On the performance of bioanalytical fluorescence correlation spectroscopy measurements in a multiparameter photon-counting microscope

Fluorescence correlation spectroscopy (FCS) data acquisition and analysis routines were developed and implemented in a home-built, multiparameter photon-counting microscope. Laser excitation conditions were investigated for two representative fluorescent probes, Rhodamine110 and enhanced green fluorescent protein (EGFP). Reliable local concentrations and diffusion constants were obtained by fitting measured FCS curves, provided that the excitation intensity did not exceed 20% of the saturation level for each fluorophore. Accurate results were obtained from FCS measurements for sample concentrations varying from pM to μM range, as well as for conditions of high background signals. These experimental constraints were found to be determined by characteristics of the detection system and by the saturation behavior of the fluorescent probes. These factors actually limit the average number of photons that can be collected from a single fluorophore passing through the detection volume. The versatility of our setup and the data analysis capabilities were tested by measuring the mobility of EGFP in the nucleus of Drosophila cells under conditions of high concentration and molecular crowding. As a bioanalytical application, we studied by FCS the binding affinity of a novel peptide-based drug to the cancer-regulating STAT3 protein and corroborated the results with fluorescence polarization analysis derived from the same photon data.     

Real time automatic detection of Orcinus orca vocalizations in a controlled environment

The use of passive acoustic observation is a useful tool in bioacoustic studies of cetaceans, such as killer whales (Orcinus orca). Such studies require the recording, detection, and classification of vocalizations of animals during long periods of time. The manual processing of the recordings is an extremely time consuming task because of the large amount of data to process. Automatic detection and classification techniques are useful to improve the processing, increase the amount of information and, as a consequence, provide information for the conservation of these species.The Orca Ocean facilities of Loro Parque in the Canary Islands, Spain, were used as an experimental platform for developing devices to perform bioacoustic studies. Detection methods with low computational complexity were tested in order to capture vocalizations of four O. orca specimens in real time. The algorithms were also tested in other scenarios in order to determine their global performance. The sensitivity to noise was also tested. The most accurate method in this study was implemented and integrated with a continuous recording system generating an event database in real time, downsizing the storage demand to 7%. This allowed the storage of all the sound events produced in about one month on a standard computer harddisk and the generation of basic statistics on vocal activity of the animals.     

Characterization of new types of stationary phases for fast and ultra-fast liquid chromatography by signal processing based on AutoCovariance Function: A case study of application to Passiflora incarnata L. extract separations

In this study, a comparative investigation was performed of HPLC Ascentis^® (2.7 μm particles) columns based on fused-core particle technology and Acquity^® (1.7 μm particles) columns requiring UPLC instruments, in comparison with Chromolith™ RP-18e columns. The study was carried out on mother and vegetal tinctures of Passiflora incarnata L. on one single or two coupled columns. The fundamental attributions of the chromatographic profiles are evaluated using a chemometric procedure, based on the AutoCovariance Function (ACVF). Different chromatographic systems are compared in terms of their separation parameters, i.e., number of total chemical components (m_tot), separation efficiency (σ), peak capacity (n_c), overlap degree of peaks and peak purity. The obtained results show the improvements achieved by HPLC columns with narrow size particles in terms of total analysis time and chromatographic efficiency: comparable performance are achieved by Ascentis^® (2.7 μm particle) column and Acquity^® (1.7 μm particle) column requiring UPLC instruments. The ACVF plot is proposed as a simplified tool describing the chromatographic fingerprint to be used for evaluating and comparing chemical composition of plant extracts by using the parameters D% – relative abundance of the deterministic component – and c_EACF – similarity index computed on ACVF.     

Synthesis of 2-substituted 2H-chromenes using potassium vinyltrifluoroborates

2H-Chromenes were synthesized from salicylaldehyde using potassium vinylic borates in the presence of secondary amines. We synthesized these 2H-chromene derivatives as a part of an ongoing project to develop inhibitors for TGF-β receptors. Potassium vinyl trifluoroborates react with salicylaldehydes at 80 °C in the presence of a secondary amine and produced 2-substituted 2H-chromene derivatives with a 70-90% yield.     

A fast and sensitive enzyme immunoassay for brain natriuretic peptide based on micro-magnetic probes strategy

In this paper, a simple, rapid and low-cost method for the high-sensitivity detection of brain natriuretic peptide (BNP) was developed, which adopted three amplification steps: (a) biotin-streptavidin amplification; (b) micro-magnetic probe amplification; (c) HRP (horseradish peroxidase) signal amplification. In the present strategy, the streptavidin-coated micro-magnetic particles (MMPs) were first conjugated with biotin-labeled capture antibody via the biotin-streptavidin interaction, which formed bio-functional micro-magnetic probes. Then, the analyte (antigen) is sandwiched by HRP-labeled antibody and capture antibody bound to MMPs. Finally, the HRP at the surface of sandwich structures catalytically oxidized the substrate and generated optical signals that reflected the amount of the target BNP. The influence of some important parameters such as the size of magnetic particles, the working concentration of HRP-labeled BNP antibody, the stability of magnetic probes, and the assay medium of serum BNP, etc. on the detection ability of present method was investigated in details. It is found that the detection limit of the proposed method could reach 10 pg/mL for BNP, which is much lower than that of sandwich-type ELISA. Furthermore, this detection time for the proposed method just takes about 30 min (two reaction steps and one wash step), which is faster than that of conventional sandwich-type ELISA (taking about 4 h, three reaction steps and three wash steps). Inspired by these advantages, it is expected that this method can probably be applicable to the detection of other hormones and tumor markers that are present in only low concentrations within the human body.     

A method based on stochastic resonance for the detection of weak analytical signal

An effective method for detection of weak analytical signals with strong noise background is proposed based on the theory of stochastic resonance (SR). Compared with the conventional SR-based algorithms, the proposed algorithm is simplified by changing only one parameter to realize the weak signal detection. Simulation studies revealed that the method performs well in detection of analytical signals in very high level of noise background and is suitable for detecting signals with the different noise level by changing the parameter. Applications of the method to experimental weak signals of X-ray diffraction and Raman spectrum are also investigated. It is found that reliable results can be obtained.     

等离子体原子荧光光谱仪激发光源空心阴极灯脉冲发生和信号处理控制系统的研制和评估

本文用带３８６处理器的微型计算机,通过自行研制的接口,在ＢａｉｒｄＰｌａｓｍａＡＦＳ－２０００原子荧光光谱仪上实现了荧光激发光源空心阴极灯供电脉冲发生和信号处理的控制。典型元素的检测限、相对标准偏差和线性相关系数表明,系统性能良好。新系统增强了仪器的功能。