微流控液滴技术及其应用的研究进展

微液滴具有体积小、比表面积大,速度快、通量高,大小均匀、体系封闭,内部稳定等特性,在药物控释、病毒检测、颗粒材料合成、催化剂等领域中均有重要应用.微流控技术的发展为微液滴生成中实现尺寸规格、结构形貌和功能特性等的可控设计和精确操控提供了全新平台.本文概述了微流控液滴技术的基本原理、液滴生成方式及其基本操控,比较分析了微液滴的传统制备法与微流控合成法的异同,介绍了近年来微流控液滴技术在功能材料合成、生物医学和食品加工等领域中的研究新进展,探讨并展望了微流控液滴技术的潜在价值和未来发展方向.     

Analytical detection techniques for droplet microfluidics—A review

In the last decade, droplet-based microfluidics has undergone rapid progress in the fields of single-cell analysis, digital PCR, protein crystallization and high throughput screening. It has been proved to be a promising platform for performing chemical and biological experiments with ultra-small volumes (picoliter to nanoliter) and ultra-high throughput. The ability to analyze the content in droplet qualitatively and quantitatively is playing an increasing role in the development and application of droplet-based microfluidic systems. In this review, we summarized the analytical detection techniques used in droplet systems and discussed the advantage and disadvantage of each technique through its application. The analytical techniques mentioned in this paper include bright-field microscopy, fluorescence microscopy, laser induced fluorescence, Raman spectroscopy, electrochemistry, capillary electrophoresis, mass spectrometry, nuclear magnetic resonance spectroscopy, absorption detection, chemiluminescence, and sample pretreatment techniques. The importance of analytical detection techniques in enabling new applications is highlighted. We also discuss the future development direction of analytical detection techniques for droplet-based microfluidic systems.     

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100?000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.     

Lab-chip HPLC with integrated droplet-based microfluidics for separation and high frequency compartmentalisation

We demonstrate the integration of a droplet-based microfluidic device with high performance liquid chromatography (HPLC) in a monolithic format. Sequential operations of separation, compartmentalisation and concentration counter were conducted on a monolithic chip. This describes the use of droplet-based microfluidics for the preservation of chromatographic separations, and its potential application as a high frequency fraction collector.     

Droplet microfluidics: recent developments and future applications

We report recent advances in the field of droplet-based microfluidics. Specifically, we highlight the unique features of such platforms for high-throughput experimentation; describe functional components that afford complex analytical processing and report on applications in synthesis, high-throughput screening, cell biology and synthetic and systems biology. Issues including the integration of high-information content detection methods, long term droplet stability and opportunities for large scale and intelligent biological experimentation are also discussed.     

Surface-enhanced Raman scattering in nanoliter droplets: towards high-sensitivity detection of mercury (II) ions

We report a new method for the trace analysis of mercury (II) ions in water. The approach involves the use of droplet-based microfluidics combined with surface-enhanced Raman scattering (SERS) detection. This novel combination provides both fast and sensitive detection of mercury (II) ions in water. Specifically, mercury (II) ion detection is performed by using the strong affinity between gold nanoparticles and mercury (II) ions. This interaction causes a change in the SERS signal of the reporter molecule rhodamine B that is a function of mercury (II) ion concentration. To allow both reproducible and quantitative analysis, aqueous samples are encapsulated within nanoliter-sized droplets. Manipulation of such droplets through winding microchannels affords rapid and efficient mixing of the contents. Additionally, memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, are removed since the aqueous droplets are completely isolated by a continuous oil phase. Quantitative analysis of mercury (II) ions was performed by calculating spectral peak area of rhodamine B at 1,647 cm⁻¹. Using this approach, the calculated concentration limit of detection was estimated to be between 100 and 500 ppt. Compared with fluorescence-based methods for the trace analysis of mercury (II) ions, the detection sensitivities were enhanced by approximately one order of magnitude. The proposed analytical method offers a rapid and reproducible trace detection capability for mercury (II) ions in water.     

Droplet microfluidics

Droplet-based microfluidic systems have been shown to be compatible with many chemical and biological reagents and capable of performing a variety of "digital fluidic" operations that can be rendered programmable and reconfigurable. This platform has dimensional scaling benefits that have enabled controlled and rapid mixing of fluids in the droplet reactors, resulting in decreased reaction times. This, coupled with the precise generation and repeatability of droplet operations, has made the droplet-based microfluidic system a potent high throughput platform for biomedical research and applications. In addition to being used as microreactors ranging from the nano- to femtoliter range; droplet-based systems have also been used to directly synthesize particles and encapsulate many biological entities for biomedicine and biotechnology applications. This review will focus on the various droplet operations, as well as the numerous applications of the system. Due to advantages unique to droplet-based systems, this technology has the potential to provide novel solutions to today's biomedical engineering challenges for advanced diagnostics and therapeutics.     

微液滴微流控芯片:微液滴的形成、操纵和应用

微流控芯片中形成的微液滴粒径均一、可控,与传统的连续流体系相比,具有能实现试剂的快速混合、通量更高等优点.本文介绍了微流控芯片中由微通道控制的微液滴的形成、分裂、合并、混合、分选和捕获等微液滴操纵技术,以及微液滴技术在纳米粒子、聚合物微粒的合成、纳米粒子自组装、蛋白质结晶研究和DNA、细胞分析等领域的研究进展.     

High‐Performance Nucleic Acid Sensors for Liquid Biopsy Applications

Circulating tumour nucleic acids (ctNAs) are released from tumours cells and can be detected in blood samples, providing a way to track tumors without requiring a tissue sample. This “liquid biopsy” approach has the potential to replace invasive, painful, and costly tissue biopsies in cancer diagnosis and management. However, a very sensitive and specific approach is required to detect relatively low amounts of mutant sequences linked to cancer because they are masked by the high levels of wild‐type sequences. This review discusses high‐performance nucleic acid biosensors for ctNA analysis in patient samples. We compare sequencing‐ and amplification‐based methods to next‐generation sensors for ctDNA and ctRNA (including microRNA) profiling, such as electrochemical methods, surface plasmon resonance, Raman spectroscopy, and microfluidics and dielectrophoresis‐based assays. We present an overview of the analytical sensitivity and accuracy of these methods as well as the biological and technical challenges they present.     

Recent advances in droplet microfluidics for microbiology

Advances in microbiology rely on innovations in technology. Droplet microfluidics, as a versatile and powerful technique that allows high-throughput generation and manipulation of subnanoliter volume droplets, has become an indispensable tool shifting experimental paradigms in microbiology. Droplet microfluidics has opened new avenues to various microbiological research, from resolving single-cell heterogeneity to investigating spatiotemporal dynamics of microbial communities, from precise quant...     

Droplet-based microreactor for the production of micro/nano-materials

Droplet-based microreactors are of great interest to researchers due to their incredible ability in the synthesis of micro/nano-materials with multi-function and complex geometry. In recent years, a broad range of micro/nano-materials has been synthesized in droplet-based microreactors, which provide apparent advantages, such as better reproducibility, reliable automation, and accurate manipulation. In this review, we give a comprehensive and in-depth insight into droplet-based microreactors, covering fundamental research from droplet generation and manipulation to the applications of droplet-based microreactors in micro/nano-material generation. We also explore the outlook for droplet-based microreactors and challenges that lie ahead and give a possible effort direction. We hope this review will promote communications among researchers and entrepreneurs.     

Optical and electrochemical detection techniques for cell-based microfluidic systems

The ability to fabricate microfluidic systems with complex structures and with compatible dimensions between the microfluidics and biological cells have attracted significant attention in the development of microchips for analyzing the biophysical and biochemical functions of cells. Just as cell-based microfluidics have become a versatile tool for biosensing, diagnostics, drug screening and biological research, detector modules for cell-based microfluidics have also undergone major development over the past decade. This review focuses on detection methods commonly used in cell-based microfluidic systems, and provides a general survey and an in-depth look at recent developments in optical and electrochemical detection methods for microfluidic applications for biological systems, particularly cell analysis. Selected examples are used to illustrate applications of these detection systems and their advantages and weaknesses.     

Droplet microfluidics for the study of artificial cells

In this review, we describe recent advances in droplet-based microfluidics technology that can be applied in studies of artificial cells. Artificial cells are simplified models of living cells and provide valuable model platforms designed to reveal the functions of biological systems. The study of artificial cells is promoted by microfluidics technologies, which provide control over tiny volumes of solutions during quantitative chemical experiments and other manipulations. Here, we focus on current and future trends in droplet microfluidics and their applications in studies of artificial cells.     

纳米粒子在食源性致病菌检测中的应用进展

食品安全事关人民群众的身体健康和生命安全,而食源性致病菌是食品安全的主要影响因素。由食源性致病菌引起的疾病和死亡持续威胁着全球的公共卫生安全。因此,开发快速、准确且灵敏的食源性致病菌检测方法是预防食源性疾病暴发和确保食品安全的关键。常规检测方法费时费力,需要昂贵的设备和专业的人员,应用受限。近年来,随着纳米技术的快速发展,纳米粒子凭借其小尺寸、高比表面积和高反应活性等理化特性成为食源性致病菌检测领域的研究热点。此外,将识别元件修饰于纳米粒子表面并结合新颖的分析技术,能提高检测的特异性和灵敏度。该综述主要总结和比较了磁性纳米粒子、贵金属纳米粒子、荧光纳米粒子和二氧化硅纳米粒子在食源性致病菌检测中的应用,以期为食源性致病菌的快速分析提供思路。     

A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels

A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry.     

Applications of Modular Microfluidics Technology

Microfluidics has been widely used in the life science, analytical chemistry, environmental science and other fields in the recent years. Traditional microfluidics systems usually use a highly integrated system with multiple components for handling the fluid in the micro/nano scale. The design and fabrication of integrated microfluidics usually require highly sophisticated instruments and operation professionals. With the experience inherited from integrated circuit and micro electro mechanical system, the modular microfluidics system has been experienced a rapid development in recent years. Modular microfluidics system is a combination of a series of individual modules to achieve complicated liquid handling functions. Compared with conventional microfluidics approach, the modular microfluidics method has the potential in significantly reducing the fabrication cost by using the massive production of single chip, besides, it is easy to be operated, and the user can easily assembly the modules to obtain their customized microfluidics system. The concept of modular microfluidics also indicates the future development path for the standardization of microfluidics system and also provides a promising approach for the industrial massive production of microfluidics. However, the study of modular microfluidics is still in an early stage. Although lots of studies have been conducted with varies materials, fabrication methods and interface technologies, issues like modular interface still restricted the further development of microfluidics. In this paper, a comprehensive review for the latest research on the modular microfluidics and applications in biological and medical fields is provided, and the future research trends of modular microfluidics is also discussed.     

Opto-fluidic micro-ring resonator for sensitive label-free viral detection

We have demonstrated sensitive label-free virus detection using the opto-fluidic ring resonator (OFRR) sensor. The OFRR is a novel sensing platform that integrates the microfluidics and photonic sensing technology with a low detection limit and small volume. In our experiment, filamentous bacteriophage M13 was used as a safe model system. Virus samples were flowed through the OFRR whose surface was coated with M13-specific antibodies. We studied the sensor performance by monitoring in real-time the virus and antibody interaction. It is shown that OFRR can detect M13 with high specificity and sensitivity. The detection limit is approximately 2.3 x 10(3) pfu mL(-1) and the detection dynamic range spanned seven orders of magnitude. Theoretical analysis was also carried out to confirm the experimental results. Our study will lead to development of novel OFRR-based, sensitive, rapid, and low-cost micro total analysis devices for virus detection.     

A colorimetric sensor array for detection and discrimination of biothiols based on aggregation of gold nanoparticles

Developments of sensitive, rapid, and cheap systems for identification of a wide range of biomolecules have been recognized as a critical need in the biology field. Here, we introduce a simple colorimetric sensor array for detection of biological thiols, based on aggregation of three types of surface engineered gold nanoparticles (AuNPs). The low-molecular-weight biological thiols show high affinity to the surface of AuNPs; this causes replacement of AuNPs’ shells with thiol containing target molecules leading to the aggregation of the AuNPs through intermolecular electrostatic interaction or hydrogen-bonding. As a result of the predetermined aggregation, color and UV–vis spectra of AuNPs are changed. We employed the digital mapping approach to analyze the spectral variations with statistical and chemometric methods, including hierarchical cluster analysis (HCA) and principal component analysis (PCA). The proposed array could successfully differentiate biological molecules (e.g., cysteine, glutathione and glutathione disulfide) from other potential interferences such as amino acids in the concentration range of 10–800 μmol L⁻¹.     

Rapid estimation of bacteria by a fluorescent gold nanoparticle-polythiophene composite

Herein we present a facile method for rapid quantitation of bacterial cells over several logarithmic dilutions. The quantitation is based on loss of the fluorescence intensity of a positively charged Au nanoparticle-polythiophene composite in the presence of bacterial cells. The present method allowed estimation of both Gram-positive and Gram-negative bacteria with cells as low as 1000. Transmission electron microscopic investigations revealed attachment of the composite with bacteria with no discernible change in the morphology of the cells. Further, dynamic light scattering experiments indicated preferential attachment of smaller composite particles over larger ones, which were also attached at higher bacterial concentrations. The ease of operation with minimal sample manipulation steps, high sensitivity, quantitative detection, and its generality offer specific advantages over conventional methods.     

细菌的核酸适配体筛选的研究进展

核酸适配体(aptamer)是通过指数富集配体系统进化技术(SELEX)筛选的能够以高亲和力和高特异性识别靶标分子或细胞的核糖核酸(RNA)和单链脱氧核糖核酸(ssDNA)。作为化学抗体,核酸适配体的制备和合成比抗体的成本更低。核酸适配体的靶标范围极其广泛,包括小分子、生物大分子、细菌和细胞等。针对细菌靶标筛选的适配体,目前主要应用于食品、医药和环境中的细菌检测。细菌的核酸适配体筛选可以通过离心法将菌体-适配体复合物与游离的适配体分离,并通过荧光成像、荧光光谱分析、流式细胞仪分选、DNA捕获元件、酶联适配体分析等方法表征适配体与靶标的相互作用。筛选出的适配体可结合生物、化学检测方法用于细菌检测。本文介绍了细菌适配体的筛选和表征方法以及基于适配体的检测方法的最新进展,分析了不同检测方法的利弊,并列出了2011~2015年筛选的细菌的核酸适配体。