Comparative microRNA detection from precursor-microRNA-transfected hepatocellular carcinoma cells by capillary electrophoresis with dual-color laser-induced fluorescence

A dual-LIF (dLIF) setup combined with CE for microRNA (miRNA) detection is proposed in this study. An argon ion laser (488 nm) and a solid state laser (640 nm) were chosen to excite the fluorescent dye-labeled DNA probe after splinted ligation of miRNA. The crosstalk of emission spectrum of Alex Fluor 488 and Alex Fluor 647 is minimized with a zero crosstalk matrix for Alex Fluor 647 to 488 channels. The linear ranges of the device for the fluorescent dye-labeled DNA probe were both from 1.0 nM to 0.1 pM. The limits of detection for Alexa Fluor 488-labeled DNA and Alex Fluor 647-labeled DNA were 9.3 and 31 fM, respectively. The detection of specific miRNA has been accomplished by combining splinted ligation with the fluorescent dye-labeled oligonucleotides. The linear range for the synthetic miRNA is from 1.0 nM to 1.0 pM. Without PCR amplification, CE-dLIF was applied to discriminate a pre-miR-10b*-transfected cells (contains precursor miR-10b*) from hepatocellular carcinoma cell (control cells). Therefore, this result indicates CE-dLIF has great potential to provide a rapid comparative assay for miRNAs detection.     

毛细管电泳-荧光/非接触电导组合型检测器的研制

报道了一种毛细管电泳-荧光／非接触电导组合型检测器。该检测器共用非接触电导检测池,实现了双检测器响应同步。优化了非接触电导检测系统中激发电压信号及其频率;荧光检测是用发光二极管作为激发光源,用光纤收集并传输荧光信号至光电倍增管。用无机金属离子和异硫氰酸荧光素评价该体系,结果表明,该检测器达到了任一单类型检测器性能指标。     

The potential of autofluorescence for the detection of single living cells for label-free cell sorting in microfluidic systems

A novel method for studying unlabeled living mammalian cells based on their autofluorescence (AF) signal in a prototype microfluidic device is presented. When combined, cellular AF detection and microfluidic devices have the potential to facilitate high-throughput analysis of different cell populations. To demonstrate this, unlabeled cultured cells in microfluidic devices were excited with a 488 nm excitation light and the AF emission (> 505 nm) was detected using a confocal fluorescence microscope (CFM). For example, a simple microfluidic three-port glass microstructure was used together with conventional electroosmotic flow (EOF) to switch the direction of the fluid flow. As a means to test the potential of AF-based cell sorting in this microfluidic device, granulocytes were successfully differentiated from human red blood cells (RBCs) based on differences in AF. This study demonstrated the use of a simple microfabricated device to perform high-throughput live cell detection and differentiation without the need for cell-specific fluorescent labeling dyes and thereby reducing the sample preparation time. Hence, the combined use of microfluidic devices and cell AF may have many applications in single-cell analysis.     

Water-soluble AIE-active Fluorescent Organic Nanoparticles: Design,Preparation and Application for Specific Detection of Cyanide in Water and Food Samples

A dilactosyl-dicyanovinyl-functionalized tetraphenylethene ( TPELC ) was designed, synthesized and used for ratiometric sensing of cyanide. TPELC was comprised of three moieties (tetraphenylethylene, dicyanovinyl group and lactose unit) in one molecule, making TPELC water-soluble and aggregation-induced emission (AIE)-active and selectively reactive to cyanide. Compared with other reported fluorescent probes containing dicyanovinyl group, TPELC is the first AIE luminogen to be assembled as fluorescent organic nanoparticles (FONs) for sensing of cyanide in water without the use of surfactant or the help of organic solvents based on the nucleophilic addition reaction. The detection mechanism was verified by liquid chromatograph mass spectrometry experiments and by protonation of cyanide to reduce the nucleophilicity of cyanide. In addition, TPELC was used for detection of the cyanide content of food samples and test strips were developed to simplify the detection procedure.     

FRET‐based Fluorescent and Colorimetric Probe for Selective Detection of Hg(II) and Cu(II) with Dual‐mode

A dual‐function fluorescence resonance energy transfer (FRET)‐based fluorescent and colorimetric probe was rationally fabricated from an energy donor coumarin moiety and an energy acceptor rhodamine moiety linked by a thiohydrazide arm for selective detection of Hg²⁺ and Cu²⁺. Two distinct mechanisms were used for the selective detection. Results revealed that probe 1 showed high fluorescent selectivity towards Hg²⁺ and evident colorimetric selectivity for Cu²⁺, which was suitable for ‘naked‐eye’ detection.     

Multiplex immunoassays of equine virus based on fluorescent encoded magnetic composite nanoparticles

A new detection format for multiplexed analysis based on fluorescent encoded magnetic composite nanoparticles is presented. Two kinds of virus were analyzed by this new method: equine influenza virus (EIV) and equine infectious anemia virus (EIAV). Firstly, EIV antigen and EIAV antigen were conjugated to two kinds of fluorescent encoded magnetic composite nanoparticles, while the green-emitting CdTe quantum dots (QDs) were attached to the antibody of EIV and EIAV. Then both green-emitting CdTe QD-labeled antibodies and antigens labeled with fluorescent encoded magnetic composite nanoparticles were used to form an immunoassay system for the detection of EIV and EIAV antigens. The method is time-saving and has higher sensitivity (1.3 ng mL⁻¹ for EIV antigens and 1.2 ng mL⁻¹ for EIAV antigens) than the conventional methods. A competitive immunoassay method based on this analysis system was used to detect EIV and EIAV antigens in spiked serum samples with satisfactory results.     

一种可用于检测2,4,6-三硝基苯酚的杂环芳族卤化物小分子荧光探针

以2,6-二甲氧基苯甲酸为起始原料得到氨基噻二唑衍生物,通过Gattermann反应合成一种杂环芳族卤化物2-氯-5-(3-氯-2,6-二甲氧基苯基)-1,3,4-噻二唑(L),利用核磁共振波谱仪(NMR)和高分辨率质谱仪(HRMS)等测试手段确定了结构,并将其用作小分子荧光探针检测2,4,6-三硝基苯酚(TNP),系统地研究了其荧光特性,并且结合理论计算,探究了其可能的猝灭机制。 研究结果表明,探针L对TNP具有高选择性、高灵敏度、抗干扰能力强,在较宽的pH值范围内,仍然表现出良好的荧光性能。 具有较低的检测限(4.2×10^-7 mol/L),可用于实际水样中TNP的检测。     

G-quadruplex DNAzyme as the turn on switch for fluorimetric detection of genetically modified organisms

A novel fluorescent sensor for detection of genetically modified organisms was developed, and in the sensor G-quadruplex DNAzyme (G-quadruplex-hemin complex) was used as the turn on switch.     

A fluorescent polymer dots positive readout fluorescent quenching lateral flow sensor for ractopamine rapid detection

A fluorescent polymer dots positive readout and sensitive lateral flow assay (LFA) based on fluorescent quenching has been developed to detect ractopamine (Rac), a chemical residue in food, harmful to human health. Compared with traditional LFA strips, these fluorescent quenching LFA (FQLFA) strips provide a positive correlation method that allows users to obtain results from a weak fluorescent signal. The immunoassay strip scheme is based on the fact that fluorescent polymer dots (FPDs) in close proximity to gold nanoparticles (AuNPs) represent a strong fluorescent quenching. We show that the FQLFA strips can be used as a source to quantitatively analyze Rac in phosphate buffers (PB), swine urine and muscle tissue samples. The lowest detection limitation of the FQLFA was 0.16 ng mL⁻¹. Our results indicated that this novel scheme was more suitable for rapid detection of small molecules.     

Synthesis and evaluation of fluorogenic reagents for simultaneous detection of peptides and proteins by HPLC in two different samples

To find the pairs of fluorogenic reagents having similar retention times in HPLC but with different fluorescent characteristics, six fluorogenic reagents bearing benzoxadiazole or benzoselenadiazole skeletons were synthesized. The resultant derivatives obtained from the reaction of peptides and proteins with reagents which have a benzoselenadiazole skeleton showed different fluorescence characteristics from those with a benzoxadiazole skeleton. Since each corresponding derivatives of trypsin inhibitor and BSA with DAABD-Cl and 7-fluoro-N-[2-(diethylamino)ethyl]-2,1,3-benzoselenadiazole-4-sulfonamide (DEAEABSeD-F) have similar retention times, the pair of reagents was adopted for the sensitive simultaneous detection of proteins in two different samples. When the soluble fraction of mouse hippocampus was divided into the two samples (A and B), each was reacted with DEAEABSeD-F for A and DAABD-Cl for B, respectively. The two reaction solutions were combined and subjected to HPLC analysis with two fluorescent detectors in series (excitation and emission at different wavelengths for A and B, respectively). The resultant two chromatograms had quite similar patterns for each other. The new pair of fluorogenic reagents (DAABD-Cl and DEAEABSeD-F) would be applicable to proteomics studies using the previously reported FD-LC-MS/MS method.     

Continuous wave-based multiphoton excitation fluorescence for capillary electrophoresis

It was reported that a novel detection method, continuous wave (CW)-based multiphoton excitation (MPE) fluorescence detection with diode laser (DL), has been firstly proposed for capillary electrophoresis (CE). Special design of end-column detection configuration proved to be superior to on-column type, considering the detection sensitivity. Three different kinds of fluorescent tags that were widely used as molecular label in bio-analysis, such as small-molecule dye, fluorescent protein and nano particle or also referred to as quantum dot (QD), have been evaluated as samples for the constructed detection scheme. Quantitative analyses were also performed using rhodamine species as tests, which revealed dynamic linear range over two orders of magnitude, with detection limit down to zeptomole-level. Simultaneous detection of fluorescent dyestuffs with divergent excitation and emission wavelengths in a broad range showed advantage of this scheme over conventional laser-induced fluorescence (LIF) detection. Further investigations on CW-MPE fluorescence detection with diode laser for capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) separations of fluorescein isothiocyanate (FITC) labeled amino acids indicated good prospect of this detection approach in various micro or nano-column liquid phase separation technologies.     

Analysis of the performance of antibody capture methods using fluorescent peptides with capillary zone electrophoresis with laser-induced fluorescence

A method to analyze the performance of an antibody capture method using fluorescent peptides by capillary zone electrophoresis using laser-induced fluorescence (CZE-LIF) for detection has been developed. Fluorescent peptides from the prion protein were synthesized and the corresponding antibodies were produced in rabbits against these peptides. The antibodies were used to capture the fluorescent peptides. The antibodies were then bound to protein A Sepharose. After elution, the amount of fluorescent peptide that was captured vs. the total amount placed in the assay was evaluated by CZE-LIF. Of the three peptides used in this evaluation, it was found that the recovery was approximately 25-35%. When the abnormal prion protein was prepared from scrapie-infected brain samples from hamsters and a sheep using the previously described extraction method and this method, the amount of abnormal prion protein that was measured in the fluorescence immunoassay correlated with amounts estimated from Western blot. We conclude that this method can be used to detect abnormal prion protein in a tissue sample.     

反相高效液相色谱法测定荧光增白剂CBS中N,N-二甲基甲酰胺的残留量

建立了反相高效液相色谱外标法检测荧光增白剂CBS(即CF-351)中N,N-二甲基甲酰胺(DMF)残留量的方法。使用Accurasil C18(150 mm×4.6 mm,5 μm)反相液相色谱柱,以甲醇-水为流动相梯度洗脱,流速为1.0 mL/min,检测波长为205 nm,柱温为30 ℃,进样量为5 μL。在上述条件下,在DMF质量浓度为0.19～141 mg/L范围内,其峰面积与质量浓度的线性关系良好。在加标水平（用质量分数表示）为0.2344%和0.4678%时,回收率为98.4%～107.3%。本方法最低检出限(LOD)为0.019%（质量分数）,峰面积的相对标准偏差(RSD)为1.73%。此方法可快速、准确地测定出荧光增白剂CBS中DMF的残留量。     

Comparative quantification of nucleic acids using single-molecule detection and molecular beacons

This paper reports a highly sensitive homogenous method for comparative quantification of nucleic acids based on single-molecule detection (SMD) and molecular beacons (MBs). Two different color MBs were used to perform a separation-free comparative hybridization assay for simultaneous quantification of both target and control strands. A fluorescent burst, emitted from a single hybrid when it passes through a minuscule laser-focused region, is detected with high signal-to-noise ratio (SNR) by using single-molecule fluorescence spectroscopy. Targets are quantified via counting of discrete fluorescent bursts. The high SNR achieved in both detection channels overcame the complications of fluorescent variability usually observed in dual-color ensemble measurements. In comparison with the conventional ensemble methods, this method improved the detection limit by 3 orders of magnitude and reduced the probe consumption by 6 orders of magnitude, facilitating a highly sensitive approach for comparative quantification of nucleic acids and offering great promise for genomic quantification without amplification.     

Simultaneous particle counting and detecting on a chip

This paper reports a lab-on-a-chip device that performs particle detection and number counting by coupling the fluorescent detection and particle counting simultaneously. The particle number counting is realized by a resistive pulse sensor (RPS) and fluorescent particle detection is achieved by a miniaturized laser-fiber optic detection system. By using a single microfluidic channel with two detecting arm channels placed at the two ends of the sensing section, the RPS signal-to-noise ratio is improved significantly. Two-stage differential amplification is used to further increase the signal-to-noise ratio for both the RPS and fluorescent signals. This method is also highly sensitive, so that we were able to realize the RPS and fluorescent detection of 0.9 microm (mean diameter) fluorescent particles. Excellent agreement was achieved by comparing the results obtained by our system with the results from a commercial flow cytometer for a variety of samples of mixed fluorescent and non-fluorescent particles. The method described in this paper is simple and can be applied to develop a compact device without the need of lock-in amplifier or similar bulky supplemental equipment.     

Thiourea functionalized CdSe/CdS quantum dots as a fluorescent sensor for mercury ion detection

CdSe/CdS quantum dots(QDs) functionalized by thiourea(TU) were synthesized and used as a fluorescent sensor for mercury ion detection.The TU-functionalized QDs were prepared by bonding TU via electrostatic interaction to the core/shell CdSe/CdS QDs after capping with thioglycolic acid(TGA).It was observed that the fluorescence of the functionalized QDs was quenched upon the addition of Hg~(2+).The quantitative detection of Hg~(2+) with this fluorescent sensor could be conducted based on the linear relationship between the extent of quenching and the concentration of Hg~(2+) added in the range of1-300 μg L~(-1).A detection limit of 0.56 μg L~(-1) was achieved.The sensor showed superior selectivity for Hg~(2+) and was successfully applied to the determination of mercury in environmental samples with satisfactory results.     

Fluorescent sensor for Cu2+ with a tunable emission wavelength

A concept of fluorescent metal ion sensing with an easily tunable emission wavelength is presented and its principle demonstrated by detection of Cu(2+). A fluorescein dye was chemically modified with a metal chelating group and then attached to the terminus of ss-DNA. This was combined with a complementary ss-DNA modified with another fluorescent dye (ATTO 590), emitting at a longer wavelength. In the assembled duplex, fluorescence resonance energy transfer (FRET) between the fluorescein donor (excited at 470 nm) and the ATTO 590 acceptor (emitting at 624 nm) is observed. Proper positioning within the rigid DNA double helix prevents intramolecular contact quenching of the two dyes. Coordination of paramagnetic Cu(2+) ions by the chelating unit of the sensor results in direct fluorescence quenching of the fluorescein dye and indirect (by loss of FRET) quenching of the ATTO 590 emission at 624 nm. As a result, emission of the acceptor dye can be used for monitoring of the concentration of Cu(2+), with a 20 nM detection limit. The emission wavelength is readily tuned by replacement of ATTO-DNA by other commercially available DNA-acceptor dye conjugates. Fluorescent metal ion sensors emitting at >600 nm are very rare. The possibility of tuning the emission wavelength is important with respect to the optimization of this sensor type for application to biological samples, which usually show broad autofluorescence at <550 nm.     

Hexaplex PCR assay and liquid bead array for detection of stacked genetically modified cotton event 281-24-236×3006-210-23

A hexaplex system based on multiplex polymerase chain reaction (PCR) coupled with liquid bead array was developed to assist detection of stacked genetically modified (GM) cotton event 281-24-236 × 3006-210-23 (Widestrike) expressing two kinds of endotoxin from Bacillus thuringiensis (Bt). The efficiency of this multiplex detection system was assessed. Specific primer sets for simultaneous detection of six targets in the stacked GM cotton event were constructed and used for the PCR assay. Each of the six targets was amplified, and the amplicons could be separated as discrete bands by agarose gel electrophoresis. A liquid bead array assay for the stacked GM cotton was performed using the hexaplex PCR products followed by hybridization between the biotinylated targets and anti-tagged microsphere beads. The hybridization products produced fluorescent signals that were detected by the Luminex system. Signal strengths were analyzed by their median fluorescent intensity values. Comparison of the assays showed that results from the liquid bead array using specific probes agreed with those from the PCR, and detection of the different target elements was found to be very specific with no cross-reaction. Therefore, the combination of hexaplex PCR and liquid bead array for detection of stacked GM events can be a useful and efficient system for screening and analyzing multiple transgenes for simultaneous qualitative analysis.     

Modification of aniline containing proteins using an oxidative coupling strategy

A new bioconjugation reaction has been developed based on the chemoselective modification of anilines through an oxidative coupling pathway. Aryl amines were installed on the surface of protein substrates through lysine acylation reactions or through the use of native chemical ligation techniques. Upon exposure to NaIO4 in aqueous buffer, the anilines coupled rapidly to the aromatic rings of N,N-dialkyl-N'-acyl-p-phenylenediamines. The identities of the reaction products were confirmed using ESI-MS and through comparison to small molecule analogs. Control experiments indicated that none of the native amino acids participated in the reaction. The resulting bioconjugates were found to be stable toward hydrolysis from pH 4 to pH 11 and in the presence of many commonly used oxidants, reductants, and nucleophiles. A fluorescent phenylenediamine reagent was synthesized for the selective detection of aniline labeled proteins in mixtures, and the reaction was used to append the C-terminus of the green fluorescent protein with a single PEG chain. When combined with techniques for the incorporation of unnatural amino acids into proteins, this bioorthogonal coupling method should prove useful for a number of applications requiring a high degree of labeling specificity.     

Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel

A microfluidic and optical system was created for the detection and analysis of single molecules in solution. Fluidic channels with submicrometer dimensions were used to isolate, detect and identify individual quantum dots conjugated with organic fluorophores. The channels were fabricated in fused silica with a 500 nm square cross section. The resulting focal volume of approximately 500 aL reduced fluorescent background and increased the signal to noise ratio of single molecule detection. The channels also enabled the rapid detection of 99% of quantum dots and organic fluorophores traversing the focal volume. Conjugates were driven through the channels electrokinetically at 2.3 kV cm(-1), excited with a single 476 nm wavelength laser and detected with a confocal microscope. Fluorescence emission was collected simultaneously from green (500-590 nm) and red (610-680 nm) regions of the spectrum. Signal rejection was minimized by the narrow and symmetric emission spectra of the quantum dots. To demonstrate efficient multicolor detection and characterization of single molecule binding, Qdot 655 Streptavidin Conjugates were bound to Alexa Fluor 488 molecules and individually detected. Photon counting histogram analysis was used to quantify coincident detection and degree of binding. Fluorescence correlation spectroscopy was used to measure the mobility of bound and unbound species. The union of fluidic channels with submicrometer dimensions and quantum dots as fluorescent labels resulted in efficient and rapid multiplexed single molecule detection and analysis.