Affinity depletion of albumin from human cerebrospinal fluid using Cibacron-blue-3G-A-derivatized photopatterned copolymer in a microfluidic device

In the context of proteomic research, affinity separations for the prefractionation of complex mixtures, such as cell lysates or human tissues, have become increasingly important. Microfluidic devices have shown significant potential to achieve fast analysis and low sample consumption. Here, we demonstrate the use of a microfluidic device to achieve affinity capture of albumin from human cerebrospinal fluid. Traditional photolithography and wet etching techniques were used to fabricate devices from borosilicate glass wafers. Monolithic porous polymer was prepared in a microfluidic channel by photopolymerization of glycidyl methacrylate and trimethylolpropane trimethacrylate. After derivatization with Cibacron-blue-3G-A, the modified polymer was used to achieve affinity capture of lysozyme and human albumin. Both fluorescence detection and matrix-assisted laser desorption ionization time of flight mass spectrometry were used to validate the results.     

Microfluidic immunoaffinity separations for bioanalysis

Microfluidic devices often rely on antibody-antigen interactions as a means of separating analytes of interest from sample matrices. Immunoassays and immunoaffinity separations performed in miniaturized formats offer selective target isolation with minimal reagent consumption and reduced analysis times. The introduction of biological fluids and other complicated matrices often requires sample pretreatment or system modifications for compatibility with small-scale devices. Miniaturization of external equipment facilitates the potential for portable use such as in patient point-of-care settings. Microfluidic immunoaffinity systems including capillary and chip platforms have been assembled from basic instrument components for fluid control, sample introduction, and detection. The current review focuses on the use of immunoaffinity separations in microfluidic devices with an emphasis on pump-based flow and biological sample analysis.     

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.     

Lateral phase separations and perpendicular transport in membranes

A valinomycin-mediated potassium conductivity has been studied using a glass U-tube in which two aqueous compartments are separated by a fritted glass filter impregnated with valinomycin and one or more pure phospholipids. This system can be used to detect the beginning and end of lateral phase separations in binary lipid mixtures, and also demonstrates a pronounced maximum in electrical conductivity of dipalmitoyl phosphatidylcholine at the transition temperature, 41°C.     

Microfluidic Pneumatic Cages: A Novel Approach for In-chip Crystal Trapping,Manipulation and Controlled Chemical Treatment

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.     

New tools and new biology: Recent miniaturized systems for molecular and cellular biology

Recent advances in applied physics and chemistry have led to the development of novel microfluidic systems. Microfluidic systems allow minute amounts of reagents to be processed using μm-scale channels and offer several advantages over conventional analytical devices for use in biological sciences: faster, more accurate and more reproducible analytical performance, reduced cell and reagent consumption, portability, and integration of functional components in a single chip. In this review, we introduce how microfluidics has been applied to biological sciences. We first present an overview of the fabrication of microfluidic systems and describe the distinct technologies available for biological research. We then present examples of microsystems used in biological sciences, focusing on applications in molecular and cellular biology.     

Application of microfluidic chip with integrated optics for electrophoretic separations of proteins

This paper describes the fabrication, the characterization and the applications of a capillary electrophoresis microchip. This hybrid device (glass/PDMS) features channels and optical waveguides integrated in one common substrate. It can be used for electrophoretic separation and fluorimetric detection of molecules. The microfluidic performance of the device is demonstrated by capillary zone and gel electrophoresis of proteins.     

Emulsion liquid membranes for chiral separations: Selective extraction of rac-phenylalanine enantiomers

We describe the use of emulsion liquid membrane technology to perform chiral separations on low molecular weight species. We have reviewed liquid membrane technology in the context of existing process scale chiral separations. We illustrate the potential of this new technique by presenting our results on the selective extraction of phenylalanine enantiomers, using copper (II) N-decyl-(L)-hydroxyproline as a chiral selector in an emulsion liquid membrane configuration. This is compared with an analogous batch solvent extraction system. Initial batch enantiomeric excesses of greater than 40% were observed with the emulsion liquid membrane system compared with around 25% for the solvent extraction system. It was also noted that the system is not limited by the equilibrium capacity constraints of the solvent extraction system. We have shown that kinetic chiral liquid membrane technology offers high productivity and flexibility compared with analogous process scale chiral technologies. Recent transfer of highly specific chiral reversed-phase high-performance liquid chromatographic chemistries have shown that “one-stop” enantiomeric excesses of commercial interest (>95%) are achievable using kinetic chiral liquid membrane systems. Solvent and temperature selection strategies also have been outlined as means of increasing the enantioselectivity of existing liquid membrane extraction chemistries. Chirality 9:261–267, 1997. © 1997 Wiley-Liss, Inc.     

3D printing in biotechnology—An insight into miniaturized and microfluidic systems for applications from cell culture to bioanalytics

Since its invention in the 1980s, 3D printing has evolved into a versatile technique for the additive manufacturing of diverse objects and tools, using various materials. The relative flexibility, straightforwardness, and ability to enable rapid prototyping are tremendous advantages offered by this technique compared to conventional methods for miniaturized and microfluidic systems fabrication (such as soft lithography). The development of 3D printers exhibiting high printer resolution has enabled the fabrication of accurate miniaturized and microfluidic systems—which have, in turn, substantially reduced both device sizes and required sample volumes. Moreover, the continuing development of translucent, heat resistant, and biocompatible materials will make 3D printing more and more useful for applications in biotechnology in the coming years. Today, a wide variety of 3D‐printed objects in biotechnology—ranging from miniaturized cultivation chambers to microfluidic lab‐on‐a‐chip devices for diagnostics—are already being deployed in labs across the world. This review explains the 3D printing technologies that are currently used to fabricate such miniaturized microfluidic devices, and also seeks to offer some insight into recent developments demonstrating the use of these tools for biotechnological applications such as cell culture, separation techniques, and biosensors.     

Microfluidic device‐assisted etching of p‐HEMA for cell or protein patterning

The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2‐hydroxyethyl methacrylate) (p‐HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p‐HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p‐HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p‐HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p‐HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:243–248, 2018     

Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG–Salt Aqueous Two‐Phase Systems for Rapid Screening Applications

Aqueous two‐phase extraction (ATPE) has been showing significant potential in the biopharmaceutical industry, allowing the selective separation of high‐value proteins directly from unclarified cell culture supernatants. In this context, effective high‐throughput screening tools are critical to perform a rapid empirical optimization of operating conditions. In particular, microfluidic ATPE screening devices, coupled with fluorescence microscopy to continuously monitor the partition of fluorophore‐labeled proteins, have been recently demonstrated to provide short diffusion distances and rapid partition, using minimal reagent volumes. Nevertheless, the currently overlooked influence of the labeling procedure on partition must be carefully evaluated to validate the extrapolation of results to the unlabeled molecule. Here, three fluorophores with different global charge and reactivity selected to label immunoglobulin G (IgG) at degrees of labeling (DoL) ranging from 0.5 to 7.6. Labeling with BODIPY FL maleimide (DoL = 0.5), combined with tris(2‐carboxyethyl) phosphine (TCEP) to generate free thiol groups, is the most promising strategy to minimize the influence of the fluorophore on partition. In particular, the partition coefficient (K_p) measured in polyethylene glycol (PEG) 3350–phosphate systems with and without the addition of NaCl using microtubes (batch) or microfluidic devices (continuous) is comparable to those quantified for the native protein.     

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.     

Fast Benchtop Fabrication of Laminar Flow Chambers for Advanced Microscopy Techniques

Background

Fluid handling technology is acquiring an ever more prominent place in laboratory science whether it is in simple buffer exchange systems, perfusion chambers, or advanced microfluidic devices. Many of these applications remain the providence of laboratories at large institutions with a great deal of expertise and specialized equipment. Even with the expansion of these techniques, limitations remain that frequently prevent the coupling of controlled fluid flow with other technologies, such as coupling microfluidics and high-resolution position and force measurements by optical trapping microscopy.

Method

Here we present a method for fabrication of multiple-input laminar flow devices that are optically clear [glass] on each face, chemically inert, reusable, inexpensive, and can be fabricated on the benchtop in approximately one hour. Further these devices are designed to allow flow regulation by a simple gravity method thus requiring no specialized equipment to drive flow. Here we use these devices to perform total internal reflection fluorescence microscopy measurements as well as position sensitive optical trapping experiments.

Significance

Flow chamber technology needs to be more accessible to the general scientific community. The method presented here is versatile and robust. These devices use standard slides and coverslips making them compatible with nearly all types and models of light microscopes. These devices meet the needs of groups doing advanced optical trapping experiments, but could also be adapted by nearly any lab that has a function for solution flow coupled with microscopy.     

Tobacco protoplast culture in a polydimethylsiloxane-based microfluidic channel

Summary. Several advances have been made in the use of microfluidic devices for insect and mammalian cell cultures, but no reports of their use for plant cell cultures have been published. We, therefore, conducted a plant cell culture in a microfluidic device using polydimethylsiloxane. Nicotiana tabacum protoplasts were cultured in a variously shaped polydimethylsiloxane channel containing Nitsch medium supplemented with 0.5 g of NLN-13 vitamin mixture, 2.0 mg of α-naphthaleneacetic acid, and 0.5 mg of 6-benzyladenine per liter and 9% mannitol. Protoplasts in the polydimethylsiloxane channel showed cell division and microcolony formation within 4 weeks. The use of a microfluidic channel is a novel technique in the field of plant cell culture. The results of this study will encourage the utilization of polydimethylsiloxane-based microfluidic devices in plant cell engineering and cell analysis. Correspondence and reprints: Department of Biology, Dankook University, 29 San Anseo-dong, Cheonan 300-714, South Korea.     

Aminoalkylsilane-treated glass slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions

Summary Attempts to investigate the cellular localization of keratin mRNAs byin situ hybridization with specific [³⁵S]-labelled cDNA probes to mouse epithelia have been seriously impeded by the uncontrollable detachment of frozen tissue sections on conventionally coated glass slides (i.e. those coated with egg white, gelatine, collagen). Similarly, a variety of other coating and attachment devices have proved to be unsatisfactory or impracticable for large scale investigations. These difficulties were completely overcome andin situ hybridization was possible after a short immersion of the glass slides in a 2% solution of 3-aminopropyltriethoxysilane in acetone. This treatment provides the glass surface with aminoalkyl groups which are apparently able to react covalently with aldehyde or ketone functions of frozen tissue sections. The resulting firm adhesion of the sections enabled us to investigate the influence of different fixation and prehybridization procedures on the quality of thein situ hybridization. It was found that especially harsh prehybridization, involving hydrochloric acid, heat and proteinase K treatment, drastically reduces the morphological integrity of the sections, thus rendering a reliable assignment of the label difficult. In contrast, a mild prehybridization, consisting mainly of a rehydration of the sections in phosphate-buffered saline and equilibration in 0.1 M glycine, leaves the morphology intact and leads to a highly efficient and specificin situ hybridization.     

Superparamagnetic hydrophobic particles as shell material for digital microfluidic droplets and proof‐of‐principle reaction assessments with immobilized laccase

In the field of biotechnology and molecular biology, the use of small liquid volumes has significant advantages. In particular, screening and optimization runs with acceptable amounts of expensive and hardly available catalysts, reagents, or biomolecules are feasible with microfluidic technologies. The presented new microfluidic system is based on the inclusion of small liquid volumes by a protective shell of magnetizable microparticles. Hereby, discrete aqueous microreactor drops with volumes of 1–30 μL can be formed on a simple planar surface. A digital movement and manipulation of the microreactor is performed by overlapping magnetic forces. The magnetic forces are generated by an electrical coil matrix positioned below a glass plate. With the new platform technology, several discrete reaction compartments can be moved simultaneously on one surface. Due to the magnetic fields, the reactors can even be merged to initiate reactions by mixing or positioned above surface‐immobilized catalysts and then opened by magnetic force. Comparative synthesis routes of the magnetizable shell particles and superhydrophobic glass slides including their performance and stability with the reaction platform are described. The influence of diffusive mass transport during the catalyzed reaction is discussed by evaluation finite element model of the microreactor. Furthermore, a first model dye reaction of the enzyme laccase has been established.     

Electrophoresis today and tomorrow: Helping biologists' dreams come true

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two‐dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High‐throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser‐induced fluorescence detection of labeled DNA strands have been of key importance for the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH‐driven mobilization, provides efficient separations and on‐line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single‐cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single‐molecule detection.     

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 µm to 50 µm in diameter while using large channels that are robust against fouling and clogging.     

Fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridisation

Fluorescence in situ hybridisation (FISH) is a rapid and reliable technique for chromosomal investigations that is used for a wide variety of cytogenetic purposes at present. This molecular-cytogenetic method has been developed continuously for many years. As a consequence, various modifications with different kinds of fluorescently labelled probes have been introduced to optimise the detection of DNA and RNA sequences. This review articlepaper presents the general principles of in situ hybridisation, probe labelling and examples of proper use of different kinds of probes. In addition, some newer FISH methods and their usefulness in human molecular cytogenetics are described.     

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.