高分辨阿达玛变换显微荧光图像分析(Ⅰ)——系统的成像能力及其在单个肿瘤细胞分析中的应用

阿达玛变换(Hadamard transform, HT)是一种类似于傅里叶变换的光谱调制技术, 具有多通道同时检测和多通道成像能力. 实现高分辨HT成像的关键在于阿达玛模板的制作, 阿达玛模板有两种, 即移动式机械编码模板(Movable mechanical mask)和固定式光电模板(Stationary electro-optic mask). 在实际成像方面, 移动模板和固定模板各有优缺点: 前者一般用石英玻璃制作, 对光信号不会因模板吸收而导致信号损失, 因此数据很可靠, 而且模板的制作也较为容易, 但由于采用步进电机驱动而容易导致机械故障, 难以实现快速编码; 后者无移动部件, 无机械故障, 因此系统比较紧凑, 但由于它是由液晶材料制成的(可导致信号损失), 从而限制了其在某些光谱区域的使用. 此外, 它对系统的软件设计要求比前者高, 实现高分辨成像更加困难. 正是由于上述原因, 实现快速、高分辨HT成像具有一定难度, 最近有关HT成像技术的报道极少.     

A prototypic system of parallel electrophoresis in multiple capillaries coupled with microwell arrays

We present a microfluidic system that can be directly coupled with microwell array and perform parallel electrophoresis in multiple capillaries simultaneously. The system is based on an array of glass capillaries, fixed in a polydimethylsiloxane (PDMS) microfluidic scaffold, with one end open for interfacing with microwells. In this capillary array, every two adjacent capillaries act as a pair to be coupled with one microwell; samples in the microwells are introduced and separated by simply applying voltage between two electrodes that are placed at the other ends of capillaries; thus no complicated circuit design is required. We evaluate the performance of this system and perform multiple CE with direct sample introduction from microwell array. Also with this system, we demonstrate the analysis of cellular contents of cells lysed in a microwell array. Our results show that this prototypic system is a promising platform for high-throughput analysis of samples in microwell arrays.     

Trapping cells on a stretchable microwell array for single-cell analysis

There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.     

Measurement of neuropeptides in clinical samples using chip-based immunoaffinity capillary electrophoresis

The current interest in micro-fabrication has extended to the clinical arena where there is a growing lobby for promoting these for point-of-care purposes. The advantages of such devices are their relative speed of analysis, lower reagent costs, and their application to clinical screening and diagnosis. Two chip-based capillary electrophoresis systems have been designed and their performance evaluated for rapidly measuring the concentrations of inflammatory neuropeptides in tissue fluids of patients with neuropeptide-associated muscle pain. Both chips were manufactured to fit a commercially available chip electrophoresis system. One chip was designed to perform electrokinetic flow immunoassays while the other utilized an immunoaffinity port, containing an array of immobilized antibodies, to capture the analytes of interest. Comparison of the results to commercially available high-sensitivity immunoassays demonstrated that both chip-based systems could provide a relatively fast, accurate procedure for studying inflammatory biomarkers in complex biological fluids. However, the immunoaffinity capture system proved the superior of the two chips. Using this system, twelve different inflammation-associated mediators could be determined in approximately 2 min as compared to 30 min when using the flow immunoassay chip. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different analyses.     

Uniform‐sized neurosphere‐mediated motoneuron differentiation in microwell arrays

We developed the photocrosslinkable hydrogel microwell arrays for uniform‐sized neurosphere‐mediated motoneuron differentiation. Neural stem cells (NSCs) were obtained from embryonic cerebral cortex and spinal cord. To generate uniform‐sized neurospheres in a homogeneous manner, the dissociated cells were cultured in the hydrogel microwell arrays for 3 days. Uniform‐sized neurospheres harvested from microwell arrays were replated into laminin‐coated substrate. In parallel, uniform‐sized neurospheres cultured in microwell arrays were encapsulated by photocrosslinkable gelatin methacrylate hydrogels in a three‐dimensional manner. We demonstrated the effect of hydrogel microwell sizes (e.g., 50, 100, 150 μm in diameter) on motoneuron differentiation, showing that the largest uniform‐sized neurospheres derived from embryonic spinal cord efficiently differentiated into motoneurons. Therefore, this hydrogel microwell array could be a powerful array to regulate the uniform‐sized neurosphere‐mediated motoneuron differentiation.     

Optimization of critical factors affecting the performance of an allergen chip for the analysis of an allergen-specific human IgE in serum.

A sensitive and multiplexed assay of allergen-specific human immunoglobulin E (IgE) is of great significance in the precise diagnosis of allergies. We report on the optimization of critical factors for chip-based analysis of IgE in human serum with a high reliability. Extracts of two mite species were used as model allergens, and were spotted onto a glass slide for the construction of an allergen chip. Respective allergen-specific IgE in human serum was analyzed by using biotinylated anti-human IgE and a streptavidin-Cy3 conjugate. Factors affecting the performance of the allergen chip were investigated and optimized. Especially, the effect of additives, the concentrations of biotinylated anti-human IgE and the streptavidin-Cy3 conjugate, the serum dilution factor, and the concentration of allergen extract as a capturing agent were examined in detail. Under the optimized conditions, a chip-based analysis for sera from 43 patients revealed a reliable and reproducible diagnosis of respective allergies, showing a good correlation with a conventional CAP assay.     

Analysis of inflammatory biomarkers from tissue biopsies by chip-based immunoaffinity CE

To aid in the biochemical analysis of human skin biopsies, a semiautomatic chip-based CE system has been developed for measuring inflammatory biomarkers in microdissected areas of the biopsy. Following solubilization of the dissected tissue, the desired biomarkers were isolated by immunoaffinity capture using a panel of 12 antibodies, immobilized on a disposable glass fiber disk, within the extraction port of the chip. The captured analytes were labeled with a 635 nm light-emitting laser dye and electroeluted into the separation channel. Electrophoretic separation of all of the analytes was achieved in 2.2 min with quantification of each peak being performed by online LIF detection and integration of each peak area. Comparison of the results obtained from the chip-based system to those obtained using commercially available high-sensitivity immunoassays demonstrated that the chip-based assay provides a fast, accurate procedure for studying the concentrations of inflammatory biomarkers in complex biological materials. The degree of accuracy and precision achieved by the chip-based CE is comparable to conventional immunoassays and the system is capable of analyzing circa six samples per hour. With the ever-expanding array of antibodies that are commercially available, this chip-based system can be applied to a wide variety of different biomedical analyses.     

Evaluation of cell-free protein synthesis using PDMS-based microreactor arrays.

A living cell has numerous proteins, only a few thousand of which have been identified to date. Cell-free protein synthesis is a useful and promising technique to discover and produce various proteins that might be beneficial for biotechnological, pharmaceutical, and medical applications. For this study, we evaluated the performance and the general applicability of our previously developed microreactor array chip to cell-free protein synthesis by comparisons with a commercially available system. The microreactor array chip comprises a temperature control chip made of glass and a disposable reaction chamber chip made of polydimethylsiloxane (PDMS). For evaluation of the microreactor array chip, rat adipose-type fatty acid binding protein, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin, and firefly luciferase were synthesized from their respective DNA templates using a cell-free extract prepared from Escherichia coli. All these proteins were synthesized in the microreactor array chip, and their respective amounts and yields were investigated quantitatively.     

Instrumentation of a PLC-regulated temperature cycler with a PID control unit and its use for miniaturized PCR systems with reduced volumes of aqueous sample droplets isolated in oil phase in a microwell

We have developed a temperature cycler for polymerase chain reaction (PCR) in a microwell fabricated on a polymer/glass chip. The entire system consisted of three subsystems, which included (1) a thermal conditioner, (2) a proportional-integral-derivative (PID) control signal conditioner and (3) a data acquisition subsystem. The subsystems were regulated coordinately by a ladder logic program written for the programmable logic control (PLC) so that an actual sample temperature could be timed, changed and maintained according to the programmed temperature cycles. The present temperature control system showed high accuracy, stability and minimum overshoot with reduced heating and cooling transition rates. Applicability of the temperature controller to the miniaturized PCR system with reduced volumes of aqueous sample droplets isolated in an oil phase was confirmed by successful amplifications of a target DNA sequence in the microwell.     

MEMS microwell and microcolumn arrays: novel methods for high-throughput cell-based assays

Although the cell-based assay is becoming more popular for high throughput drug screening and the functional characterization of disease-associated genes, most researchers in these areas do not use it because it is a complex and expensive process. We wanted to create a simple method of performing an on-chip cell-based assay. To do this, we used micro-electro-mechanical systems (MEMS) to fabricate a microwell array chip comprised of a glass substrate covered with a photoresist film patterned to form multiple microwells and tested it in two reverse transfection experiments, an exogenous gene expression study and an endogenous gene knockdown study. It was used effectively in both. Then, using the same MEMS technology, we fabricated a complementary microcolumn array to be used as a drug carrier device to topically apply drugs to cells cultured in the microwell array. We tested the effectiveness of microwell-microcolumn on-chip cell-based assay by using it in experiments to identify epidermal growth factor receptor (EGFR) activity inhibitors, for which it was found to provide effective high throughput and high content functional screening. In conclusion, this new method of cell-based screening proved to be a simple and efficient method of characterizing gene function and discovering drug leads.     

A microchip-based flow injection-amperometry system with mercaptopropionic acid modified electroless gold microelectrode for the selective determination of dopamine

A novel chip-based flow injection analysis (FIA) system has been developed for automatic, rapid and selective determination of dopamine (DA) in the presence of ascorbic acid (AA). The system is composed of a polycarbonate (PC) microfluidic chip with an electrochemical detector (ED), a gravity pump, and an automatic sample loading and injection unit. The selectivity of the ED was improved by modification of the gold working microelectrode, which was fabricated on the PC chip by UV-directed electroless gold plating, with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA). Postplating treatment methods for cleaning the surface of electroless gold microelectrodes were investigated to ensure the formation of high quality SAMs. The effects of detection potential, flow rate, and sampling volume on the performance of the chip-based FIA system were studied. Under optimum conditions, a detection limit of 74 nmol L⁻¹ for DA was achieved at the sample throughput rate of 180 h⁻¹. A RSD of 0.9% for peak heights was observed for 19 runs of a 100 μmol L⁻¹ DA solution. Interference-free determination of DA could be conducted if the concentration ratio of AA–DA was no more than 10.     

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.     

Substrate selection for optimum qualitative and quantitative single atmospheric particles analysis using nano-manipulation,sequential thin-window electron probe X-ray microanalysis and micro-Raman spectrometry

Sequential electron probe X-ray microanalysis using thin-window energy dispersive X-ray detection (TW-EDX-EPMA) and micro-Raman spectrometry (MRS) on the same atmospheric particle using nano-manipulation, is demonstrated. The advantageous combination of these two techniques allows information on the morphology, size, elemental and molecular composition, as well as the molecular structure of the same individual particle with a diameter as small as 500 nm. The use of an ultra-thin atmospheric window and a cold stage in EPMA enables qualitative and quantitative analysis of low-Z elements like C, N, and O as well as higher-Z elements. The work illustrates substrate optimisation and subsequent application in the analysis of atmospheric particles. Particle relocation was achieved by manipulative transfer onto transmission electron microscope grids, in an environmental scanning electron microscope, using 100 nm glass tips. A moderate correlation between the elemental composition obtained by TW-EDX-EPMA and the molecular fingerprint obtained by MRS is illustrated and its useful application in the interpretation of indoor air quality is discussed.     

Spatial confinement of avidin domains in microwell arrays

Microwell arrays were chemically etched across the distal faces of coherent fiber-optic bundles. A typical 1.6 mm diameter array comprised approximately 3000 individual microwells that were approximately 1-14 microm deep and approximately 22 microm wide. A methodology involving organosilane functionalized microwell surfaces and site-selective photobiotin chemistry was developed to partially fill microwells with a thin avidin layer. Avidin microwell arrays were characterized using charge coupled device optical microscopy and scanning electron microscopy. The avidin microwell arrays had individual well volumes that were six orders of magnitude smaller and up to 30-fold more numerous than commercially available avidin-coated microtiter plates. Preliminary results indicated that individual avidin microwells were ideally suited to house single biological cells. Using standard epifluorescence microscope optics and a mercury-arc lamp, an individual 22 microm wide microwell could be optically addressed and selectively filled with avidin without the use of a photolithographic mask. The ability to control both the size and position of avidin domains on the microwell array surface demonstrates the utility of this methodology towards fabricating a single microwell array with multianalyte sensing capabilities.     

Directed positioning of single cells in microwells fabricated by scanning probe lithography and wet etching methods

Scanning probe microscopy has emerged as a powerful technique for mapping the surface morphology of biological specimens, including proteins and cells. In addition to providing measurements of topographic images, it enables the fabrication of micro-/nanostructures with a high spatial resolution. Herein, we demonstrate a simple and reliable method for the preparation of single Escherichia coli bacterial cell arrays using pre-fabricated microwell structures. Using a <100>-oriented silicon substrate, microwell arrays with inclined sidewalls were fabricated by scanning probe lithography and sequential chemical wet etching. The trapping efficiency of single cells was optimized by controlling the geometries of the microwells. These data suggest that single-cell arrays may be applicable in a variety of areas, including drug testing and toxicology, as well as basic cell biology.     

微流控芯片流动注射气体扩散分离光度测定系统的研究

提出了纳升级进样量的微流控芯片流动注射气体扩散分离光度检测系统. 制作三层结构微流控芯片, 在玻璃片上加工微反应通道, 用聚二甲基硅氧烷[Poly(dimethylsiloxane), PDMS]加工气体渗透膜和具有接收气体微通道的底片, 实现了生成气体的化学反应、气-液分离和检测在同一微芯片上的集成化. 采用缝管阵列纳升流动注射进样系统连续进样, 用吸光度法测定NH+4以验证系统性能. 结果表明， 该系统对NH+4的检出限为140 μmol/L(3σ), 峰高精度为3.7%(n=9). 在进样时间12 s、注入载流48 s和每次进样消耗200 nL试样条件下, 系统分析通量可达60样/h. 若加大样品量到800 nL, 使接收溶液停流1 min, 该系统对NH+4的检出限可达到35 μmol/L(3σ), 但分析通量降低到20样/h.     

Quantitative DNA imaging in breast tumor cells by a Hadamard transform fluorescence imaging microscope.

This paper presents a novel Hadamard transform (HT) fluorescence imaging microscope by combining multiplexed imaging technique with a conventional upright fluorescence microscope for single-cell imaging and quantitative cellular analysis. The HT imaging microscope can provide 511 x 512-pixel single-cell image with high sensitivity within 21 s. In this study, the high potential value of the microscope in biomedical analysis has been demonstrated by using it to evaluate the malignancy degree of thirty cases of human breast tumors based on the measurements of cellular DNA contents, with conclusions highly accordant with pathological diagnosis. The results show that the HT microscope has the ability to analyze very small specimens and the capability of detecting very high ploidy cells, which are advantages over flow cytometry. The microscope was also successfully applied to cellular morphological analysis, and it was demonstrated that a significant linear relationship exists between tumor nuclear DNA contents and the nuclear area, and malignant and benign tumors are significantly different in both DNA contents and nuclear area. The reliability of the HT microscope in cellular DNA measurements was also investigated.     

Functional integration of DNA purification and concentration into a real time micro-PCR chip

Microfluidic devices for on-chip amplification of DNA from various biological and environmental samples have gained extensive attention over the past decades with many applications including molecular diagnostics of disease, food safety and biological warfare testing. But the integration of sample preparation functions into the chip remains a major hurdle for practical application of the chip-based diagnostic system. We present a PCR-based molecular diagnostic device comprised of a microfabricated chip and a centrifugal force assisted liquid handling tube (CLHT) that is designed to carry out concentration and purification of DNA and subsequent amplification of the target gene in a single chip. The reaction chamber of the chip contains an array of pillar structures to increase the surface area for capturing DNA from a raw sample of macro volume in the presence of kosmotropic agents. The CLHT was designed to provide an effective interface between sample preparation and the microfluidic PCR chip. We have characterized the effect of various fluidic parameters including DNA capture, amplification efficiency and centrifugal pressure generated upon varying sample volume. We also evaluated the performance of this system for quantitative detection of E. coli O157:H7. From the samples containing 10(1) to 10(4) cells per mL, the C(T) value linearly increased from 25.1 to 34.8 with an R(2) value greater than 0.98. With the effectiveness and simplicity of operation, this system will provide an effective interface between macro and micro systems and bridge chip-based molecular diagnosis with practical applications.     

微流控芯片测定单细胞内化学组分的进展

细胞是生命的基本单元。由于细胞的个体差异,传统分析群体细胞的方法难以得到单细胞的重要信息。准确可靠地测定单细胞内化学组分的含量能大大提高从正常细胞中辨别不正常细胞的能力,为进一步研究和发展生物化学、医学和临床检验等领域奠定基础。近年来,用微流控芯片进行单细胞分析已引起广泛的兴趣。微流控芯片可以集成单细胞进样、溶膜、电泳分离胞内化学组分和高灵敏度测定等一系列操作步骤,为分析单细胞内的化学组分提供了新的技术平台。本文主要综述了近年来微流控芯片测定单细胞内化学组分的进展。重点在于利用电渗流、压力结合电渗流和激光镊子等技术操控单细胞在微流控芯片上完成单细胞进样、溶膜、细胞内化学组分的电泳分离和高灵敏度测定等一系列操作步骤。对在微流控芯片上的衍生技术也做了较为详细的阐述。     

Rapid assessment of drug response in cancer cells using microwell array and molecular imaging

Selection of personalized chemotherapy regimen for individual patients has significant potential to improve chemotherapy efficacy and to reduce the deleterious effects of ineffective chemotherapy drugs. In this study, a rapid and high-throughput in vitro drug response assay was developed using a combination of microwell array and molecular imaging. The microwell array provided high-throughput analysis of drug response, which was quantified based on the reduction in intracellular uptake (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose) (2-NBDG). Using this synergistic approach, the drug response measurement was completed within 4 h, and only a couple thousand cells were needed for quantification. The broader application of this microwell molecular imaging approach was demonstrated by evaluating the drug response of two cancer cell lines, cervical (HeLa) and bladder (5637) cancer cells, to two distinct classes of chemotherapy drugs (cisplatin and paclitaxel). This approach did not require an extended cell culturing period, and the quantification of cellular drug response was 4–16 times faster compared with other cell-microarray drug response studies. Moreover, this molecular imaging approach had comparable sensitivity to traditional cell viability assays, i.e., the MTT assay and propidium iodide labeling of cellular nuclei;and similar throughput results as flow cytometry using only 1,000–2,000 cells. Given the simplicity and robustness of this microwell molecular imaging approach, it is anticipated that the assay can be adapted to quantify drug responses in a wide range of cancer cells and drugs and translated to clinical settings for a rapid in vitro drug response using clinically isolated samples.