PDMS-glass hybrid microreactor array with embedded temperature control device. Application to cell-free protein synthesis

A microreactor array was developed which enables high-throughput cell-free protein synthesis. The microreactor array is composed of a temperature control chip and a reaction chamber chip. The temperature control chip is a glass-made chip on which temperature control devices, heaters and temperature sensors, are fabricated with an ITO (indium tin oxide) resistive material. The reaction chamber chip is fabricated by micromolding of PDMS (polydimethylsiloxane), and is designed to have an array of reaction chambers and flow channels for liquid introduction. The microreactor array is assembled by placing the reaction chamber chip on the temperature control chip. The small thermal mass of the reaction chamber resulted in a short thermal time constant of 170 ms for heating and 3 s for cooling. The performance of the microreactor array was examined through the experiments of cell-free protein synthesis. By measuring the fluorescence emission from the products, it was confirmed that GFP (Green Fluorescent Protein) and BFP (Blue Fluorescent Protein) were successfully synthesized using Escherichia coli extract.     

微流控芯片中高效合成苯基二吡咯-2-基甲烷的研究

微流控芯片是近年发展起来的一种新型的微反应器.在微流控芯片中以HCl为催化剂常温下合成了苯基二吡咯-2-基甲烷,考察了反应物流速、催化剂浓度、吡咯与苯甲醛的比例对苯基二吡咯-2-基甲烷产率的影响.通过在催化剂溶液中加入离子液体[bmim]BF4明显地提高了反应效率,苯基二吡咯-2-基甲烷的产率可以达到95%以上.采用本法大大减少了试剂用量和条件试验阶段的实验成本.     

Amphiphilic polysaccharide nanogels as artificial chaperones in cell-free protein synthesis

Cell-free protein synthesis is a promising technique for the rapid production of proteins. However, the application of the cell-free systems requires the development of an artificial chaperone that prevents aggregation of the protein and supports its correct folding. Here, nanogel-based artificial chaperones are introduced that improve the folding efficiency of rhodanese produced in cell-free systems. Although rhodanese suffers from rapid aggregation, rhodanese was successfully expressed in the presence of the nanogel and folded to the enzymatically active form after addition of cyclodextrin. To validate the general applicability, the cell-free synthesis of ten water-soluble proteins was examined. It is concluded that the nanogel enables efficient expression of proteins with strong aggregation tendency.     

Selective deposition and self-assembly of triblock copolymers into matrix arrays for membrane protein production

To improve the stability of cell membrane mimics, there has been growing interest in the use of block copolymers. Here, we present an easy approach to create an array of planar polymeric matrices capable of hosting membrane proteins. The array of polymeric matrices was formed by the selective deposition of triblock copolymers onto an array of hydrophilic islands situated within a hydrophobic background. The thickness of these matrices corresponds to the length of a single polymer chain. These polymeric matrices were used to host cell-free expressed membrane proteins, and offers a prototype from which a membrane protein array can be created for diagnostics or drug discovery purposes.     

Synthesis of AgInS2 QDs in droplet microreactors: Online fluorescence regulating through temperature control

We designed a temperature-controllable droplet microreactor with more precisely temperature control and shorter synthesis time for water-soluble AgInS₂ QDs synthesis. When reaction temperature increased from 30 ℃ to 70 ℃, QDs fluorescence peak constantly red-shifted from 590 nm to 720 nm along with enhanced fluorescence QY and intensity, we can get products with the maximum fluorescence intensity and the QY of 8.8% at 70 ℃.     

Multistage Microfluidic Platform for the Continuous Synthesis of III–V Core/Shell Quantum Dots

We present a fully continuous chip microreactor‐based multistage platform for the synthesis of quantum dots with heterostructures. The use of custom‐designed chip reactors enables precise control of heating profiles and flow distribution across the microfluidic channels while conducting multistep reactions. The platform can be easily reconfigured by reconnecting the differently designed chip reactors allowing for screening of various reaction parameters during the synthesis of nanocrystals. III–V core/shell quantum dots are chosen as model reaction systems, including InP/ZnS, InP/ZnSe, InP/CdS and InAs/InP, which are prepared in flow using a maximum of six chip reactors in series.     

Measuring reaction kinetics in a lab-on-a-chip by microcoil NMR

A microfluidic chip with an integrated planar microcoil was developed for Nuclear Magnetic Resonance (NMR) spectroscopy on samples with volumes of less than a microliter. Real-time monitoring of imine formation from benzaldehyde and aniline in the microreactor chip by NMR was demonstrated. The reaction times in the chip can be set from 30 min down to ca. 2 s, the latter being the mixing time in the microfluidic chip. Design rules will be described to optimize the microreactor and detection coil in order to deal with the inherent sensitivity of NMR and to minimize magnetic field inhomogeneities and obtain sufficient spectral resolution.     

Easy Production of “Difficult Peptides” Using Cell-Free Protein Synthesis and a New Methionine Analogue as a Latent Peptide Cleavage Site

Aliphatic γ-chloro-α-amino acids incorporated in place of their canonical analogues through cell-free protein synthesis act as heat-labile linkers, offering a useful strategy for the straightforward production of target peptides as fusion proteins, from which the targets are readily released. Until now, the natural abundance of aliphatic amino acids in peptides has limited the scope of the method, as it leads to undesired cleavage sites in synthesized products, but here the authors report the development of a new cleavable chloro amino acid that incorporates in place of the relatively rare amino acid methionine, thus greatly expanding the scope of producible targets. This new strategy is employed for simplified peptide synthesis with a methionine-free fusion partner, allowing single-site incorporation of the cleavable linker for clean release and easy purification of the target peptide. Its utility is demonstrated through the straightforward preparation of two peptides reported to be challenging targets and not accessible through standard solid-phase chemical methodologies, as well as analogues.     

Synthesis of a three-member array of cycloadducts in a glass microchip under pressure driven flow

The synthesis of a series of cycloadduct products carried out in a glass microchip under pressure driven flow is presented. Initially, four different compounds were synthesized individually with conversions similar to those obtained in the corresponding batch macroscale reactions. Using identical experimental conditions for all four compounds, the results were highly predictable and reproducible, without the need for individual optimization. A multi-reaction experiment was then carried out in a single chip achieving the synthesis of a three-member array in a single run.     

Surface plasmon resonance-enabled mass spectrometry arrays

Biosensors that utilize surface plasmon resonance (SPR) as a method of detection of protein interactions can be used for selective separation of proteins prior to MS analysis. The combination of SPR and MS results in a unique multiplexed detection technology capable of both quantitative and qualitative protein analysis. To further the development of a high-throughput SPR-MS approach, the possibility of arraying binding ligands on SPR chips for affinity capture of proteins and their MS analysis was explored. Antibodies to beta-2-microglobulin, cystatin C, transferrin, and insulin-like growth factors I and II were arrayed on a large number of SPR chips. Human plasma samples were injected over the antibody array chips in an SPR Biosensor, after which on-chip MS analysis was performed to detect the bound proteins. Signals from the targeted proteins were observed for each antibody-derivatized chip, indicating successful antibody immobilization and protein capture. The SPR-MS arrays are robust, highly reproducible, and are capable of high-throughput analysis.     

Droplet synthesis of well-defined block copolymers using solvent-resistant microfluidic device

Well-defined diblock copolymers were synthesized via an exothermic RAFT route by a droplet microfluidic process using a solvent-resistant and thermally stable fluoropolymer microreactor fabricated by a non-lithographic embedded template method. The resulting polymers were compared to products obtained from continuous flow capillary reactor and conventional bulk synthesis. The droplet based microreactor demonstrated superior molecular weight distribution control by synthesizing a higher molecular weight product with higher conversion and narrow polydispersity in a much shorter reaction time. The high quality of the as-synthesized block copolymer PMMA-b-PS led to a generation of micelles with a narrow size distribution that could be used as a template for well-ordered mesoporous silica with regular frameworks and high surface areas.     

Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry

A novel monolithic enzymatic microreactor was prepared in the fused-silica capillary by in situ polymerization of acrylamide (AA), N-acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, which could offer very low back pressure, enabling the fast digestion of proteins. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at high flow rate, and the comparisons between the in-solution digestion and on-column reaction were made by a nano-high performance liquid chromatography-mass spectrometry (nano-HPLC-MS) system. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at the fast flow rate of 1 microL/min, which afforded a residence time of 7s, yielding a sequence coverage of 54.81% using strict multiple database searching thresholds. Future more, a mixture of four standard proteins was digested and analyzed using the on-line digestion and nano-HPLC-MS system. The results showed the promising of such a system in the analysis of protein mixture.     

Versatile protein biotinylation strategies for potential high-throughput proteomics

We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo.     

Geno-Tox: Cell Array Biochip for Genotoxicity Monitoring and Classification

In vitro genotoxicity tests detect carcinogens that are thought to act primarily via a mechanism involving direct genetic damage. In this study, we constructed a Geno-Tox cell array chip for genotoxicity testing; eight recombinant bioluminescent bacteria were used to successfully fabricate a Geno-Tox cell array chip. Four well-characterized DNA damage chemicals were selected to determine the capabilities of this Geno-Tox array chip, and each strain of the chip was distinctly responsive, according to the specific mode of genotoxic action. Therefore, this Geno-Tox cell array chip could be implemented to characterize and understand the genotoxic modes of impact; thus, it could be used to provide the genotoxic mechanism of action for new drugs or unknown or newly synthesized chemicals in food and the environment.     

Dealing with misfolded proteins: examining the neuroprotective role of molecular chaperones in neurodegeneration

Human neurodegenerative diseases arise from a wide array of genetic and environmental factors. Despite the diversity in etiology, many of these diseases are considered "conformational" in nature, characterized by the accumulation of pathological, misfolded proteins. These misfolded proteins can induce cellular stress by overloading the proteolytic machinery, ultimately resulting in the accumulation and deposition of aggregated protein species that are cytotoxic. Misfolded proteins may also form aberrant, non-physiological protein-protein interactions leading to the sequestration of other normal proteins essential for cellular functions. The progression of such disease may therefore be viewed as a failure of normal protein homeostasis, a process that involves a network of molecules regulating the synthesis, folding, translocation and clearance of proteins. Molecular chaperones are highly conserved proteins involved in the folding of nascent proteins, and the repair of proteins that have lost their typical conformations. These functions have therefore made molecular chaperones an active area of investigation within the field of conformational diseases. This review will discuss the role of molecular chaperones in neurodegenerative diseases, highlighting their functional classification, regulation, and therapeutic potential for such diseases.     

Cell immobilization induces changes in the protein response of Escherichia coli K-12 to a cold shock

We have compared the protein expression of gel-entrapped Escherichia coli cells submitted to a cold shock at 4 degrees C with those of exponential- and stationary-phase free-floating counterparts. Autoradiograms of two-dimensional gel electrophoresis patterns of proteins radiolabeled with L-[35S]methionine were compared using computing scanning densitometry. The levels of 203 proteins synthesized during the temperature shift were significantly and reproducibly higher than those corresponding to synthesis at 37 degrees C. A principal component analysis (PCA) was performed on the synthesis levels of these 203 proteins in the different incubation conditions tested. This study showed that the protein response of immobilized cells after the cold shock was significantly different from those of exponential- and stationary-phase free-floating organisms. For instance, protein SSB was specifically overexpressed by shocked immobilized organisms. Such induction of specific molecular mechanisms in immobilized bacteria might explain the high resistance of sessile-like organisms to stresses.     

Microsized 2D protein arrays immobilized by micro-stamps and micro-wells for disease diagnosis and drug screening

A novel method of protein array immobilization, using micro stamps to pick up proteins from micro wells and deposit them on to a bio-absorption chip, has been developed. This method can potentially transfer several protein spots on to an organized array for applications such as disease diagnosis and drug screening by parallel biological or chemical processes. Fabrication of the micro stamp and the micro well arrays involves thick-photoresist lithography, bulk micromachining, and a molding process, whereas fabrication of the bio-absorption chip involves amino-modification by use of APTS (aminopropyItrimethoxysilane) and surface activation by use of BS3 (bis-sulfosuccinimidyl suberate). Successful transfer of protein on to the bio-absorption surface using the micro stamp-well array has been demonstrated. The size variation between different stamping spots has been shown to be less than 10%, and the APTS-BS3 surface has also been proved to bind the protein efficiently. Appreciable protein retention was achieved during 6-h washing, which shows the binding strength of the bio-absorption surface is sufficient for protein processing.     

Microspheres of Mixed Proteins

This paper describes the synthesis of mixed proteinaceous microspheres (MPMs) by the sonochemical method. The current fundamental research follows the research of Suslick and co‐workers who have developed a method by which high‐intensity ultrasound is used to make aqueous suspensions of proteinaceous microcapsules filled with water‐insoluble liquids. 1 By using high‐intensity ultrasound, we have synthesized microspheres made of a few different proteins. The three proteins used in the current experiments are bovine serum albumin (BSA), green fluorescent protein (GFP), and cyan fluorescent protein–glucose binding protein–yellow fluorescent fused protein (CFP‐GBP‐YFP). The two synthesized microspheres made of mixed proteins are BSA‐GFP and BSA‐(CFP‐GBP‐YFP). This paper presents the characterization of the sonochemically produced microspheres of mixed proteins. It also provides an estimate of the efficiency of the sonochemical process in converting the native proteins to microspheres.     

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling

An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29?μg, was analyzed, and sequence coverages for BSA, myoglobin, β-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.