生物分析中近红外荧光探针进展

本文评述了10余年来方酸菁类、花菁类、噻嗪类、E嗪类、罗丹明类、BODIPY及酞菁等近红外荧光探针的发展。按其种类分别讨论了试剂结构与性能之间的关系,并介绍了它们在生物分析中的某些应用。引用文献42篇。     

Two new techniques for sample preparation in bioanalysis: Microextraction in packed sorbent (MEPS) and use of a bonded monolith as sorbent for sample preparation in polypropylene tips for 96-well plates

Analytical methods providing high throughput are required for the ever increasing number of samples in bioanalysis. Currently, the method of choice in bioanalysis is LC-MS-MS. This method is quite rapid and thereby the focus has been directed to sample preparation as being a bottleneck in total analysis systems. It has become necessary to develop sample preparation techniques to a new improved level. This development has been based on a systematic and scientific approach. The key factors in this development have been miniaturization, integration, and automation of the techniques. This review provides a short overview of recent developments. Special emphasis is on two techniques: microextraction in packed syringe (MEPS) and use of a monolithic acrylamide plug as sorbent in polypropylene tips primarily intended for use with 96-well plate systems.     

FloDots: luminescent nanoparticles

Luminescent dye-doped silica nanoparticles (FloDots) have been developed for ultrasensitive bioanalysis and diagnosis in the past several years. Those novel nanoparticles are highly luminescent and extremely photostable. In this paper, we review the preparation, characterization, bioconjugation and bioapplication of FloDots. All the results clearly demonstrated that FloDots have many advantages over currently used luminescent probes, such as traditional fluorophores and quantum dots.     

Liquid chromatographic methods for the quantification of catecholamines and their metabolites in several biological samples—A review

The measurement of catecholamines and their metabolites in biological samples remains a current analytical challenge, in spite of the great diversity of methodologies that have been developed throughout the years. High-performance liquid chromatography is the standard method for their separation and quantification in biological samples, either coupled with electrochemical, fluorescence, chemiluminescence or mass spectrometry detection. This review summarizes the most important physicochemical properties of catecholamines, the wide panoply of sample preparation techniques and the main issues to consider during the development of chromatographic methods. The major difficulties encountered during the optimization of these procedures are related with the high tendency of catecholamines to oxidize and the very low quantities at which they exist in biological matrices. Herein, the most important aspects that ought to be considered during collection, treatment and storage of fluid and tissue samples intended for catecholamine analysis are underlined, the chromatographic conditions are compared and the technical advantages and limitations of each detection system are discussed.     

Comparison of a new high‐resolution monolithic column with core‐shell and fully porous columns for the analysis of retinol and α‐tocopherol in human serum and breast milk by ultra‐high‐performance liquid chromatography†

The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high‐throughput assay methods in bioanalysis. It can be achieved either by ultra‐high‐performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction—monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second‐generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core‐shell and fully porous sub‐2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice.     

UV-ablation nanochannels in micro/nanofluidics devices for biochemical analysis

This paper presents a simple and cost-effective UV-ablation technique for fabrication of size-tunable nanofluidics devices via photochemical decomposition reaction. UV-irradiation through a PET photomask results in continuous decomposition of poly(carbonate) (PC), forming nanochannel and carboxyl groups on the surface of the etched PC. This photochemical decomposition process occurs at molecular scale, therefore, the depth of nanochannels can be controlled at nanometer level. The etching rate is estimated to be ca. 0.015 nm s⁻¹. To demonstrate the potential application of the present UV-ablation technique, a nanochannel was fabricated and integrated with microchannels to form a micro/nanofluidics chip for protein concentration. Using this device, about 10³-10⁵ fold protein concentration can be achieved within 10 min. The present approach offers a simple and practical solution to fabricate nanofluidics devices at low-cost, and the resulting device could provide ideal platforms for μTAS towards various applications in biology and chemistry.     

Relative matrix effects: A step forward using standard line slopes and ANOVA analysis

One of the alternative methods to identify and study the matrix effect is by determination of “relative” matrix effect. In this experiment % coefficient of variance of standard line slopes are calculated. First, six standard lines are prepared from single plasma lot. In another experiment standard line slopes are compared from six different lots of plasma. All these standard curves are prepared by using three different types of IS (internal standard). From all these experiments it is observed that using SIL-IS (stable isotope labeled-internal standard) is one of the best approach in methods having matrix effects. Alternatively, analog IS is a cost effective approach. After comparing a large number of calibration curve slopes, it can be recommended that during every bioanalytical method validation, where the sample size is >50, scientist should perform the “relative” matrix effect experiment by standard line slope method. In selected cases, the precision of standard line slopes in six different lots of a biofluid was compared with precision values determined six times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 5% for the method to be considered reliable and free from the relative matrix effect liability.     

Analytical and bioanalytical applications of carbon dots

Carbon dots (CDs) comprise a recently discovered class of strongly fluorescent, emission-color-tuning and non-blinking nanoparticles with great analytical and bioanalytical potential. Raw CDs can be obtained by laser ablation or electrochemical exfoliation of graphite, from soot, or thermal carbonization, acid dehydration or ultrasonic treatment of molecular precursors. Passivation of raw CDs makes them fluorescent and their functionalization confers reactivity towards selected targets. CDs can be excited by single-photon (ultraviolet or near-ultraviolet) and multi-photon (red or near-infrared) excitation, and their luminescence properties are due to surface defects. CDs are being proposed as bioimaging probes because they comprise non-toxic elements and are biocompatible. Passivated and functionalized CDs can be made to sense pH, metal ions and molecular substances.     

Bioanalytical applications of fast capillary electrophoresis

Capillary electrophoresis is unique among liquid-phase separations in its utility for fast separations. Development of technology such as optical-gating, flow-gating, and microfabrication has allowed separations on the millisecond time scale to be developed. The fast separation times place great demands on the detector systems, frequently requiring detection limits below 1 amol to be practical. The development of such fast separations has opened many new applications not previously feasible for separations-based methods. This has included real time chemical monitoring, detection of short-lived species such as protein conformers or non-covalent complexes, and rapid multi-dimensional separations. Other applications currently being developed include high-throughput assays for clinical laboratories or screening combinatorial libraries. This review covers recent developments in the instrumentation for fast CE and some of the applications.