200-fold excess of labeled probe molecules.     

Fluorescence anisotropy based single liposome assay to measure molecule-membrane interactions

Nanometer-scaled liposomes are used frequently for research, therapeutic, and analytical applications as carriers for water-soluble molecules. Recent technical advances allow the monitoring of single liposomes, which provides information on heterogeneous properties that were otherwise hidden due to ensemble averaging. Recent observations demonstrated that the efficiency of entrapping water-soluble molecules increases with decreasing vesicle size. The molecular mechanism behind this observation is not clear, but enhanced molecule-membrane interactions due to the increase of the surface area-to-volume ratio could play an important role. To investigate this hypothesis, we extended our single liposome assay based on confocal fluorescence imaging by implementation of fluorescence anisotropy. This combination has not been widely exploited, and confocal fluorescence anisotropy imaging in particular has seldom been used. We investigated different small dye molecules and were able to determine if these molecules interact or not with the liposome membrane. We confirm the liposome size-dependent entrapment of molecules whereas the molecule-membrane interactions appear to be independent of liposome size. Our fluorescence anisotropy assay can be used as a general method to investigate molecule-membrane interactions or molecule-molecule interactions in a high-throughput manner in nanometer-scaled containers like liposomes.     

Microfluidic one-step synthesis of alginate microspheres immobilized with antibodies

Micrometre- and submicrometre-size functionalized beads are frequently used to capture targets of interest from a biological sample for biological characterizations and disease diagnosis. The main challenge of the microbead-based assay is in the immobilization of probe molecules onto the microbead surfaces. In this paper, we report a versatile droplet microfluidics method to fabricate alginate microspheres while simultaneously immobilizing anti-Mycobacterium tuberculosis complex IgY and anti-Escherichia coli IgG antibodies primarily on the porous alginate carriers for specific binding and binding affinity tests. The binding affinity of antibodies is directly measured by fluorescence intensity of stained target bacteria on the microspheres. We demonstrate that the functionalized alginate microspheres yield specificity comparable with an enzyme-linked immunosorbent assay. The high surface area-to-volume ratio of the functionalized porous alginate microspheres improves the detection limit. By using the droplet microfluidics, we can easily modify the size and shape of alginate microspheres, and increase the concentration of functionalized alginate microspheres to further enhance binding kinetics and enable multiplexing.     

Single-molecule analysis of DNA immobilized on microspheres

The formation and analysis of single molecules of fluorescently labeled DNA immobilized on polystyrene microspheres is described. Analysis by confocal fluorescence microscopy revealed single-step photobleaching, characteristic of a single fluorophore. Microspheres provide a means of locating single molecules by bright-field microscopy, prior to single-molecule detection. This allows the interrogation of single molecules without suffering the limitations of premature photobleaching. Statistical analysis of fluorescence intensities for >100 microspheres suggests attachment of DNA to micropsheres to be consistent with Poisson statistics.     

Manipulating Nanoscale Features on the Surface of Dye‐Loaded Microbubbles to Increase Their Ultrasound‐Modulated Fluorescence Output

The nanoscale surface features of lipid‐coated microbubbles can dramatically affect how the lipids interact with one another as the microbubble diameter expands and contracts under the influence of ultrasound. During microbubble manufacturing, the different lipid shell species naturally partition forming concentrated lipid islands. In this study the dynamics of how these nanoscale islands accommodate the expansion of the microbubbles are monitored by measuring the fluorescence intensity changes that occur as self‐quenching lipophilic dye molecules embedded in the lipid layer change their distance from one another. It was found that when the dye molecules were concentrated in islands, less than 5% of the microbubbles displayed measurable fluorescence intensity modulation indicating the islands were not able to expand sufficiently for the dye molecules to separate from one another. When the microbubbles were heated and cooled rapidly through the lipid transition temperature the islands were melted creating an even distribution of dye about the surface. This resulted in over 50% of the microbubbles displaying the fluorescence‐modulated signal indicating that the dye molecules could now separate sufficiently to change their self‐quenching efficiency. The separation of the surface lipids in these different formations has significant implications for microbubble development as ultrasound and optical contrast agents.     

Nanopatterned monolayers of an adsorbed chromophore

A simple lift-off process was developed to rapidly fabricate nanopatterned photofunctional surfaces. Dye molecules of a perylene derivative (PDID) were adsorbed irreversibly on clean silicon through the holes of an electron-beam lithographied polymer mask. The subsequent removal of the mask in a proper solvent results in PDID nanosized regions of width as small as 30?nm for stripes and of diameter as small as 120?nm for dots. Numerical analyses of atomic force microscopy and laser-scanning confocal microscopy images show that the dye molecules are confined to the regions defined by the lithographic process, with the integrated fluorescence intensity being essentially proportional to the size of the nanofeatures. This demonstrates that a simple organic lift-off process compatible with clean-room technology, and not involving any chemical step, is able to produce photofunctional nanopatterned surfaces, even though the dye is not chemically bonded to the silicon surface.     

High-throughput flow cytometric DNA fragment sizing

The rate of detection and sizing of individual fluorescently labeled DNA fragments in conventional single-molecule flow cytometry (SMFC) is limited by optical saturation, photon-counting statistics, and fragment overlap to approximately 100 fragments/s. We have increased the detection rate for DNA fragment sizing in SMFC to approximately 2000 fragments/s by parallel imaging of the fluorescence from individual DNA molecules, stained with a fluorescent intercalating dye, as they passed through a planar sheet of excitation laser light, resulting in order of magnitude improvements in the measurement speed and the sample throughput compared to conventional SMFC. Fluorescence bursts were measured from a fM solution of DNA fragments ranging in size from 7 to 154 kilobase pairs. A data acquisition time of only a few seconds was sufficient to determine the DNA fragment size distribution. A linear relationship between the number of detected photons per burst and the DNA fragment size was confirmed. Application of this parallel fluorescence imaging method will lead to improvements in the speed, throughput, and sensitivity of other types of flow-based analyses involving the study of single molecules, chromosomes, cells, etc.     

染料前体对光热敏微胶囊光聚合的影响

利用界面聚合技术获得了新型光信息记录材料光热敏微胶囊。借助荧光光谱及红外光谱技术研究了染料前体ODB-2对光引发剂激发态及光热敏微胶囊内部光聚合特性的影响。实验结果表明染料前体在紫外的光吸收竞争造成光引发剂荧光激发谱与发射光谱强度的降低,引起光引发剂激发态分子数的减少,进而影响到光引发剂初级自由基的量子产率以及光聚合的反应过程;在光热敏微胶囊内部发现单体光聚合速度与最终聚合程度随染料前体浓度的增加均减小,且不同种类染料前体对微胶囊内部光聚合的影响程度也不相同。     

Platinum plasmonic nanostructure arrays for massively parallel single-molecule detection based on enhanced fluorescence measurements

We fabricated platinum bowtie nanostructure arrays producing fluorescence enhancement and evaluated their performance using two-photon photoluminescence and single-molecule fluorescence measurements. A comprehensive selection of suitable materials was explored by electromagnetic simulation and Pt was chosen as the plasmonic material for visible light excitation near 500 nm, which is preferable for multicolor dye-labeling applications like DNA sequencing. The observation of bright photoluminescence (λ = 500-600 nm) from each Pt nanostructure, induced by irradiation at 800 nm with a femtosecond laser pulse, clearly indicates that a highly enhanced local field is created near the Pt nanostructure. The attachment of a single dye molecule was attempted between the Pt triangles of each nanostructure by using selective immobilization chemistry. The fluorescence intensities of the single dye molecule localized on the nanostructures were measured. A highly enhanced fluorescence, which was increased by a factor of 30, was observed. The two-photon photoluminescence intensity and fluorescence intensity showed qualitatively consistent gap size dependence. However, the average fluorescence enhancement factor was rather repressed even in the nanostructure with the smallest gap size compared to the large growth of photoluminescence. The variation of the position of the dye molecule attached to the nanostructure may influence the wide distribution of the fluorescence enhancement factor and cause the rather small average value of the fluorescence enhancement factor.     

Quantitating Fluorescence Intensity From Fluorophore: Assignment of MESF Values

A procedure is presented to convert the comparison of measured fluorescence signals into a comparison of fluorescence yields (FY). The fluorescence yield, which is a property of a solution or a suspension, is defined as the product of the fluorophore concentration and the molecular quantum yield. The paper revises the measurement model which relates the measured fluorescence signal to the FY. The equality of FY of two solutions provides an equivalence between the concentrations of fluorophore in the two solutions. The equivalence is the basis for quantitation in terms of molecules of equivalent soluble fluorophore (MESF). The quantitation procedure starts with the measurement of fluorescence signals from a serial dilution of fluorescein solutions to obtain a calibration of a fluorometer. The fluorometer is used to measure the fluorescence signal of a suspension of microspheres with immobilized fluorescein isothiocyanate (FITC). The calibration is used to obtain the concentration of soluble fluorophores which gives the same fluorescence signal as the microsphere suspension. The number concentration of microspheres is measured and the equality of fluorescence yields is used to obtain the number of soluble fluorescein molecules equivalent to a single microsphere.     

Optical and electrical properties of merocyanine dye LB films by various time of UV irradiation and heat treatment

The Langmuir-Blodgett (LB) technique provides many possibilities for the control of film thickness, dimensions, and molecular structures on the nanometer scale. Various kinds of dye molecules have been found to form the J-aggregation which has been used as sensitizers of silver halide photography for long time. In recent years, they attract attention as model systems for investigating the ultra-fast exciton dynamics, materials for ultra-fast nonlinear optical devices, fluorescence probes for mitochondrial membranes. We fabricated the merocyanine dye LB films with arachidic acid (AA). In order to observe the J-aggregation of the merocyanine dye LB films, CdCl2 and KHCO3 solutions were added in subphase. From the optical absorption spectra of the mixed dye LB films (6Me-Ds:AA = 1:2) at different layers, the optical absorption peak was about 520 nm. However, the optical absorption peak of the LB films was shifted to 600 nm, when CdCl2 and KHCO3 solutions were added. This is the consequence result to the J-aggregation of the merocyanine dye. We also investigated the optical absorption peak of the LB films according to various time at 60 degrees C and 275 nm UV. We measured the STM morphology of the merocyanine dye LB film (1 layer) before UV irradiation and heat treatment. The morphology size of the LB film on HOPG was 5 nm. The roughness and molecular size were about 66.163 pm and 0.176 nm, respectively. The J-aggregation of this type was also accompanied by large morphological changes. We analyze the morphology and electrical properties of the LB films by the scanning tunneling microscopy (STM).