In-column field-amplified sample stacking of biogenic amines on microfabricated electrophoresis devices

A novel method for performing in-column field-amplified sample stacking (FASS) in chip-based electrophoretic systems is presented. The methodology involves the use of a narrow sample channel (NSC) injector. NSC injectors allow sample plugs to be introduced directly into the separation channel, and subsequent stacking and separation can proceed without any need for leakage control. More importantly, stacking and separation occur in a single step negating the requirement for complex channel geometries and voltage switching to control sample plugs during the stacking procedure. The chip is composed of six paralleled systems. Using the NSC injector design, the number of reservoirs in the multiplexed chip is reduced to N + 2, where N is the number of paralleled systems. This design feature radically reduces the complexity in chip structures and associated chip operation. The approach is applied to the analysis of fluorescently labelled biogenic amines affording detection at concentrations down to 20 pM.     

Electrophoretic chip for high-fidelity fractionation of double-stranded DNA

We report the high fidelity, on-chip fractionation of selected segments from an electrophoretic flow of separated fragments. dsDNA fragments (10-330 base pairs (bp)) were initially separated using a 6.5 cm long channel with an electric field strength of 150 V/cm. As an example of the fractionation process, a target fragment of 20 bp was selected and extracted from the separation channel. The extraction was confirmed and evaluated by fluorescence imaging. High resolution and extraction fidelity were achieved by introducing new procedures for (i) extraction channel-blocking and (ii) segment transfer with cleaning. These procedures are necessary for the development of a practical, fully automated multitarget fractionation electrophoretic chip. A kind of CCD image processing method was introduced to monitor, control, and evaluate the procedure of fractionation. The resolution limits of the separation and extraction are discussed.     

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.     

A thin cover glass chip for contactless conductivity detection in microchip capillary electrophoresis

A microfabricated thin glass chip for contactless conductivity detection in chip capillary electrophoresis is presented in this contribution. Injection and separation channels were photolithographed and chemically etched on the surface of substrate glass, which was bonded with a thin cover glass (100 μm) to construct a new microchip. The chip was placed over an independent contactless electrode plate. Owing to the thinness between channel and electrodes, comparatively low excitation voltage (20–110 V in V_p–p) and frequency (40–65 kHz) were suitable, and favorable signal could be obtained. This microchip capillary electrophoresis device was used in separation and detection of inorganic ions, amino acids and alkaloids in amoorcorn tree bark and golden thread in different buffer solutions. The detection limit of potassium ion was down to 10 μmol/L. The advantages of this microchip system exist in the relative independence between the microchip and the detection electrodes. It is convenient to the replacement of chip and other operations. Detection in different position of the channel would also be available.     

Electrophoretic chip for fractionation of selective DNA fragment

A microchannel chip has been used to fractionate selected segments from an electrophoretic flow of separated fragments. A sample, which covers the size from 35 to 670 bp, was initially separated using an 8.8-cm-long channel at the electric field strength of 100 V/cm. The target fragment of 318 bp was selected and extracted from the separation channel. High-resolution fractionation was achieved by introducing new procedures for blocking, extraction, and segment transfer. Fractionation quality with and without blocking were compared using a 310 Genetic Analyzer (Applied Biosystems). The results show that no contamination was found in the sample, which was fractionated with blocking; however, a contamination by short segments was found in the sample, which was fractionated without blocking. Furthermore, fractionation by the chip was found to be of higher fidelity than that by the polyacrylamide slab gel, which displayed a small overlapped peak after the target peak. Compared with the traditional method, our chips enable faster and high-fidelity fractionation, thus providing a new tool for bioanalysis and other applications.     

A simultaneous space sampling method for DNA fraction collection using a comb structure in microfluidic devices

Fraction collection of selected components from a complex mixture plays a critical role in biomedical research, environmental analysis, and biotechnology. Here, we introduce a novel electrophoretic chip device based on a signal processing theorem that allows simultaneous space sampling for fractionation of ssDNA target fragments. Ten parallel extraction channels, which covered 1.5-mm-long sampling ranges, were used to facilitate the capturing of fast-moving fragments. Furthermore, the space sampling extraction made it possible to acquire pure collection, even from partly overlapping fragments that had been insufficiently separated after a short electrophoretic run. Fragments of 180, 181, and 182 bases were simultaneously collected, and then the recovered DNA was PCR amplified and assessed by CE analysis. The 181-base target was shown to be isolated in a 70-mm-long separation length within 10 min, in contrast to the >50 min required for the 300-mm-long separation channel in our previous study. This method provides effective combination of time and space, which is a breakthrough in the traditional concept of fraction collection on a chip.     

微流控芯片操纵传输及实时监测单细胞量子释放

微流控芯片技术用于细胞生化分析已引起了广泛关注.Harrison等首次在微流控芯片上对细胞群体进行操纵、传输及反应.yang等在微流控芯片上操纵细胞群体的排列,并用荧光检测细胞群体摄取钙的反应.至今还未见到微流控芯片对单个细胞进行操纵传输、定位及实时监测的报道.单细胞受激释放的监测对探索生物体神经传导具有重要意义.     

Numerical analysis of a dielectrophoresis field‐flow fractionation device for the separation of multiple cell types

In this study, a dielectrophoresis field‐flow fractionation device was analyzed using a numerical simulation method and the behaviors of a set of different cells were investigated. By reducing the alternating current frequency of the electrodes from the value used in the original setup configuration and increasing the number of exit channels, total discrimination in cell trajectories and subsequent separation of four cell types were achieved. Cells were differentiated based on their size and dielectric response that are represented in their real part of Clausius–Mossotti factor at different frequencies. A number of novel designs were also proposed based on the original setup configuration. It was seen that by reducing the length of the main channel and the number of electrodes at low frequencies and not changing the inlet flow velocities, cell separation was still achieved successfully, although with a slightly larger electrode voltage. The shorter main channel decreased the residence time for the cells on the chip and also reduced the overall size of the device—these were improvements over the original design. The obtained results can be used to analyze other cell types by knowing their size and dielectric properties to design geometries that can ensure separation.     

Quantification and evaluation of Joule heating in on-chip capillary electrophoresis

We present the use of a novel, picoliter volume interferometer to measure, for the first time, the extent of Joule heating in chip-scale capillary electrophoresis (CE). The simple optical configuration for the on-chip interferometric backscatter detector (OCIBD) consists of an unfocused laser, an unaltered silica chip with a half-cylinder channel and a photodetector. Using OCIBD for millidegree-level noninvasive thermometry, temperature changes associated with Joule heating (2.81 degrees C above ambient) in on-chip CE have been observed in 90 microm wide and 40 microm deep separation channels. The temporal response of Joule heating in isotropically etched channels was exponential, with it taking an excess of 2.7 s to reach equilibrium. Buffer viscosity changes have also been derived from empirical on-chip thermometry data, allowing for the determination of diffusion coefficients for solutes when separated in heated buffers. In addition, OCIBD has allowed the reduction in separation efficiency to be estimated in the absence of laminar flow and due to increased molecular diffusion and lower buffer viscosity. A 7% reduction in separation efficiency was determined for a high current drawing buffer such as Tris-boric acid under an applied field of just 400 V/cm. Results indicate that heating effects in on-chip CE have been underestimated and there is a need to readdress the theoretical model.     

Multistep liquid-phase lithography for fast prototyping of microfluidic free-flow-electrophoresis chips

We present a fast and versatile method to produce functional micro free-flow electrophoresis chips. Microfluidic structures were generated between two glass slides applying multistep liquid-phase lithography, omitting troublesome bonding steps or cost-intensive master structures. Utilizing a novel spacer-less approach with the photodefinable polymer polyethyleneglycol dimethacrylate (PEG-DA), microfluidic devices with hydrophilic channels of only 25 μm in height were generated. The microfluidic chips feature ion-permeable segregation walls between the electrode channels and the separation bed and hydrophilic surfaces. The performance of the chip is demonstrated by free-flow electrophoretic separation of fluorescent xanthene dyes and fluorescently labeled amino acids.     

Rapid analysis of genetically modified organisms by in-house developed capillary electrophoresis chip and laser-induced fluorescence system

A microfabricated, inexpensive, reusable glass capillary electrophoresis chip and a laser-induced fluorescence system were developed in-house for the rapid DNA-based analysis of genetically modified organisms (GMOs). The 35S promoter sequence of cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens were both detected since they are present in most genetically modified organisms. The detection of genetically modified soybean in the presence of unaltered soybean was chosen as a model. Lectin, a plant-specific gene, was also detected for confirmation of the integrity of extracted DNA. The chip was composed of two glass plates, each 25 x 76 mm, thermally bonded together to form a closed structure. Photomasks with cross-topology were prepared rapidly by using polymeric material instead of chrome plates. The widths of the injection and separation channels were 30 and 70 microm, respectively, the effective separation length 4.5 cm. The glass slide was etched to a depth of 30 microm for both the injection and separation channel. The cost of the chip was less than 1 $ and required 2 days for photomask preparation and microfabrication. The separation and detection of polymerase chain reaction-amplified NOS, 35S, and lectin sequences (180, 195, and 181 bp, respectively) was completed in less than 60 s. As low as 0.1% GMO content was detectable by the proposed system after 35 and 40 amplification cycles for 35S and NOS, respectively, using 25 ng of extracted DNA as starting material. This corresponds to only 20 genome copies of genetically modified soybean.     

无死体积蚀刻多孔膜接口的制作及其在二维毛细管电泳分离系统中的应用

介绍了一种在毛细管柱上原位腐蚀而成的多孔膜接口的制作方法,并用该接口构建了一类毛细管电泳二维分离技术平台。柱上原位腐蚀刻成的多孔膜接口具有零死体积、制作过程简易、成本低廉、耐用、柱间切换便捷等优点,特别适合作为基于毛细管柱的二维及多维电泳联用中的接口,是目前二维及多维毛细管柱联用中一类较为新型、实用、理想的接口。以鹿茸冻干粉可溶物样品为例,验证了该接口在二维毛细管电泳联用系统中的可行性和分离效能。实验结果表明：鹿茸冻干粉可溶物整个二维分离分析的时间在1 h内完成,二维分离系统的分辨率和总峰容量都比一维的高。     

Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip electrophoresis

A novel comprehensive two-dimensional (2-D) separation system coupling capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is demonstrated. Reversed-phase cHPLC was used as the first dimension, and chip CE acted as the second dimension to perform fast sample transfers and separations. A valve-free gating interface was devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made confocal laser-induced fluorescence detector was used to perform on-chip high-sensitive detection. The cHPLC effluents were continuously delivered to the chip and pinched injections of the effluents every 20 seconds were employed for chip CE separation. Gradient elution of cHPLC was carried out to obtain the high-efficiency separation. Free-zone electrophoresis was performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system was proved to be effective. The relative standard deviations for migration time and peak height of rhodamine B in 150 sample transfers were 3.2% and 9.8%, respectively. Peptides of the fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin were fairly resolved and detected with this comprehensive 2-D system.     

Microfluidic device for capillary electrochromatography-mass spectrometry

A novel microfabricated device that integrates a monolithic polymeric separation channel, an injector, and an interface for electrospray ionization-mass spectrometry detection (ESI-MS) was devised. Microfluidic propulsion was accomplished using electrically driven fluid flows. The methacrylate-based monolithic separation medium was prepared by photopolymerization and had a positively derivatized surface to ensure electroosmotic flow (EOF) generation for separation of analytes in a capillary electrochromatography (CEC) format. The injector operation was optimized to perform under conditions of nonuniform EOF within the microfluidic channels. The ESI interface allowed hours of stable operation at the flow rates generated by the monolithic column. The dimensions of one processing line were sufficiently small to enable the integration of 4-8 channel multiplexed structures on a single substrate. Standard protein digests were utilized to evaluate the performance of this microfluidic chip. Low- or sub-fmol amounts were injected and detected with this arrangement.     

Capillary liquid chromatography-microchip atmospheric pressure chemical ionization-mass spectrometry

A miniaturized nebulizer chip for capillary liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (capillary LC-microchip APCI-MS) is presented. The APCI chip consists of two wafers, a silicon wafer and a Pyrex glass wafer. The silicon wafer has a DRIE etched through-wafer nebulizer gas inlet, an edge capillary insertion channel, a stopper, a vaporizer channel and a nozzle. The platinum heater electrode and pads for electrical connection were patterned on to the Pyrex glass wafer. The two wafers were joined by anodic bonding, creating a microchip version of an APCI-source. The sample inlet capillary from an LC column is directly connected to the vaporizer channel of the APCI chip. The etched nozzle in the microchip forms a narrow sample plume, which is ionized by an external corona needle, and the formed ions are analyzed by a mass spectrometer. The nebulizer chip enables for the first time the use of low flow rate separation techniques with APCI-MS. The performance of capillary LC-microchip APCI-MS was tested with selected neurosteroids. The capillary LC-microchip APCI-MS provides quantitative repeatability and good linearity. The limits of detection (LOD) with a signal-to-noise ratio (S/N) of 3 in MS/MS mode for the selected neurosteroids were 20-1000 fmol (10-500 nmol l(-1)). LODs (S/N = 3) with commercial macro APCI with the same compounds using the same MS were about 10 times higher. Fast heat transfer allows the use of the optimized temperature for each compound during an LC run. The microchip APCI-source provides a convenient and easy method to combine capillary LC to any API-MS equipped with an APCI source. The advantages and potentials of the microchip APCI also make it a very attractive interface in microfluidic APCI-MS.     

Microchip separations of protein biotoxins using an integrated hand-held device

We report the development of a hand-held instrument capable of performing two simultaneous microchip separations (gel and zone electrophoresis), and demonstrate this instrument for the detection of protein biotoxins. Two orthogonal analysis methods are chosen over a single method in order to improve the probability of positive identification of the biotoxin in an unknown mixture. Separations are performed on a single fused-silica wafer containing two separation channels. The chip is housed in a microfluidic manifold that utilizes o-ring sealed fittings to enable facile and reproducible fluidic connection to the chip. Sample is introduced by syringe injection into a septum-sealed port on the device exterior that connects to a sample loop etched onto the chip. Detection of low nanomolar concentrations of fluorescamine-labeled proteins is achieved using a miniaturized laser-induced fluorescence detection module employing two diode lasers, one per separation channel. Independently controlled miniature high-voltage power supplies enable fully programmable electrokinetic sample injection and analysis. As a demonstration of the portability of this instrument, we evaluated its performance in a laboratory field test at the Defence Science and Technology Laboratory with a series of biotoxin variants. The two separation methods cleanly distinguish between members of a biotoxin test set. Analysis of naturally occurring variants of ricin and two closely related staphylococcal enterotoxins indicates the two methods can be used to readily identify ricin in its different forms and can discriminate between two enterotoxin isoforms.     

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.     

On-chip temperature gradient interaction chromatography

This paper reports the first integrated microelectromechanical system (MEMS) HPLC chip that consists of a parylene high-pressure LC column, an electrochemical sensor, a resistive heater and a thermal-isolation structure for on-chip temperature gradient interaction chromatography application. The separation column was 8 mm long, 100 microm wide, 25 microm high and was packed with 5 microm sized, C18-coated beads using conventional slurry-packing technique. A novel parylene-enhanced, air-gap thermal isolation technology was used to reduce heater power consumption by 58% and to reduce temperature rise in the off-column area by 67%. The fabricated chip consumed 400 mW when operated at 100 degrees C. To test the chromatography performance of the fabricated system, a mixture of derivatized amino acids was chosen for separation. A temporal temperature gradient scanning from 25 to 65 degrees C with a ramping rate of 3.6 degrees C/min was applied to the column during separation. Successful chromatographic separation of derivatized amino acids was carried out using our chip. Compared with conventional temperature gradient HPLC system which incorporates "macro oven" to generate temporal temperature gradient on the column, our chip's thermal performance, i.e., power consumption and thermal response, is greatly improved without sacrificing chromatography quality.     

多功能芯片对合成样本中肝癌细胞HepG2的测试研究

设计并制作了一种集多孔流分离(Multi-orifice flow fractionation,MOFF)技术与磁捕获技术于一体的用于特异性分离和捕获合成样本中肝癌细胞HepG2的多功能微流控细胞芯片.此芯片由玻璃基片和PDMS微通道盖片组成,PDMS盖片上含有3条进样通道、MOFF分离区和六边形腔体的细胞富集检测区.其中,MOFF分离区总长20 mm,由80组长度为0.18 mm、深度为50μm、收缩区域宽度为0.06 mm、扩张区域宽度为0.20 mm的半菱形收缩/扩张重复单元组成,每组收缩/扩张重复单元间的夹角为103.0°.实验以肝癌细胞HepG2-血细胞混悬液为样本;根据磁珠表面修饰c-Met抗体能与肝癌细胞HepG2特异性结合的原理,通过表面羧基化的磁珠、EDC(1 mg/mL)、NHS(1 mg/mL)和c-Met抗体制备了浓度为50μg/mL的免疫磁珠(Anti-MNCs)悬浮液.在样本流速为50μL/min条件下,利用外加磁场实现了血细胞合成样本中微量肝癌细胞HepG2的有效捕获;采用微波加热法以柠檬酸、硫脲为原料制备了用于荧光标记HepG2的碳量子点,在芯片上实现了血液中肝癌细胞HepG2的原位荧光可视化观测.对芯片检测区捕获到的HepG2进行了显微计数分析,对500μL血细胞(107 cell/mL)中含10个HepG2细胞的合成样本,捕获效率达到88.5%±6.7%(n=20).结果表明,所设计的多模式多功能的微流控芯片具有良好的肿瘤细胞分离和检测功能.     

Enrichment of putative stem cells from adipose tissue using dielectrophoretic field-flow fractionation

We have applied the microfluidic cell separation method of dielectrophoretic field-flow fractionation (DEP-FFF) to the enrichment of a putative stem cell population from an enzyme-digested adipose tissue derived cell suspension. A DEP-FFF separator device was constructed using a novel microfluidic-microelectronic hybrid flex-circuit fabrication approach that is scaleable and anticipates future low-cost volume manufacturing. We report the separation of a nucleated cell fraction from cell debris and the bulk of the erythrocyte population, with the relatively rare (<2% starting concentration) NG2-positive cell population (pericytes and/or putative progenitor cells) being enriched up to 14-fold. This work demonstrates a potential clinical application for DEP-FFF and further establishes the utility of the method for achieving label-free fractionation of cell subpopulations.