Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet

We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 microm wide, 100 microm deep, and possessed an 8 cm effective length separation channel with a double-T injector (V(inj) = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated.     

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.     

Microfluidic chip electrophoresis for biochemical analysis

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics‐based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted.     

Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis

The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.     

基于单螺旋微流控芯片的细菌气溶胶的高效富集

设计了一种单螺旋通道的聚二甲基硅氧烷(Poly(dimethylsiloxane),PDMS)微流控芯片,用于副溶血性弧菌气溶胶的快速有效富集。该芯片的特征在于其通道呈螺旋分布,且通道内部含有均匀分布的鱼骨形结构。结果表明,在不同富集时间段内,采用该芯片方法捕获的细菌总数均远高于传统落板法。对于传统落板法无法有效捕获的低浓度样本(10~4CFU/mL)的缺陷,该方法的优势在于:芯片内部的螺旋通道可增大对气溶胶中微生物的离心力;鱼骨形结构的设计增加了待测样品与芯片内壁间的接触几率。此外,以无鱼骨形的螺旋芯片作为对照,验证了鱼骨形结构对于高效富集的意义。此芯片设计巧妙、易于制备、高效便携、富集效果较好,在气溶胶污染严重的水产加工等场所具有较大的应用前景。     

微流控芯片实验室

以作者所在课题组近年来的研究工作为基础,就芯片实验室平台建设及相应的以系统生物学为最终目标的功能化研究作一说明,对在分子和细胞层面,甚至是单分子、单细胞水平上实现以规模集成为特征的临床诊断和药物筛选的努力予以特别的关注。     

Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein identification by proteomics

Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) has become a current tool in proteomics applications increasingly used in the search for new biomarkers. A new integrated microfluidic device (HPLC-Chip), coupled to ion trap mass spectrometry (ITMS), appears as an innovative and robust tool for improving the identifications commonly performed by nanoLC/MS/MS. We tested this device for the identification of proteins obtained from two-dimensional gel electrophoresis or chromatography. The chip allows the measurement of reproducible retention times that, in association with m/z ratios, was found useful for identifying peptide sequences without ambiguity. A sensitivity increase of a factor of at least 5-fold is obtained compared to the results obtained previously in our laboratory by conventional nanoLC/MS/MS on the same ion trap. We conclude that this recently available microfluidic device can be a valuable tool during biomarker discovery programs, particularly identifying low-abundance proteins.     

Hot embossing and thermal bonding of poly(methyl methacrylate) microfluidic chips using positive temperature coefficient ceramic heater

As a self-regulating heating device, positive temperature coefficient ceramic heater was employed for hot embossing and thermal bonding of poly(methyl methacrylate) microfluidic chip because it supplied constant-temperature heating without electrical control circuits. To emboss a channel plate, a piece of poly(methyl methacrylate) plate was sandwiched between a template and a microscopic glass slide on a positive temperature coefficient ceramic heater. All the assembled components were pressed between two elastic press heads of a spring-driven press while a voltage was applied to the heater for 10 min. Subsequently, the embossed poly(methyl methacrylate) plate bearing negative relief of channel networks was bonded with a piece of poly(methyl methacrylate) cover sheet to obtain a complete microchip using a positive temperature coefficient ceramic heater and a spring-driven press. High quality microfluidic chips fabricated by using the novel embossing/bonding device were successfully applied in the electrophoretic separation of three cations. Positive temperature coefficient ceramic heater indicates great promise for the low-cost production of poly(methyl methacrylate) microchips and should find wide applications in the fabrication of other thermoplastic polymer microfluidic devices.     

A capillary electrophoresis chip with hydrodynamic sample injection for measurements from a continuous sample flow

A microchip-based capillary electrophoresis device supported by a microfluidic network made of poly(dimethylsiloxane), used for measuring target analytes from a continuous sample flow, is presented. The microsystem was fabricated by means of replica molding in combination with standard microfabrication technologies, resulting in microfluidic components and an electrochemical detector. A new hydrodynamic sample injection procedure is introduced, and the maximum number of consecutive measurements that can be made with a poly(dimethylsiloxane) capillary electrophoresis chip with amperometric detection is investigated with respect to reproducibility. The device features a high degree of functional integration, so the benefits associated with miniaturized analysis systems apply to it.     

On-chip chiral separation based on bovine serum albumin-conjugated carbon nanotubes as stationary phase in a microchannel

A novel method of chiral separation based on protein-stationary phase immobilized in a poly(methyl methacrylate) microfluidic chip was developed. BSA conjugated with the shortened carboxylic single-walled carbon nanotubes (SWNTs) was employed as the chiral selector. Successful separation of tryptophan enantiomers was achieved in less than 70 s with a resolution factor of 1.35 utilizing a separation length of 32 mm. This is the first example of chiral separation based on SWNTs-BSA conjugates as stationary phase immobilized in microchip channel. The stability of the stationary phase in the channel was examined by microchip electrophoresis with laser-induced fluorescence detection. Factors that influenced the chiral separation resolution were examined. Under the optimized conditions, the proposed modified chip revealed adequate repeatability concerning run-to-run. These results show that the use of SWNTs-BSA conjugates within microfluidic channels hold great promise for a variety of analytical schemes.     

Separation of DNA fragments for fast diagnosis by microchip electrophoresis using programmed field strength gradient

We evaluated a novel strategy for fast diagnosis by microchip electrophoresis (ME), using programmed field strength gradients (PFSG) in a conventional glass double-T microfluidic chip. The ME-PFSG allows for the ultrafast separation and enhanced resolving power for target DNA fragments. These results are based on electric field strength gradients (FSG) that use an ME separation step in a sieving gel matrix poly-(ethylene oxide). The gradient can develop staircase or programmed shapes FSG over the time. The PFSG method could be easily used to increase separation efficiency and resolution in ME separation of specific size DNA fragments. Compared to ME that uses a conventional and constantly applied electric field (isoelectrostatic) method, the ME-PFSG achieved about 15-fold faster analysis time during the separation of 100 bp DNA ladder. The ME-PFSG was also applied to the fast analysis of the PCR products, 591 and 1191 bp DNA fragments from the 18S rRNA of Babesia gibsoni and Babesia caballi.     

Analysis of multiplex PCR fragments with PMMA microchip

Microchip electrophoresis is a promising technique for analysis of bio-molecules. It has the advantages of fast analysis, high sensitivity, high resolution and low-cost of samples. Plastic chip has the potential of mass production for clinical use for its advantages in biocompatibility and low cost. In this work, the method for fabrication of poly(methyl methacrylate) (PMMA) chip was described, and conditions for DNA separation were investigated with the chip. The PMMA microchip was used for detection of multiplex PCR products of 18 and 36 cases with SARS and hepatitis B virus infection under optimized separation conditions. Microchip electrophoresis showed higher sensitivity, higher resolution and less time consumption when compared with gel electrophoresis. The microchip electrophoresis with PMMA chip provided a rapid, sensitive and reliable method for analysis of multiplex PCR products.     

用于细胞三维培养的集成微柱阵列的微流控芯片设计与验证

设计并验证了一种用于细胞三维培养的集成微柱阵列的微流控芯片.芯片由一片聚二甲基硅氧烷(PDMS)沟道片和一片玻璃盖片组成, 在PDMS沟道片上集成了一个由两排微柱阵列围成的细胞培养室和两条用于输送培养基的侧沟道.微柱间距直接影响了芯片的使用性能, 是整个芯片设计的关键.基于数值模拟和实验验证, 本研究对微柱间距进行了优化设计.优化后的微流控芯片可以很好地实现细胞与细胞外基质模拟材料混合液的稳定注入、培养基中营养物质向培养室内的快速扩散和细胞代谢物的及时排出.在芯片上进行了神经干细胞的三维培养, 证明了芯片上构建的细胞体外微环境的稳定性.     

溶胶凝胶微流控酶反应器用于蛋白质肽谱分析的研究

The silica-based poly(dimethylsiloxane)(PDMS)microfluidic enzymatic reactor was reported along with itsanalytical features in coupling with MALDI TOF and ESI MS.Microfluidic chip was fabricated using PDMS cast-ing and O_2-plasma techniques,and used for the preparation of enzymatic reactor.Plasma oxidation for PDMS en-abled the channel wall of microfluidics to present a layer of silanol(SiOH)groups.These SiOH groups as anchorsonto the microchannel wall were linked covalently with the hydroxy groups of trypsin-encapsulated sol matrix.As aresult,the leakage of sol-gel matrix from the microchannel was effectively prevented.On-line protein analysis wasperformed with the microfluidic enzymatic reactor by attachment of stainless steel tubing electrode and replaceabletip.The success of trypsin encapsulation was investigated by capillary electrophoresis(CE)detection,and MALDITOF and ESI MS analysis.The lab-made device provided excellent extent of digestion even at the fast flow rate of7.0 μL/min with very short residence time of ca.2 s.In addition,the encapsulated trypsin exhibits increased stabil-ity even after continuous use.These features are the most requisite for high-throughput protein identification.     

Chip-based CE for rapid separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS) derivatized glycans

Fluorescently labeled carbohydrates released from glycoproteins were separated using a commercially available microfluidic chip electrophoresis system. While the instrumentation was primarily designed for DNA analysis it was found that the application base can be easily expanded using the development software provided by the manufacturer. The carbohydrates were released by enzymatic digestion (PNGase F) from glycoproteins present in human plasma after boronic acid - lectin affinity enrichment. After fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonic acid the carbohydrates were separated based on capillary gel electrophoresis mechanism and detected by a fluorescence detector using a blue (470 nm) LED. The separation was completed in 40 s in a microfluidic channel of 14 mm length. Glucose ladder carbohydrate oligomers differing by one glucose unit were baseline separated up to a 20-mer with the main limitation being the detection sensitivity. As expected, the observed resolution in these experiments did not approach that of standard CE with 20 times longer separation distance; however, the chip-based analysis excelled in the speed of the separation. Similar electrophoretic profiles of glycans released from plasma glycoproteins were obtained using a standard CE equipment with 35 cm separation length and microfluidic chips with a separation distance of only 14 mm.     

Rapid fabrication of a poly(dimethylsiloxane) microfluidic capillary gel electrophoresis system utilizing high precision machining

In this work, we demonstrate a rapid protocol to address one of the major barriers that exists in the fabrication of chip devices, creating the micron-sized structures in the substrate material. This approach makes it possible to design, produce, and fabricate a microfluidic system with channel features >10 microm in poly(dimethylsiloxane)(PDMS) in under 8 hours utilizing instrumentation common to most machine shops. The procedure involves the creation of a master template with negative features, using high precision machining. This master is then employed to create an acrylic mold that is used in the final fabrication step to cast channel structures into the PDMS substrate. The performance of the microfluidic system prepared using this fabrication procedure is evaluated by constructing a miniaturized capillary gel electrophoresis (micro-CGE) system for the analysis of DNA fragments. Agarose is utilized as the sieving medium in the micro-CGE device and is shown to give reproducible (RSD (n= 34) approximately 5.0%) results for about 34 individual separations without replenishing the gel. To demonstrate the functionality of the micro-CGE device, a DNA restriction ladder (spanning 26-700 base pairs) and DNA fragments generated by PCR are separated and detected with laser-induced fluorescence (LIF). The microchip is shown to achieve a separation efficiency of 2.53 x 10(5) plates m(-1).     

2D separations on a 1D chip: gradient elution moving boundary electrophoresis-chiral capillary zone electrophoresis

A new method is described for two-dimensional (2D) separations using a microfluidic chip normally employed for single dimension electrophoresis. The method employs a combination of gradient elution moving boundary electrophoresis (GEMBE) and chiral capillary zone electrophoresis (CZE). The simplicity of the first dimension GEMBE method enables its implementation in the injection channel of a conventional electrophoresis chip, simplifying the design and operation of the device. The method was used for high resolution 2D chiral separations of a mixture of amino acids considered as possible signatures of extant or extinct life for solar system exploration. The enantiomers of aspartic acid, glutamic acid, serine, alanine, and valine were all resolved as well as glycine (achiral) and several unidentified impurities, giving an estimated peak capacity of 35 for the region between valine and glycine. The results highlight the need for high peak capacity separations for chiral amino acid analysis if accurate enantiomeric ratios are to be determined.     

Recent advances in surface-enhanced Raman scattering detection technology for microfluidic chips

Microfluidic chip devices and their application to sensitive chemical and biological analyses have attracted significant attention over the past decade. The miniaturization of reaction systems offers practical advantages over conventional benchtop systems. In this case, however, a highly sensitive on-chip detection method is important for the monitoring of chemical reactions as well as for the detection of analytes inside the channel because the detection volume in a micrometer-size channel is extremely small. Recently, a surface-enhanced Raman scattering (SERS) technique is being regarded as a potential candidate for the highly sensitive detection of analytes in a microfluidic chip. This review provides a general survey and an in-depth look at recent developments in SERS techniques for the biological/environmental analysis of minute analytes in a microfluidic chip.     

Coupling microchip electrophoresis with MALDI-TOF-MS based on a freezing technique

A freezing technique protocol was proposed for coupling microchip electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALD1-TOF-MS).The microfluidic flow was frozen immediately after electrophoresis on microfluidic chip and the separated analyte molecules were kept in their zone pattern in the electrophoresis.Then,the frozen-chip was lyophilized and sent into TOF-MS instrument as a MALDI target,and the analyte molecules in the microfluidic channels were subjected to analysis by mass spectrometry.This approach could eliminate sample cross-contamination, providing a new interface for microchip electrophoresis and MALDI-MS.