Two-dimensional electrophoresis on a microfluidic chip for quantitative amino acid analysis

Analysis of complex biological samples requires the use of high-throughput analytical tools. In this work, a microfluidic two-dimensional electrophoresis system was developed with mercury-lamp-induced fluorescence detection. Mixtures of 20 standard amino acids were used to evaluate the separation performance of the system. After fluorescent labeling with fluorescein isothiocyanate, mixtures of amino acids were separated by micellar electrokinetic chromatography in the first dimension and by capillary zone electrophoresis in the second. A double electrokinetic valve system was employed for the sample injection and the switching between separation channels. Under the optimized conditions, 20 standard amino acids were effectively separated within 20 min with high resolution and repeatability. Quantitative analysis revealed linear dynamic ranges of over three orders of magnitudes with detection limits at micromolar range. To further evaluate the reliability of the system, quantitative analysis of a commercial nutrition supplement liquid was successfully demonstrated. Figure       

Injection by hydrostatic pressure in conjunction with electrokinetic force on a microfluidic chip

A simple method was developed for injecting a sample on a cross-form microfluidic chip by means of hydrostatic pressure combined with electrokinetic forces. The hydrostatic pressure was generated simply by adjusting the liquid level in different reservoirs without any additional driven equipment such as a pump. Two dispensing strategies using a floating injection and a gated injection, coupled with hydrostatic pressure loading, were tested. The fluorescence observation verified the feasibility of hydrostatic pressure loading in the separation of a mixture of fluorescein sodium salt and fluorescein isothiocyanate. This method was proved to be effective in leading cells to a separation channel for single cell analysis.     

Spatially selective reagent delivery into cancer cells using a two-layer microfluidic culture system

In this work, we demonstrate a two-layer microfluidic system capable of spatially selective delivery of drugs and other reagents under low shear stress. Loading occurs by hydrodynamically focusing a reagent stream over a particular region of the cell culture. The system consisted of a cell culture chamber and fluid flow channel, which were located in different layers to reduce shear stress on cells. Cells in the center of the culture chamber were exposed to parallel streams of laminar flow, which allowed fast changes to be made to the cellular environment. The shear force was reduced to 2.7 dyn cm⁻² in the two-layer device (vs. 6.0 dyn cm⁻² in a one-layer device). Cells in the side of the culture chamber were exposed to the side streams of buffer; the shear force was further reduced to a greater extent since the sides of the culture chamber were separated from the main fluid path. The channel shape and flow rate of the multiple streams were optimized for spatially controlled reagent delivery. The boundaries between streams were well controlled at a flow rate of 0.1 mL h⁻¹, which was optimized for all streams. We demonstrated multi-reagent delivery to different regions of the same culture well, as well as selective treatment of cancer cells with a built in control group in the same well. In the case of apoptosis induction using staurosporine, 10% of cells remained viable after 24 h of exposure. Cells in the same chamber, but not exposed to staurosporine, had a viability of 90%. This chip allows dynamic observation of cellular behavior immediately after drug delivery, as well as long-term drug treatment with the benefit of large cell numbers, device simplicity, and low shear stress.     

Drug cytotoxicity and signaling pathway analysis with three-dimensional tumor spheroids in a microwell-based microfluidic chip for drug screening

Currently, there has been a growing need for developing in vitro models to better reflect organism response to chemotherapy at tissue level. For this reason, a microfluidic platform was developed for mimicking physiological microenvironment of solid tumor with multicellular tumor spheroids (MTS) for anticancer drug screening. Importantly, the power of this system over traditional systems is that it is simple to operate and high integration in a more physiologically relevant context. As a proof of concept, long-term MTS cultures with uniform structure were realized on the microfluidic based platform. The response of doxorubicin and paclitaxel on different types of spheroids were simultaneously performed by in situ Live/Dead fluorescence stain to provide spatial distribution of dead cells as well as cytotoxicity information. In addition, the established platform combined with microplate reader was capable to determine the cytotoxicity of different sized MTS, showing a more powerful tool than cell staining examination at the end-point of assay. The HCT116 spheroids were then lysed on chip followed by signaling transduction pathway analysis. To our knowledge, the on chip drug screening study is the first to address the drug susceptibility testing and the offline detailed drug signaling pathway analysis combination on one system. Thus, this novel microfluidic platform provides a useful tool for drug screening with tumor spheroids, which is crucial for drug discovery and development.     

Highly selective acylation of ferrocene using microfluidic chip reactor

A rapid and highly selective acylation of ferrocene with various acid anhydrides using microfluidic chip as the reactor is described. The pressure driven glass microreactor was fabricated by standard photolithography and wet etching techniques. High conversions of ferrocene to the corresponding acylferrocenes were achieved at 25 °C and no diacylferrocene was observed in any case.     

A new flow-type cell by the application of magnetic microfluidic chip

Using a magnetically formed channel called a magnetic channel, a new flow-type cell is proposed. The magnetic channel consists of magnetic walls that are formed by heterogeneous distributions of magnetic flux density around a ferromagnetic track under a magnetic field. The magnetic wall separates the paramagnetic oxidant solution from the diamagnetic reductant solution at a liquid–liquid interface without any solid membranes. In the magnetic channel formed on the cathode, the oxidant solution flows in a quasi-frictionless mode. The anode is placed in the reductant solution surrounding the magnetic channel. Such a geometrical configuration between the oxidant and reductant solutions is interchangeable depending on the magnetism of the solutions. To examine this concept, a Daniel cell system was adopted, where the copper ion in copper sulfate solution is employed as the oxidant and the zinc atom of zinc electrode as the reductant. The copper ion is paramagnetic, so that 1 mol dm⁻³ copper sulfate solution is injected into the magnetic channel formed on the copper cathode. Zinc sulfate solution (1 mol dm⁻³; diamagnetic) together with the zinc anode are placed surrounding the magnetic channel. The performance of this flow-type battery was examined up to a current density of 22 mA cm⁻². This paper was presented at the International Symposium on Magneto-Science 2005, Yokohama, 2005. Contribution to the special issue “Magnetic Field Effects in Electrochemistry.”     

Automatic detecting and counting magnetic beads‐labeled target cells from a suspension in a microfluidic chip

A novel microfluidic method of continually detecting and counting beads‐labeled cells from a cell mixture without fluorescence labeling was presented in this paper. The detection system is composed of a microfluidic chip (with a permanent magnet inserted along the channel), a signal amplification circuit, and a LabView® based data acquisition device. The microfluidic chip can be functionally divided into separation zone and detection zone. By flowing the pre‐labeled sample solution, the target cells will be sequentially separated at the separation zone by the permanent magnet and detected and counted at the detection zone by a microfluidic resistive pulse sensor. Experiments of positive separation and detection of T‐lymphocytes and negative separation and detection of cancer cells from the whole blood samples were carried out to demonstrate the effectiveness of this method. The methodology of utilizing size difference between magnetic beads and cell‐magnetic beads complex for beads‐labeled cell detection is simple, automatic, and particularly suitable for beads‐based immunoassay without using fluorescence labeling.     

A microfluidic chip based liquid-liquid extraction system with microporous membrane

A robust and simple approach for microfabricated chip based liquid-liquid extraction was developed for on-chip sample pretreatment. The chip based extraction system was composed of two microfabricated glass plates with a microporous membrane sandwiched in between. A simple bonding approach using epoxy was used to achieve bonding and sealing of the L-L extraction chip. Gravity was employed to drive the aqueous and organic flows through separate channels in the extraction system, separated by the membrane. During extraction, the analyte in an aqueous sample stream was transferred through the membrane into the organic stream. The fluorescence intensity of the analyte extracted into the organic stream was monitored in situ by a laser induced fluorescence detection system. The performance of the system was demonstrated using an aqueous solution of butyl rhodamine B (BRB) and isobutanol as sample and extractant, respectively. The system proved to be an efficient means for achieving chip based microporous membrane liquid-liquid extraction. The precision of fluorescence measurements was 1.5% R.S.D. (n = 4). A linear response range of 1 × 10⁻⁷ to 1 × 10⁻⁴ M BRB was obtained with a regression equation: I = 8.00 × 10⁶ C + 4.91. An enrichment factor of ca. 3 was obtained with an extraction efficiency of 69%.     

氯丙嗪分子印迹化学发光微流控传感器芯片的研究

以氯丙嗪分子印迹聚合物为识别物质,以鲁米诺-K3Fe(CN)6化学发光体系,建立了一种新型的氯丙嗪化学发光微流控分子印迹传感器芯片的检测方法。利用二氧化碳激光在聚甲基丙烯酸甲酯材质上刻蚀出200μm宽,150μm深的微通道,8 mm长,1 mm宽,0.5 mm深的微检测池。微检测池中填充50μm粒径大小的热聚合得到的氯丙嗪分子印迹聚合物作为识别物质,在线富集氯丙嗪,富集的氯丙嗪可以增强鲁米诺和K3Fe(CN)6的化学发光强度,以化学发光强度定量氯丙嗪量。该传感器的响应值与0.02~0.4μg/mL氯丙嗪呈良好的线性关系,检出限为8 ng/mL(3σ)。该微流控传感器芯片已用于测定人尿液中的氯丙嗪。     

Dielectrophoretic separation of microalgae cells in ballast water in a microfluidic chip

The composition of the ship's ballast water is complex and contains a large number of microalgae cells, bacteria, microplastics, and other microparticles. To increase the accuracy and efficiency of detection of the microalgae cells in ballast water, a new microfluidic chip for continuous separation of microalgae cells based on alternating current dielectrophoresis was proposed. In this microfluidic chip, one piece of 3‐dimensional electrode is embedded on one side and eight discrete electrodes are arranged on the other side of the microchannel. An insulated triangular structure between electrodes is designed for increasing the inhomogeneity of the electric field distribution and enhancing the dielectrophoresis (DEP) force. A sheath flow is designed to focus the microparticles near the electrode, so as to increase the suffered DEP force and improve separation efficiency. To demonstrate the performance of the microfluidic separation chip, we developed two species of microalgae cells (Platymonas and Closterium) and a kind of microplastics to be used as test samples. Analyses of the related parameters and separation experiments by our designed microfluidic chip were then conducted. The results show that the presented method can separate the microalgae cells from the mixture efficiently, and this is the first time to separate two or more species of microalgae cells in a microfluidic chip by using negative and positive DEP force simultaneously, and moreover it has some advantages including simple operation, high efficiency, low cost, and small size and has great potential in on‐site pretreatment of ballast water.     

Mixing characteristics of a bubble mixing microfluidic chip for genomic DNA extraction based on magnetophoresis: CFD simulation and experiment

Mixing a small amount of magnetic beads and regents with large volume samples evenly in microcavities of a microfluidic chip is always the key step for the application of microfluidic technology in the field of magnetophoresis analysis. This article proposes a microfluidic chip for DNA extraction by magnetophoresis, which relies on bubble rising to generate turbulence and microvortices of various sizes to mix magnetic beads with samples uniformly. The construction and working principle of the microfluidic chip are introduced. CFD simulations are conducted when magnetic beads and samples are irritated by the generation of gas bubbles with the variation of supply pressures. The whole mixing process in the microfluidic chip is observed through a high-speed camera and a microfluidic system when the gas bubbles are generated continuously. The influence of supply pressure on the mixing characteristics of the microfluidic chip is investigated and discussed with both simulation and experiments. Compared with magnetic mixing, bubble mixing can avoid the magnetic beads gather phenomenon caused by magnetic forces and provide a rapid and high efficient solution to realize mixing small amount of regents in large volume samples in a certain order without complex moving structures and operations in a chip. Two applications of mixing with the proposed microfluidic chip are also carried out and discussed.     

A conjoint multi metal-ion iminodiacetic acid monolith microfluidic chip for structural-based protein pre-fractionation

PDMS-based multichannel microfluidic chip was designed and fabricated in a simple approach using readily available tools. UV-initiated in situ polymerization of poly(2-hydroxy ethyl methacrylate-co-di(ethylene glycol) diacrylate-co-N,Nʹ-diallyl l -tartardiamide) in an Eppendorf tube was achieved within 40 min. This polymerization process was successfully translated to a microfluidic chip format without any further modifications. Iminodiacetic acid was successfully immobilized on aldehyde functional monoliths via Schiff base reaction and confirmed by FT-IR spectroscopy. Four transition metal ions (Co (II), Zn (II), Ni (II), and Cu (II)) were chelated individually on four IDA-monolith microfluidic chips. The conjoint metal-ion monolith microfluidic chip has displayed high permeability (9.40 × 10^–13 m²) and a porosity of 32.8%. This affinity microfluidic chip has pre-fractioned four human plasma proteins (fibrinogen, immunoglobulin, transferrin, and human serum albumin) based on their surface-exposed histidine surface topography. A protein recovery of approximately 95% (Bradford assay data) was achieved. The multimonolith microchip can be reusable even after three protein adsorption-desorption cycles.     

Simultaneous laser-induced fluorescence and contactless-conductivity detection for microfluidic chip

A combined detection system involving simultaneous LIF and contacfless-conductometric measurements at the same place of the microfluidic chip was described. The LIF measurement was designed according to the confocal principle and a moveable contactless-conduetivity detector was used in C^4D. Both measurements were mutually independent and advantageous in analyses of mixtures. Various experimental parameters affecting the response were examined and optimized. The performances were demonstrated by simultaneous detection of Rhodamine B. And the results showed that the combined detection system could be used sensitively and reliably.     

A splicing model‐based DNA‐computing approach on microfluidic chip

DNA computing is a new computation form based on DNA biochemical reactions, which is mainly composed of sticker and splicing computation models. In this work, a microfluidic chip‐based approach was established for splicing model‐based DNA computing. A finite automaton with two input symbols (a, b) and three states (S0, S1, and S2) was applied in the pattern recognition for isosceles triangles. The DNA computation processes of automaton were realized through DNA digestion, ligation, DNA separation, and detection on the microfluidic chip. The established approach is efficient, controllable, and easy to integrate, which paves the way for the building of complete biomolecular computers in the future.     

Application of adaptive pressure-driven microfluidic chip in thyroid function measurement

The improvement in accuracy of in vitro diagnosis has always been the focus of early screening of thyroid dysfunction. We constructed a microfluidic chip based on a polystyrene polymer substrate. Total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), free thyroxine (FT4), and thyrotropin (TSH) in human whole blood samples were analysed by fluorescence immunoassay to evaluate thyroid function. The results indicate that the microfluidic chip shows a good linear relationship in the detection of TT3, TT4, FT3, FT4, and TSH standards, and the correlation coefficient (r) is not less than 0.9900. In addition, the chip also has strong anti-interference (RSD% ≤ 5%) and good repeatability (CV ≤ 8%), and its inter-batch differences are small (CV ≤ 15%). The results of practical application in clinical thyroid function measurement indicated its high accuracy (r ≥ 0.9900). It provides a new method for the determination of thyroid function and lays a foundation for subsequent clinical application.     

Integrated microfluidic cell culture and lysis on a chip

We present an integrated microfluidic cell culture and lysis platform for automated cell analysis that improves on systems which require multiple reagents and manual procedures. Through the combination of previous technologies developed in our lab (namely, on-chip cell culture and electrochemical cell lysis) we have designed, fabricated, and characterized an integrated microfluidic platform capable of culturing HeLa, MCF-7, Jurkat, and CHO-K1 cells for up to five days and subsequently lysing the cells without the need to add lysing reagents. On-demand lysis was accomplished by local hydroxide ion generation within microfluidic chambers, releasing both proteinacious (GFP) and genetic (Hoescht-stained DNA) material. Sample proteins exposed to the electrochemical lysis conditions were immunodetectable (p53) and their enzymatic activity (HRP) was investigated.     

Imitation of drug metabolism in cell co-culture microcapsule model using a microfluidic chip platform coupled to mass spectrometry

In this work,a multi-functional analysis platform by coupling a microfluidic chip to a mass spectrometry(MS) detector was described.We constructed a three-dimensional tumor-endothelial co-culture model for simulating drug resistance during tumor treatment.On this specially designed integrated platform,the first step was to prepare heterogeneous cell-encapsulated alginate microcapsules for threedimensional co-culture,and the second step was to achieve on-line perfusion culture and continuous drug stimulation on chip.It facilitates cell proliferation analysis and the collection of metabolism medium.After micro solid phase extraction column(SPE) pretreatment,subsequent mass spectrometry could detect drug metabolism.The high activity of two kinds of cells(A549 and HUVEC) shows the biocompatibility of the platform.Paclitaxel was used as a model drug,the distinctions of drug absorption between the mono-culture group and co-culture group were clearly observed by electrospray ionization quadrupole time-of-flight mass spectrometry(ESI-Q-TOF MS).Therefore,the integrated platform has shown promise as a high throughput,low cost for cell metabolism research and drug screening processes.     

Ultrasensitive determination of intracellular superoxide in individual HepG2 cells by microfluidic chip electrophoresis

A microchip electrophoresis method was established for the determination of intracellular superoxide (O₂⁻) in individual HepG2 cells. Dihydroethidium (DHE) was used as the specific fluorescent probe to react with intracellular O₂⁻ to form the fluorescent 2-hydroxyethidium. Excellent resolution between 2-hydroxyethidium and ethidium cation (E⁺) can be achieved within 20 s. E⁺ was reported to be generated from photochemical oxidation of DHE and interfere the determination of O₂⁻ with fluorescence microscopic technique. An extremely low detection limit of 2.0 amol was achieved owing to the minute sample volume and insignificant dispersion effect during microfluidic chip-based electrophoretic separation. Furthermore, only 2-hydroxyethidium peak was detected with the suggested single-cell analysis method, which indicates the photooxidation of DHE to E⁺ could be blocked by isolating either oxygen or light from them.     

微流控芯片细胞实验室

以作者所在课题组近年开展的研究工作为基础,阐述了微流控芯片细胞实验室的平台特征,并从细胞个体、群体和多细胞生命体研究等三个方面概述微流控芯片细胞实验室的应用对象特征,显示其在生物医学领域的应用前景。