基于MEMS技术的毛细管电泳芯片

基于MEMS技术的毛细管电泳芯片具有体积小、成本低、便于携带、分析速度快、所需样品和试剂少等优点,是一种新型的微型分析实验装置。介绍该芯片的结构、进样方式、材料选取、制作工艺、键合方法、样品检测方法等方面的研究进展和存在的问题,展望了应用前景 。     

Isotachophoresis preconcentration integrated microfluidic chip for highly sensitive genotyping of the hepatitis B virus

The genotyping of hepatitis B virus (HBV) has become recently a valuable tool not only for epidemiological reasons but also for the clinical practice. Conventional methods for HBV genotyping typically include amplification of the target DNA sequences with a two-round nested PCR followed by separation of the amplified fragments by gel electrophoresis. A microfluidic chip that couples isotachophoresis (ITP) preconcentration and zone electrophoresis (ZE) separation may provide great advantages for sensitive, rapid and cost-effective clinical analysis. In this study, an HBV genotyping method with only one amplification round was developed by the application of the ITP-ZE chip. All the analysis steps of the ITP-ZE separation including sample injection, stacking and separation were performed continuously, controlled by sequential high-voltage switching. A 2.1cm sample plug was preconcentrated between discontinuous buffers in ITP process, followed by ZE separation. Sensitivity enhancement was obtained through the increase of sample loading volume. The average LOD value of the ITP-ZE microfluidic chip was determined to be 0.0021pg/muL. In a large-scale HBV genotyping test, single round PCR products were analyzed by ITP-ZE microfluidic chip, and the results were compared with that of the conventional method. Among the 200 cases studied, the classification rate obtained with microfluidic chip was 93%, which was 6% higher than that obtained with the conventional method. Method with ITP-ZE chip analysis provides HBV genotyping information in reduced PCR amplification time with higher detection rate when compared with conventional method. This method holds great potential for extrapolation to the abundance of similar molecular biology-based techniques in clinical diagnosis.     

Development of a high-throughput IMS-IMS-MS approach for analyzing mixtures of biomolecules

A high-throughput approach for biomolecule analysis is demonstrated for a mixture of peptides from tryptic digest of four proteins as well as a tryptic digests of human plasma. In this method a chip based electrospray autosampler coupled to a hybrid ion mobility (IMS) mass spectrometer (MS) is utilized to achieve rapid sample analysis. This high-throughput measurement is realized by exploiting the direct infusion capability of the chip based electrospray with its rapid sample manipulating capability as well as a high sensitive IMS-MS with a recently developed IMS-IMS separation technique that can be multiplexed to provide greater throughput. From replicate IMS-MS runs of known mixtures, the average uncertainty of peak intensities is determined to be +/-7% (relative standard deviation), and a detection limit in the low attomole range is established. The method is illustrated by analyzing 124 human plasma protein samples in duplicate, a measurement that required 16.5 h. Current limitations as well as implications of the high-throughput approach for complex biological sample analysis are discussed.     

基于微流控技术的微生物细胞梯度稀释分离方法

随着微流控分析技术的快速发展,集成化的微流控芯片在满足实验高通量的同时,还在微生物细胞分离领域呈现出独特的优势。本研究基于微流控技术,制备了以聚二甲基硅氧烷(PDMS)、玻片为材料的细菌细胞梯度稀释分离芯片。该芯片的核心是通过一系列复杂的梯度网络来实现对细菌悬液的连续稀释,最终被分离的细菌细胞进入通道末端的存储孔内。结果显示,该方法能分离出的最少细菌细胞数低于10个。此芯片平台操作简单、耗时短、成本低,为微生物单细胞研究提供了新的途径。     

微流控芯片技术用于精子与上皮细胞分离的研究

为研究建立使用微流控芯片技术分离精子与阴道上皮细胞的方法,选用制作工艺简单的玻璃-PDMS芯片,对混合样本进行分离。加样前分别在进、出口池中加入7此和lO此缓冲液,然后在进样口加入2此混合样本。至少静置8nlin后,从出口池中取出3此形成重力驱动的微流体后开始分离,每隔5min在进样口补加1此缓冲液。达到理想分离效果时,用移液器从出口池取出分离出的精子,核酸酶去除游离DNA,经过提取、扩增和电泳分离脸测等步骤得到精子的分型结果。结果显示,使用基于重力驱动微流体原理的微流控芯片可在30min内分离出精子,不会有上皮细胞进入分离通道;通过核酸酶对分离出的精子液的去游DNA处理,可得到单一、完整的精子分型。与传统的差异裂解法相比,这种方法在很大程度上节省了检验时间,在性侵案件中具有一定的法医物证分析价值。     

Enhancing the Detection of Giardia duodenalis Cysts in Foods by Inertial Microfluidic Separation

The sensitivity and specificity of current Giardia cyst detection methods for foods are largely determined by the effectiveness of the elution, separation, and concentration methods used. The aim of these methods is to produce a final suspension with an adequate concentration of Giardia cysts for detection and a low concentration of interfering food debris. In the present study, a microfluidic device, which makes use of inertial separation, was designed and fabricated for the separation of Giardia cysts. A cyclical pumping platform and protocol was developed to concentrate 10-ml suspensions down to less than 1 ml. Tests involving Giardia duodenalis cysts and 1.90-μm microbeads in pure suspensions demonstrated the specificity of the microfluidic chip for cysts over smaller nonspecific particles. As the suspension cycled through the chip, a large number of beads were removed (70%) and the majority of the cysts were concentrated (82%). Subsequently, the microfluidic inertial separation chip was integrated into a method for the detection of G. duodenalis cysts from lettuce samples. The method greatly reduced the concentration of background debris in the final suspensions (10-fold reduction) in comparison to that obtained by a conventional method. The method also recovered an average of 68.4% of cysts from 25-g lettuce samples and had a limit of detection (LOD) of 38 cysts. While the recovery of cysts by inertial separation was slightly lower, and the LOD slightly higher, than with the conventional method, the sample analysis time was greatly reduced, as there were far fewer background food particles interfering with the detection of cysts by immunofluorescence microscopy.     

An integrated chip for rapid, sensitive, and multiplexed detection of cardiac biomarkers from fingerprick blood

Cardiovascular diseases are the major cause of death among adults worldwide. Electrocardiogram (ECG) is a first test when a patient suffering from chest pain sees a doctor, however, it is lack of the required sensitivity. Standard assays to detect cardiac biomarkers, like enzyme-linked immunosorbent assay (ELISA) are sensitive, but suffer from important sample and reagent consumption in large-scale studies. Moreover they are performed in central laboratories of clinics and hospitals and take a long time, which is highly incompatible with the quick decisions needed to save a heart attack patient. Herein, we describe an integrated chip allowing rapid, sensitive, and simultaneous analysis of three cardiac biomarkers in fingerprick blood. The integrated chip is composed of a filtration chip for plasma separation from blood and a silicon nanowire (SiNW) array sensor chip for protein detection. These two chips are fabricated separately and bonded to form a single unit after alignment. The integrated chip is capable of reducing the dead volume of the sample by eliminating the tubing between the two chips. After the plasma is filtrated by the filtration chip, the SiNW sensor, spotted with three different antibodies, enabled us to detect three cardiac biomarkers, troponin T (cTnT), creatine kinase MM (CK-MM) and creatine kinase MB (CK-MB), simultaneously. The integrated chip is able to attain a low detection limit of 1 pg/ml for the three cardiac biomarkers from 2 μl blood in 45 min.     

Lab-on-a-chip: applications in proteomics

Recent advances in chip-based separation of proteins provide methods that are faster and more convenient than conventional gel electrophoresis. Rapid and automated protein sizing on a chip is at the commercial stage and first attempts have been made to perform two-dimensional separation on a chip. Numerous designs have been described to interface a microfluidic chip to a mass spectrometer. Impressive integration efforts are demonstrated by the ability to perform on-chip trypsin digestion, separation and injection into a mass spectrometer with a single device.     

Effect of antifungal agents on protein composition of Candida albicans studied by capillary electrophoresis and chip technology

In the present study protein profile of a Candida albicans strain had been examined by chip technology and conventional capillary electrophoresis (CE). Profiles could be characterised by the presence of ten dominating protein peaks. These proteins could be distinguished by both techniques, but their quantity showed significant differences in the electropherograms obtained by CE and chip method. Changes in the protein profile were induced by administration of different antifungal agents. Fluconazole and amphotericin B treatment was able to induce similar changes in the pattern, appearance of a 40-kDa protein and up-regulation of a 60-kDa protein was observed by chip technology. Increase in the quantity of these proteins under stress effect (antifungal treatment) might refer to their stress function in the fungal cell. Treatment of C. albicans cells with MK 94 (fused cyclic Mannich ketone) antifungal compound induced not only the previously mentioned changes, but further specific alterations, appearance of a 19-kDa protein and up-regulation of the low molecular weight proteins. This might refer to the different mode of action of this agent on the fungal cells. Conventional capillary electrophoresis was suitable to detect the appearance of the 19-kDa peak, and up-regulation of the 60 kDa protein, but the other changes could not be detected by this technique. Shorter running time, more effective and baseline separation of proteins refer to the advantages of microchip-based method in the analysis of complex biological samples.     

A chip‐based amide‐HILIC LC/MS platform for glycosaminoglycan glycomics profiling

A key challenge to investigations into the functional roles of glycosaminoglycans (GAGs) in biological systems is the difficulty in achieving sensitive, stable, and reproducible mass spectrometric analysis. GAGs are linear carbohydrates with domains that vary in the extent of sulfation, acetylation, and uronic acid epimerization. It is of particular importance to determine spatial and temporal variations of GAG domain structures in biological tissues. In order to analyze GAGs from tissue, it is useful to couple MS with an on‐line separation system. The purposes of the separation system are both to remove components that inhibit GAG ionization and to enable the analysis of very complex mixtures. This contribution presents amide–silica hydrophilic interaction chromatography (HILIC) in a chip‐based format for LC/MS of heparin, heparan sulfate (HS) GAGs. The chip interface yields robust performance in the negative ion mode that is essential for GAGs and other acidic glycan classes while the built‐in trapping cartridge reduces background from the biological tissue matrix. The HILIC chromatographic separation is based on a combination of the glycan chain lengths and the numbers of hydrophobic acetate (Ac) groups and acidic sulfate groups. In summary, chip based amide‐HILIC LC/MS is an enabling technology for GAG glycomics profiling.     

Senescence chips for ultrahigh‐throughput isolation and removal of senescent cells

Cellular senescence plays an important role in organismal aging and age‐related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high‐throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh‐throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof‐of‐principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single‐cell resolution. After scale‐up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh‐throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.     

蛋白质芯片在微生物学领域的应用进展

蛋白质芯片是蛋白质组学研究的一种新技术。具有高通量、微型化、连续化、自动化、快速和准确等特点。已成功地应用于蛋白质的鉴定、量化和基础功能及蛋白质组学的研究。在微生物的监测、检测、分离、鉴定分类和微生物资源开发等方面拥有巨大的应用前景。就蛋白质芯片技术及其在微生物领域的应用进展作了概述。     

Chemiluminescence detection with separation techniques for bioanalytical applications

Chemiluminescence detection is known to be a sensitive, selective, and versatile method that can be used in combination with separation techniques such as high-performance liquid chromatography, capillary electrophoresis, and chip electrophoresis. This article reviews the bioanalytical applications of a combination of chemiluminescence detection and separation techniques published in the literature between 1999 and 2008. Luminol chemiluminescene, peroxyoxalate chemiluminescence, and electrochemiluminescence have been mainly used for bioanalytical application. In this paper, only the applications of the method for the analysis of biosamples, serum, plasma, urine, and tissue samples are discussed.     

Versatile tool for the manipulation of electrophoresis chips

This communication describes a versatile tool allowing standard operations (i.e. washing, pre-conditioning, separation, inner surface modification of the chip channel) with capillary electrophoresis chips. Through currently designed for a chip of maximal dimensions 30 x 60 mm, other formats of the chip require only a minimum adjustment of the equipment, namely setting of the chip sliding rails and adequate arrangement of the exchangeable heads. The application of the tool is demonstrated by the separation of the standard set of inorganic cations.     

Dielectrophoretic separation of monocytes from cancer cells in a microfluidic chip using electrode pitch optimization

This study proposes a microfluidic device capable of separating monocytes from a type of cancer cell that is called T-cell acute lymphoblastic leukemia (RPMI-8402) in a continuous flow using negative and positive dielectrophoretic forces. The use of both the hydrodynamic and dielectrophoretic forces allows the separation of RPMI-8402 from monocytes based on differences in their intrinsic electrical properties and sizes. The specific crossover frequencies of monocytes and RPMI-8402 cells have been obtained experimentally. The optimum ranges of electrode pitch-to-channel height ratio at the cross sections with different electrode widths have been generally calculated by numerical simulations of the gradients of the electric field intensities and calculation their effective values (root-mean-square). In the device, the cell sorting has been conducted empirically, and then, the separation performance has been evaluated by analyzing the images before and after dielectrophoretic forces applied to the cells. In this work, the design of a chip with 77 μm gold–titanium electrode pitch was investigated to achieve high purity of monocytes of 95.2%. The proposed device can be used with relatively low applied voltages, as low as 16.5 V (peak to peak). Thus, the design can be used in biomedical diagnosis and chemical analysis applications as a lab-on-chip platform. Also, it can be used for the separation of biological cells such as bacteria, RNA, DNA, and blood cells.

     

Plastic substrates based separation channels in electromigration techniques

Three types of plastic materials (polyester, polyurethane and polymethylmethacrylate) were tested as materials for manufacturing separation columns (polyester and polyurethane capillaries were used) or separation channels (polymethylmethacrylate) in the chip format. A set of 11 fluorescein isothiocyanate amino acid derivatives was used as the test mixture. Using alpha-cyclodextrin additive to the background electrolyte in the case of the chip separation was also tested. The main problem with all plastic separation media was the selectivity of the separation. The best results, practically identical with bare fused silica capillary, were obtained with the polymethylmethacrylate chip, provided that alpha-cyclodextrin in a concentration 40 mmol/l was added to the background electrolyte. An important observation was that in SDS containing background electrolyte all the plastic materials used exhibited a distinct electroosmotic flow, which was ascribe to the sorption of the negatively charged constituents of the background electrolyte to the capillary wall. Regarding the order in which the individual components of the test mixture were brought to the detector only a single change was observed. Histidine migrated in the polystyrene and polymethylmethacrylate separation channels more slowly than in the bare silica or polyurethane based capillaries.     

微流控芯片在水环境污染分析中的应用

近年来,一种新型技术——微流控芯片技术因其分析速度快、消耗低、体积小、操作简单等特点而备受世界各国的广泛重视.该技术以微通道网络为基本特征,以微机电系统(MEMS)工艺为技术依托,将整个实验室的功能集成在微小芯片上,即构成所谓“芯片实验室”.本文从该技术的基本情况出发,介绍了微流控芯片的发展,并从仪器小型化、系统集成化、不同的芯片材料以及多种检测技术等方面,着重讨论了其在水环境污染分析方面的实际应用和发展前景,指出了它当前所面临的一些问题.随着微流控芯片的不断发展,高速多通道检测装置、低成本设备以及集成了多种方法的高通用性微流控检测芯片,都将成为未来研究的热点.     

Microfluidic components and bio-reactors for miniaturized bio-chip applications

In this paper miniaturized disposable micro/nanofluidic components applicable to bio chip, chemical analyzer and biomedical monitoring system, such as blood analysis, micro dosing system and cell experiment, etc are reported. This system includes various microfluidic components including a micropump, micromixer, DNA purification chip and single-cell assay chip. For low voltage and low power operation, a surface tension-driven micropump is presented, as well as a micromixer, which was implemented using MEMS technology, for efficient liquid mixing is also introduced. As bio-reactors, DNA purification and single-cell assay devices, for the extraction of pure DNA from liquid mixture or blood and for cellular engineering or high-throughput screening, respectively, are presented.     

硅-玻璃聚合酶链式反应微芯片对β-葡糖苷酸酶基因的扩增

聚合酶链式反应（PCR）微芯片是基于微机电系统（MEMS）制作,在微芯片上进行PCR反应,实现生物样品扩增的一项新技术.介绍了硅-玻璃PCR微芯片的设计和制作、微反应腔的清洗和表面处理、借助外置温度控制系统进行PCR扩增反应以及扩增产物在琼脂糖凝胶电泳下的检测分析,实现了对β-葡糖苷酸酶（GUS）基因的有效扩增,扩增时间由原来的90 min缩短到现在的37 min.     

Novel Sample Preparation for Mass Spectral Analysis of Complex Biological Samples

The ability to combine a selective capture strategy with on chip MALDI-TOF analysis allows for rapid, sensitive analysis of a variety of different analytes. In this overview a series of applications of capture enhanced laser desorption ionization time of flight (CELDI-TOF) mass spectrometry are described. The key feature of the assay is an off-chip capture step that utilizes high affinity bacterial binding proteins to capture a selected ligand. This allows large volumes of sample to be used and provides for a concentration step prior to transfer to a gold chip for traditional mass spectral analysis. The approach can also be adapted to utilize specific antibody as the basis of the capture step. The direct and indirect CELDI-TOF assays are rapid, reproducible and can be a valuable proteomic tool for analysis of low abundance molecules present in complex mixtures like blood plasma.