Recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices

Cells are the fundamental unit of life, and studies on cell contribute to reveal the mystery of life. However, since variability exists between individual cells even in the same kind of cells, increased emphasis has been put on the analysis of individual cells for getting better understanding on the organism functions. During the past two decades, various techniques have been developed for single-cell analysis. Capillary electrophoresis is an excellent technique for identifying and quantifying the contents of single cells. The microfluidic devices afford a versatile platform for single-cell analysis owing to their unique characteristics. This article provides a review on recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices; focus areas to be covered include sampling techniques, detection methods and main applications in capillary electrophoresis, and cell culture, cell manipulation, chemical cytometry and cellular physiology on microfluidic devices.     

微流控器件-质谱联用接口技术研究进展与应用

生物分析是生命科学研究中的重要环节,分析仪器的小型化是提高生物分析灵敏度、速度、通量和降低成本的有效途径之一.微流控技术能够方便地操纵微量样品,具有集成度高、样品耗量小、污染少等诸多其他常量流控技术难以具备的优点,适用于进行多通道样品处理和高通量分析.除广泛采用的光学和电化学检测手段外,质谱也被用作这些微流控器件的检测器,并逐渐形成了微流控器件-质谱联用技术专门研究领域,进一步促进了自动化程度好、灵敏度高、特异性强的高通量生物分析方法的迅速发展.在大量调研国内外文献的基础上,对微流控器件-质谱联用领域的研究背景和现状进行了综述,不但介绍了微流控器件的制造技术还着重介绍了微流控器件-质谱联用技术在蛋白质组学等生物质谱分析方面的应用和新近进展,评述了可能的发展趋势.     

基于分子印迹技术的细菌传感检测

分子印迹因其材料结构的稳定性及靶标物识别的特异性而被广泛应用于生化分离分析的相关领域。近年来，将具有选择性捕获、分离和富集靶标物等优势的分子印迹技术与生化传感检测技术有机结合，是目前细菌等微生物高效检测领域备受关注的研究热点。本文就分子印迹技术在细菌分析中的印迹方法、分析检测技术和典型应用等方面的最新进展进行综述。首先介绍了细菌分子印迹原理，对表面印迹的材料以及直接压印、间接印迹和电聚合等制备方法进行了总结和归纳；重点对基于荧光、电化学、石英晶体微天平（QCM）等检测模式的细菌印迹传感监测在细菌分析检测及其与微流控芯片技术耦合的应用和进展进行了综述；最后，提出了存在的挑战及发展的趋势。     

Microfluidic bio-particle manipulation for biotechnology

Microfluidics and lab-on-a-chip technology offers unique advantages for the next generation devices for diagnostic therapeutic applications. For chemical, biological and biomedical analysis in microfluidic systems, there are some fundamental operations such as separation, focusing, filtering, concentration, trapping, detection, sorting, counting, washing, lysis of bio-particles, and PCR-like reactions. The combination of these operations led to the complete analysis systems for specific applications. Manipulation of the bio-particles is the key ingredient for these applications. Therefore, microfluidic bio-particle manipulation has attracted a significant attention from the academic community. Considering the size of the bio-particles and the throughput of the practical applications, manipulation of the bio-particles is a challenging problem. Different techniques are available for the manipulation of bio-particles in microfluidic systems. In this review, some of the techniques for the manipulation of bio-particles; namely hydrodynamic based, electrokinetic-based, acoustic-based, magnetic-based and optical-based methods have been discussed. The comparison of different techniques and the recent applications regarding the microfluidic bio-particle manipulation for different biotechnology applications are presented. Finally, challenges and the future research directions for microfluidic bio-particle manipulation are addressed.     

Quantification of chemical–polymer surface interactions in microfluidic cell culture devices

Microfluidic cell culture devices have been used for drug development, chemical analysis, and environmental pollutant detection. Because of the decreased fluid volume and increased surface area to volume ratio, interactions between device surfaces and the fluid is a key element that affects the performance and detection accuracy of microfluidic devices, particularly if fluid is recirculated by a peristaltic pump. However, this issue has not been studied in detail in a microfluidic cell culture environment. In this study, chemical loss and contaminant leakage from various polymer surfaces in a microfluidic setup were characterized. The effects of hydrophilic coating with Poly (vinyl alcohol), Pluronic® F‐68, and multi‐layer ionic coating were measured. We observed significant surface adsorption of estradiol, doxorubicin, and verapamil with PharMed® BPT tubing, whereas PTFE/BPT and stainless steel/BPT hybrid tubing caused less chemical loss in proportion to the fraction of BPT tubing in the hybrid system. Contaminants leaching out of the BPT tubing were found to be estrogen receptor agonists as determined by estrogen‐induced green fluorescence expression in an estrogen responsive Ishikawa cell line and also caused interference with an estradiol enzyme‐linked immunosorbent assay (ELISA) assay. Stainless steel/BPT hybrid tubing caused the least interference with ELISA. In summary, polymer surface and chemical interactions inside microfluidic systems should not be neglected and require careful investigations when results from a microfluidic system are compared with results from a macroscale cell culture setup. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Bead-based microfluidic immunoassays: the next generation

Microfluidic devices possess many advantages like high throughput, short analysis time, small volume and high sensitivity that fulfill all the important criteria of an immunoassay used for clinical diagnoses, environmental analyses and biochemical studies. These devices can be made from a few different materials, with polymers presently emerging as the most popular choice. Other than being optically clear, non-toxic and cheap, polymers can also be easily fabricated with a variety of techniques. In addition, there are many polymer surface modification methods available to improve the efficiency of these devices. Unfortunately, current microfluidic immunoassays have limited multiplexing capability compared to flow cytometric assays. Flow cytometry employ the use of encoded microbeads in contrast with normal or paramagnetic microbeads applied in current microfluidic devices. The encoded microbead is the key in providing multiplexing capability to the assay by allowing multi-analyte analysis. Using several unique sets of code, different analytes can be detected in a single assay by tracing the identity of individual beads. The same principle could be applied to microfluidic immunoassays in order to retain all the advantages of a fluidic device and significantly improve multiplexing capability.     

Patenting trends in enzyme related microfluidic applications

The miniaturization of continuous processes has been of interest in the academia and industry which is reflected by the increase in scientific publications and patent disclosures in the last decade. The aim of this study was to evaluate the patenting trends regarding enzyme related microfluidic applications in order to observe the progress of science and technology. The mapped patents have been classified as “immobilization method”, “biomolecule screening systems”, “integrated process development” and “microreactor design”. Half of the patent disclosures were filed by academia, whereas the other half was from industrial research which complies with the shift in microfluidics from academic and industrial research to commercial applications. Immobilization procedures carried out at room temperatures such as formulation of silica matrices using sol–gel technique, incorporation of novel hybrid materials, the integration of supercritical fluids and microfluidics, employing ionic liquids as wall-less microreactors, designing low cost, high performance microfluidic devices were the highlights which can pose challenges in various life science applications. The increasing trend is expected to continue and the presented state-of-the-art in enzyme related microfluidic applications have the potential to enhance industry's capabilities for designing innovative systems which would demonstrate significant economic, societal and environmental benefits.     

Fabrication of lab-on chip platforms by hot embossing and photo patterning

In this paper, we review the approaches developed in our laboratory to fabricate polymer-based microfluidic devices to suit a range of applications in bio- or chemical analysis. Thermoplastic materials such as polycarbonate (PC) and poly(methyl methacrylate) (PMMA) are used to fabricate microfluidic devices via hot embossing. To emboss microchannels, we use hard stamps fabricated in silicon or soft stamps molded on poly(dimethylsiloxane) (PDMS). Hard stamps are fabricated on silicon wafers through photolithography and deep reactive ion etching (DRIE). Soft stamps are fabricated by casting PDMS prepolymer on silicon molds. To enclose the fluidic channels, direct fusion bonding was found to produce the highest bond strength with minimal structural deformation. One-step photolithographic methods have also been explored to produce via photochemical patterning microfluidic structures in photocurable materials. We use the photocurable capabilities of a PDMS copolymer, which incorporates a methacrylate crosslinker. Microfluidic channels are produced via one step-photopatterning processes by crosslinking the prepolymer mixture through a photomask. The smaller feature size attainable was 100 microm. Structures with higher spatial resolution are fabricated through a photoimprinting process whereby a mold is pressed against the precured mixture during UV crosslinking exposure. The application of the fabricated fluidic devices in electrophoretic ion analysis is also presented.     

细菌群体感应信号分子的光电检测技术

群体感应（quorum sensing，QS）是一种依赖菌群密度的细菌交流系统。在探究细菌群体感应系统的调控机制中，对QS信号分子的鉴别和检测是不可或缺的环节，其对生命科学、药学等领域涉及细菌等微生物的相互作用、高效检测和作用机制解析等具有重要的参考意义。本文在总结不同类型细菌QS信号分子来源和结构的基础上，对QS信号分子的光电检测方法和技术进行了综述，重点对光电传感检测的敏感介质、传感界面、传感机制及测试效果进行探讨，同时关注了将微流控芯片分析技术应用于细菌QS信号分子原位监测的相关研究进展。     

Miniaturized bioanalytical systems: enhanced performance through liposomes

Biorecognition-element labeled liposomes are simple and versatile tools used to amplify signals for the detection of analytes of environmental, clinical, food safety, and national security interest. Relying on measurement of encapsulated species via electrochemical or spectroscopic techniques, or properties inherent to liposomes themselves (such as mass, refractive index, or charge), many advances have been made in both bench-scale and microfluidic applications. Some of these measurement techniques are inherently sensitivity limited, but through the inclusion of liposomes, reduced limits of detection potentially broaden the utility towards otherwise challenging levels of analytes. Other advances took advantage of the hydrophobic environment required by many biorecognition elements to expand the target selectivity range or utilized the amphipathic nature of the lipid bilayer to provide enhanced separation capabilities. Novel handling approaches included wavelength-specific release of contents encapsulated within thermosensitive liposomes or application of electric fields to move, concentrate, and strategically lyse liposomes. These and other topics are discussed in terms of either present incorporation or adaptation to microfluidic devices.     

Development of low-cost microfluidic systems for lab-on-a-chip biosensor applications

In this work, we develop low-cost microfluidic systems based on polydimethylsiloxane (PDMS) for lab-on-a-chip applications. PDMS microfluidic structures have been fabricated by micromolding, PDMS casting, and plasma bonding processes. The micromolding technique is used to fabricate PDMS slabs with micro-sized grooves, and the complete microchannel is formed by bonding PDMS slab with glass or PDMS substrate. The molding procedure using SU-8 photoresist patterning on silicon wafer, PDMS microchannel fabrication, and PDMS surface treatment using oxygen plasma and TiO₂ coating, are discussed. The various parameters for oxygen plasma treatment including RF power and treatment time are studied in order to obtain conditions for good bonding with the glass substrate. The best condition for plasma treatment is found to be the low RF power (8 W) with 5 min treatment time. In addition, TiO₂ coating with oxygen plasma treatment has been applied to make PDMS surface more hydrophilic to improve aqueous solution compatilbility. The microfluidic channels for various applications, including sample injection cross channel, micropump channel, T and Y sample mixers, PCR thermocyling chamber and channel, capillary electrophoresis flow channel, and conductimetric systems have been fabricated. Finally, a typical application of the PDMS chip in a flow injection conductimetric system for sodium chloride detection has been demonstrated.     

Dielectrophoretic platforms for bio-microfluidic systems

Dielectrophoresis, the induced motion of polarisable particles in a nonuniform electric field, has been proven as a versatile mechanism to transport, accumulate, separate and characterise micro/nano scale bioparticles in microfluidic systems. The integration of DEP systems into the microfluidics enables the inexpensive, fast, highly sensitive, highly selective and label-free detection and analysis of target bioparticles. This review provides an in-depth overview of state-of-the-art dielectrophoretic (DEP) platforms integrated into microfluidics aimed towards different biomedical applications. It classifies the current DEP systems in terms of different microelectrode configurations and operating strategies devised to generate and employ DEP forces in such processes, and compares the features of each approach. Finally, it suggests the future trends and potential applications of DEP systems in single cell analysis, stem cell research, establishing novel devices, and realising fully DEP-activated lab-on-a-chip systems.     

Display of proteins on bacteria

Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and discusses current uses and possible future trends of these emerging technologies.     

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.     

Tissue culture on a chip: Developmental biology applications of self‐organized capillary networks in microfluidic devices

Organ culture systems are used to elucidate the mechanisms of pattern formation in developmental biology. Various organ culture techniques have been used, but the lack of microcirculation in such cultures impedes the long‐term maintenance of larger tissues. Recent advances in microfluidic devices now enable us to utilize self‐organized perfusable capillary networks in organ cultures. In this review, we will overview past approaches to organ culture and current technical advances in microfluidic devices, and discuss possible applications of microfluidics towards the study of developmental biology.     

Cancer Liquid Biopsy Using Integrated Microfluidic Exosome Analysis Platforms

Liquid biopsies serve as both powerful noninvasive diagnostic tools for early cancer screening and prognostic tools for monitoring cancer progression and treatment efficacy. Exosomes are promising biomarkers for liquid biopsies, since these nano‐sized extracellular vesicles (EVs) enrich proteins, lipids, mRNAs, and miRNAs from cells of origin, including cancer cells. Although exosomes are abundantly present in various bodily fluids, conventional exosome isolation and detection methods that rely on benchtop equipment are time‐consuming, expensive, and involve complicated non‐portable procedures. As an alternative, recently developed microfluidic platforms can perform effective exosome separation and detection for liquid biopsies using a single device. Such methods offer advantages of integrity, speed, cost‐efficiency, and portability over conventional benchtop and early microfluidic‐based single‐functional methods which can only separate or detect exosomes separately. These advances have made exosome‐based point‐of‐care (POC) applications possible. This review outlines recent integrated microfluidic‐based exosomal detection strategies to guide future development of such devices for use in liquid biopsies for early cancer screening, prognostic monitoring, and other potential POC applications.     

Single-molecule,hybridization-based strategies for short nucleic acids detection and recognition with nanopores

DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore- and hybridization-based DNA detection, and explores a range of state-of-the-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.     

Disposable microfluidic devices: fabrication, function, and application

This review article describes recent developments in microfluidics, with special emphasis on disposable plastic devices. Included is an overview of the common methods used in the fabrication of polymer microfluidic systems, including replica and injection molding, embossing, and laser ablation. Also described are the different methods by which on-chip operations--such as the pumping and valving of fluid flow, the mixing of different reagents, and the separation and detection of different chemical species--have been implemented in a microfluidic format. Finally, a few select biotechnological applications of microfluidics are presented to illustrate both the utility of this technology and its potential for development in the future.     

Finite Element Model of Oxygen Transport for the Design of Geometrically Complex Microfluidic Devices Used in Biological Studies

Red blood cells play a crucial role in the local regulation of oxygen supply in the microcirculation through the oxygen dependent release of ATP. Since red blood cells serve as an oxygen sensor for the circulatory system, the dynamics of ATP release determine the effectiveness of red blood cells to relate the oxygen levels to the vessels. Previous work has focused on the feasibility of developing a microfluidic system to measure the dynamics of ATP release. The objective was to determine if a steep oxygen gradient could be developed in the channel to cause a rapid decrease in hemoglobin oxygen saturation in order to measure the corresponding levels of ATP released from the red blood cells. In the present study, oxygen transport simulations were used to optimize the geometric design parameters for a similar system which is easier to fabricate. The system is composed of a microfluidic device stacked on top of a large, gas impermeable flow channel with a hole to allow gas exchange. The microfluidic device is fabricated using soft lithography in polydimethyl-siloxane, an oxygen permeable material. Our objective is twofold: (1) optimize the parameters of our system and (2) develop a method to assess the oxygen distribution in complex 3D microfluidic device geometries. 3D simulations of oxygen transport were performed to simulate oxygen distribution throughout the device. The simulations demonstrate that microfluidic device geometry plays a critical role in molecule exchange, for instance, changing the orientation of the short wide microfluidic channel results in a 97.17% increase in oxygen exchange. Since microfluidic devices have become a more prominent tool in biological studies, understanding the transport of oxygen and other biological molecules in microfluidic devices is critical for maintaining a physiologically relevant environment. We have also demonstrated a method to assess oxygen levels in geometrically complex microfluidic devices.     

Microreactors for peptide synthesis: looking through the eyes of twenty first century !!!

The twenty first century has witnessed several advances in synthetic chemistry, among them microreactors. It is expected that these devices will have a considerable impact on synthetic organic chemistry since they offer a wide range of applications in various fields. Perhaps the synthesis of peptides deserves mention in this regard as these molecules are emerging as therapeutics and offer several advantages over the so-called small molecules. This minireview does not aim to address microreactors in detail, but explains various peptide synthesis methods that involve microfluidic techniques, highlighting the need for further improvement and expansion of microdevices/microreactors.