化学发光检测在微流控芯片中的应用综述

综述了近年来化学发光检测在微流控芯片中的应用.指出微流控芯片(又称为"芯片实验室"或者"微型全分析系统")因具有小型化、集成化和自动化等特点而在近20年来日益受到关注,而化学发光检测具有仪器结构简单、背景噪音低、操作和维护成本低等优点,非常适合用作微流控芯片的检测手段.     

Microfluidics technology for manipulation and analysis of biological cells

Analysis of the profiles and dynamics of molecular components and sub-cellular structures in living cells using microfluidic devices has become a major branch of bioanalytical chemistry during the past decades. Microfluidic systems have shown unique advantages in performing analytical functions such as controlled transportation, immobilization, and manipulation of biological molecules and cells, as well as separation, mixing, and dilution of chemical reagents, which enables the analysis of intracellular parameters and detection of cell metabolites, even on a single-cell level. This article provides an in-depth review on the applications of microfluidic devices for cell-based assays in recent years (2002–2005). Various cell manipulation methods for microfluidic applications, based on magnetic, optical, mechanical, and electrical principles, are described with selected examples of microfluidic devices for cell-based analysis. Microfluidic devices for cell treatment, including cell lysis, cell culture, and cell electroporation, are surveyed and their unique features are introduced. Special attention is devoted to a number of microfluidic devices for cell-based assays, including micro cytometer, microfluidic chemical cytometry, biochemical sensing chip, and whole cell sensing chip.     

A coulometric flow cell for in-line generation of reagent, titrant or standard solutions

A universal and easily ON–OFF time operated coulometric flow cell for generation of chemicals is proposed. The cell can be used for generation of reagents, titrants or standard solutions, and can be coupled to analytical equipments in combination with flow systems for generation of micro-quantity amounts of chemicals in a wide concentration range. Platinum wire electrodes and other common laboratory available materials were employed in the cell assembly. The application of the cell for analytical purposes was illustrated by employing it for generating triiodide ions as a carrier in a flow injection analysis system applied to the analysis of dipyrone in pharmaceuticals, and as a titrant in a flow-batch analysis system applied for analysis of ascorbic acid in natural orange juice samples. Preliminary studies demonstrate that such a flow cell is suitable to allow the use of unstable reagents in analytical chemistry, since they can be generated in a closed system and promptly used in subsequent reactions. In addition, it offers advantages of saving chemicals, time and specially the laborious calibrating standard solutions preparation task of routine laboratories. Taking in account its analytical performance, the proposed cell promises to be an important tool in analytical chemistry with a great competition potential as compared to the other ones proposed in the literature.     

Analysis Laboratory on a Chip

Microfluidic chip offers a promising platform for chemical or biological analysis on the basis of flexible integration of various functional operation units. This article provides an overview of the recent achievements of microfluidic chip and its applications based on the works mainly carried out in the authors' lab, especially for the purpose of constructing analytical laboratory on a microfluidic chip. Different operation units and some representative applications in molecules, cell and organism analysis are described.     

Stopped-flow injection simultaneous determination of phosphate and silicate using molybdenum blue

Kinetic information for the phosphate–molybdate–ascorbic acid reaction can be obtained by making use of a very simple manually operated stopped-flow injection (FI) system. Various parameters (concentrations of reagents, flow rate, mixing coils, and volume of flow cell) were investigated for determination of phosphate. A stopped-FI system should be arranged for low degree of mixing (of reactants) and low dispersion so that good signals of rate changes will be observed. Simultaneous determination of phosphate and silicate by the stopped-FI technique is proposed, using a laboratory-made semi-automatic stopped-FI Analyzer with LED-based photometer. It is based on kinetic separation of phosphate and silicate using molybdenum blue. The proposed procedure has been demonstrated for the application to water samples. The results obtained agree with that of a standard method.     

Immunobiosensor detection of domoic acid as a screening test in bivalve molluscs: comparison with liquid chromatography-based analysis

A rapid and sensitive immuno-based screening method was developed to detect domoic acid (DA) present in extracts of shellfish species using a surface plasmon resonance-based optical biosensor. A rabbit polyclonal antibody raised against DA was mixed with standard or sample extracts and allowed to interact with DA immobilized onto a sensor chip surface. The characterization of the antibody strongly suggested high cross-reactivity with DA and important isomers of the toxin. The binding of this antibody to the sensor chip surface was inhibited in the presence of DA in either standard solutions or sample extracts. The DA chip surface proved to be highly stable, achieving approximately 800 analyses per chip without any loss of surface activity. A single analytical cycle (sample injection, chip regeneration, and system wash) took 10 min to complete. Sample analysis (scallops, mussels, cockles, oysters) was achieved by simple extraction with methanol. These extracts were then filtered and diluted before analysis. Detection limits in the ng/g range were achieved by the assay; however, the assay parameters chosen allowed the test to be performed most accurately at the European Union's official action limit for DA of 20 microg/g. At this concentration, intra- and interassay variations were measured for a range of shellfish species and ranged from 4.5 to 7.4% and 2.3 to 9.7%, respectively.     

Allergen diagnosis microarray with high-density immobilization capacity using diamond-like carbon-coated chips for profiling allergen-specific IgE and other immunoglobulins

The diagnosis of antibody-mediated allergic disorders is based on clinical findings, skin prick tests and detection of allergen-specific IgE in serum. Here, we present a new microarray technique of high-density antigen immobilization using carboxylated arms on the surface of a diamond-like carbon (DLC)-coated chip. High immobilization capacity of antigen on DLC chip at (0.94–7.82) × 10⁹ molecules mm⁻² allowed the analysis of allergen-specific immunoglobulins against not only purified proteins but also natural allergen extracts with wide assay dynamic range. The higher sensitivity of the allergen-specific IgE detection on DLC chip was observed for comparison with the UniCAP system: the DLC chip allowed lowering the limit of dilution rate in UniCAP system to further dilution at 4–8-fold. High correlations (ρ > 0.9–0.85) of allergen-specific IgE values determined by the DLC chip and UniCAP were found in most of 20 different allergens tested. The DLC chip was useful to determine allergen-induced antibodies of IgA, IgG, IgG1, and IgG4 in sera, apart from IgE, as well as secretory IgA in saliva against the same series of allergens on the chip in a minimal amount (1–2 μL) of sample.     

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.     

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.     

Determination of sulfur components in natural gas: A review

More stringent environmental regulations as well as higher demands presently being imposed on the sulfur content of natural gas feed-stocks for chemical processes necessitate the development of new analytical procedures for sulfur determination in natural gas. Only analytical procedures based on gas chromatography can meet the sensitivity and accuracy requirements dictated by environmental regulation institutions and modern chemical industry. The complexity of the natural gas matrix as well as the extremely low concentration levels at which the sulfur species occur make the development of these analytical methods a true challenge. In this review the three steps common for analytical methods for trace analysis in complex matrices, i.e. pretreatment, chromatographic separation, and detection, are discussed in detail. Possible methods for calibration of the system are discussed in the final section. Various techniques to determine sulfur in natural gas are described. Depending on the application, the most suitable system has to be selected. For example, for on-line application in a hazardous area a simple and rugged system is required, i.e. a simple gas chromatograph with a flame photometric detector, while for laboratory application a more complex instrument including preconcentration, column switching, and more exotic detection systems could be more suitable. Therefore it is crucial to define the requirements of the instrument at an early stage and use the information in this review article to develop/select a dedicated instrument/procedure for the problem at hand.     

A valve‐based microfluidic device for on‐chip single cell treatments

Assays toward single‐cell analysis have attracted the attention in biological and biomedical researches to reveal cellular mechanisms as well as heterogeneity. Yet nowadays microfluidic devices for single‐cell analysis have several drawbacks: some would cause cell damage due to the hydraulic forces directly acting on cells, while others could not implement biological assays since they could not immobilize cells while manipulating the reagents at the same time. In this work, we presented a two‐layer pneumatic valve‐based platform to implement cell immobilization and treatment on‐chip simultaneously, and cells after treatment could be collected non‐destructively for further analysis. Target cells could be encapsulated in sodium alginate droplets which solidified into hydrogel when reacted with Ca²⁺. The size of hydrogel beads could be precisely controlled by modulating flow rates of continuous/disperse phases. While regulating fluid resistance between the main channel and passages by the integrated pneumatic valves, on‐chip capture and release of hydrogel beads was implemented. As a proof of concept for on‐chip single‐cell treatments, we showed cellular live/dead staining based on our devices. This method would have potential in single cell manipulation for biochemical cellular assays.     

Creating Complex Polyacrylamide Hydrogel Structures Using 3D Printing with Applications to Mechanobiology

Due to its favorable physical and chemical properties, including chemical inertness, low fouling by biological molecules, high porosity and permeability, optical transparency, and adjustable elasticity, polyacrylamide has found a wide range of biomedical and non‐biomedical applications. To further increase its versatility, this communication describes a simple method, using readily available reagents and equipment, for 3D printing polyacrylamide hydrogels at a resolution of 100–150 μm to create complex structures. As a demonstration of the application, the method is used for creating a lab‐on‐a‐chip cell culture surface with micropatterned stiffness, which then leads to the discovery of stiffness‐guided collective cell segregation distinct from durotaxis. The present technology is expected to unleash new applications such as the construction of biocompatible elastic medical devices and artificial organs.     

Some recent developments on cost-effective flow-based analysis

This paper reviews some recent developments on cost-effective flow-based analysis. They include the newly developed Lab-at-Valve (LAV), concepts in using the stopped-flow injection approach, on-line sample pretreatment systems, including bead injection–flow injection and flow injection–ion-chromatography, systems for size-based speciation, and cost-effective reagents. Applications and advantages of such techniques are discussed.     

Development of a PDMS‐based microchip electrophoresis device for continuous online in vivo monitoring of microdialysis samples

A PDMS‐based microfluidic system for online coupling of microdialysis sampling to microchip electrophoresis with fluorescence detection for in vivo analysis of amino acid neurotransmitters using naphthalene‐2,3‐dicarboxaldehyde and sodium cyanide as the derivatization reagents is described. Fabricating chips from PDMS rather than glass was found to be simpler and more reproducible, especially for chips with complex designs. The microchip incorporated a 20‐cm serpentine channel in which sample plugs were introduced using a “simple” injection scheme; this made fluid handling and injection on‐chip easier for the online system compared with gated or valve‐based injection. The microchip was evaluated offline for the analysis of amino acid standards and rat brain microdialysis samples. Next, precolumn derivatization was incorporated into the chip and in vivo online microdialysis‐microchip electrophoresis studies were performed. The system was employed for the continuous monitoring of amino acid neurotransmitters in the extracellular fluid of the brain of an anesthetized rat. Fluorescein was dosed intravenously and monitored simultaneously online as a marker of in vivo blood–brain barrier permeability. The microdialysis‐microchip electrophoresis system described here will be employed in the future for simultaneous monitoring of changes in blood–brain barrier permeability and levels of amino acid neurotransmitters in the rat stroke model.     

Droplet-based microfluidics for dose–response assay of enzyme inhibitors by electrochemical method

A simple but robust droplet-based microfluidic system was developed for dose–response enzyme inhibition assay by combining concentration gradient generation method with electrochemical detection method. A slotted-vials array and a tapered tip capillary were used for reagents introduction and concentration gradient generation, and a polydimethylsiloxane (PDMS) microfluidic chip integrated with microelectrodes was used for droplet generation and electrochemical detection. Effects of oil flow rate and surfactant on electrochemical sensing were investigated. This system was validated by measuring dose–response curves of three types of acetylcholinesterase (AChE) inhibitors, including carbamate pesticide, organophosphorus pesticide, and therapeutic drugs regulating Alzheimer's disease. Carbaryl, chlorpyrifos, and tacrine were used as model analytes, respectively, and their IC₅₀ (half maximal inhibitory concentration) values were determined. A whole enzyme inhibition assay was completed in 6 min, and the total consumption of reagents was less than 5 μL. This microfluidic system is applicable to many biochemical reactions, such as drug screening and kinetic studies, as long as one of the reactants or products is electrochemically active.     

A microfluidic-based system for analysis of single cells based on Ca2+ flux

A microfluidic format-based system has been developed for in situ monitoring of the calcium flux response to agonists using Chinese hamster ovary (CHO) cells. The assay is based on measuring the fluorescent intensity of the calcium-sensitive indicator, Fluo-4 AM, and was performed in a modified glass chip channel, whose surface was functionalised using a silanisation method with 3-aminopropyltriethoxysilane (APTS) (enabling the cells to be immobilised on the channel surface). CHO cells calcium flux response was measured for different agonists over a range of concentrations. Cells and reagents were introduced into the chip in a continuous flow as a series of plugs in a given sequence.     

Synthesis and analysis of combinatorial libraries performed in an automated micro reactor system

This paper presents the synthesis of combinatorial libraries performed on a single-channel glass micro reactor under hydrodynamic flow control. The experiments were carried out in a non-well based micro chip and consisted of the preparation of libraries of pyrazoles by means of a Knorr reaction of 1,3-dicarbonyl compounds with hydrazines. The aim of this work is to investigate the capabilities of an automated micro reactor based system to synthesise sequentially multiple analogue reactions. Small slugs of reactants were introduced automatically by an autosampler in a serpentine-etched glass chip. The mobility of the reagents and products was achieved using hydrodynamic driven flow. Reaction slug dilution and UV slug detection took place at the outlet. A sample of the slug was analysed by using an on-line LC-UV-MS system. The degree of conversion was quantified using the UV signal and comparing with standards of starting materials and final products. After the LC-UV-MS analysis, the automated system proceeds to inject the slugs to carry out the next reaction programmed. The results suggest that the micro reactor system is capable of repeating the process of injection, mixing and reaction in an automated manner as many times as required.     

微流控技术在外泌体分离分析中的研究进展

外泌体是一类由细胞分泌的含有脂质、蛋白、核酸等多种物质的纳米级囊泡,主要参与细胞间的物质交换及信息传导,与多种疾病的发生发展密切相关。对外泌体进行深入研究,理解其生物学功能,对疾病诊断与治疗具有重要意义。由于外泌体尺寸较小且密度和体液接近,想要对复杂生物样本中的外泌体进行分离与分析十分困难。传统的外泌体分离方法如超速离心、超滤等大都需要借助大型仪器设备,且耗时长、操作复杂。因此迫切需要开发高效、便捷的外泌体分离检测手段。微流控技术因其微型化、高通量、可集成等特点,为外泌体的分离分析提供了一个新的平台。该文主要对近年来微流控技术在外泌体分离分析相关领域的研究进展进行了综述。重点从外泌体物理特性和生化特性两个角度出发,介绍了微流控芯片技术用于外泌体分离领域的主要原理、策略和方法。此外,还介绍了微流控技术与荧光、电化学传感、表面等离子体共振等多模态检测方法结合,实现外泌体一体化分析的新进展。最后,该文分析了目前微流控技术用于外泌体分离检测存在的挑战,并对其发展趋势和前景进行了展望。随着微流控外泌体分离分析装置的不断微型化、集成化、自动化,微流控芯片技术将在外泌体分离、生化检测、机制研究等方面将发挥越来越重要的作用。     

Integrated electrofluidic circuits: pressure sensing with analog and digital operation functionalities for microfluidics

Microfluidic technology plays an essential role in various lab on a chip devices due to its desired advantages. An automated microfluidic system integrated with actuators and sensors can further achieve better controllability. A number of microfluidic actuation schemes have been well developed. In contrast, most of the existing sensing methods still heavily rely on optical observations and external transducers, which have drawbacks including: costly instrumentation, professional operation, tedious interfacing, and difficulties of scaling up and further signal processing. This paper reports the concept of electrofluidic circuits - electrical circuits which are constructed using ionic liquid (IL)-filled fluidic channels. The developed electrofluidic circuits can be fabricated using a well-developed multi-layer soft lithography (MSL) process with polydimethylsiloxane (PDMS) microfluidic channels. Electrofluidic circuits allow seamless integration of pressure sensors with analog and digital operation functions into microfluidic systems and provide electrical readouts for further signal processing. In the experiments, the analog operation device is constructed based on electrofluidic Wheatstone bridge circuits with electrical outputs of the addition and subtraction results of the applied pressures. The digital operation (AND, OR, and XOR) devices are constructed using the electrofluidic pressure controlled switches, and output electrical signals of digital operations of the applied pressures. The experimental results demonstrate the designed functions for analog and digital operations of applied pressures are successfully achieved using the developed electrofluidic circuits, making them promising to develop integrated microfluidic systems with capabilities of precise pressure monitoring and further feedback control for advanced lab on a chip applications.     

Accurate dispensing of volatile reagents on demand for chemical reactions in EWOD chips

Digital microfluidic chips provide a new platform for manipulating chemicals for multi-step chemical synthesis or assays at the microscale. The organic solvents and reagents needed for these applications are often volatile, sensitive to contamination, and wetting, i.e. have contact angles of <90° even on the highly hydrophobic surfaces (e.g., Teflon? or Cytop?) typically used on digital microfluidic chips. Furthermore, often the applications dictate that the processes are performed in a gas environment, not allowing the use of a filler liquid (e.g., oil). These properties pose challenges for delivering controlled volumes of liquid to the chip. An automated, simple, accurate and reliable method of delivering reagents from sealed, off-chip reservoirs is presented here. This platform overcomes the issues of evaporative losses of volatile solvents, cross-contamination, and flooding of the chip by combining a syringe pump, a simple on-chip liquid detector and a robust interface design. The impedance-based liquid detection requires only minimal added hardware to provide a feedback signal to ensure accurate volumes of volatile solvents are introduced to the chip, independent of time delays between dispensing operations. On-demand dispensing of multiple droplets of acetonitrile, a frequently used but difficult to handle solvent due to its wetting properties and volatility, was demonstrated and used to synthesize the positron emission tomography (PET) probe [(18)F]FDG reliably.