Hand-held microanalytical instrument for chip-based electrophoretic separations of proteins

The design, fabrication, and demonstration of a hand-held microchip-based analytical instrument for detection and identification of proteins and other biomolecules are reported. The overall system, referred to as muChemLab, has a modular design that provides for reliability and flexibility and that facilitates rapid assembly, fluid and microchip replacement, troubleshooting, and sample analysis. Components include two independent separation modules that incorporate interchangeable fluid cartridges, a 2-cm-square fused-silica microfluidic chip, and a miniature laser-induced fluorescence detection module. A custom O-ring sealed manifold plate connects chip access ports to a fluids cartridge and a syringe injection port and provides sample introduction and world-to-chip interface. Other novel microfluidic connectors include capillary needle fittings for fluidic connection between septum-sealed fluid reservoirs and the manifold housing the chip, enabling rapid chip priming and fluids replacement. Programmable high-voltage power supplies provide bidirectional currents up to 100 microAlpha at 5000 V, enabling real-time current and voltage monitoring and facilitating troubleshooting and methods development. Laser-induced fluorescence detection allows picomolar (10(-11) M) detection sensitivity of fluorescent dyes and nanomolar sensitivity (10(-9) M) for fluorescamine-labeled proteins. Migration time reproducibility was significantly improved when separations were performed under constant current control (0.5-1%) as compared to constant voltage control (2-8%).     

Microfluidic chip for low-flow push-pull perfusion sampling in vivo with on-line analysis of amino acids

Multilayer soft lithography was used to prepare a poly(dimethylsiloxane) microfluidic chip that allows for in vivo sampling of amino acid neurotransmitters by low-flow push-pull perfusion. The chip incorporates a pneumatically actuated peristaltic pump to deliver artificial cerebrospinal fluid to a push-pull perfusion probe, pull sample from the probe, perform on-line derivatization with o-phthaldialdehyde, and push derivatized amino acids into the flow-gated injector of a high-speed capillary electrophoresis-laser-induced fluorescence instrument. Peristalsis was achieved by sequential actuation of six, 200 microm wide by 15 microm high control valves that drove fluid through three fluidic channels of equal dimensions. Electropherograms with 100,000 theoretical plates were acquired at approximately 20-s intervals. Relative standard deviations of peak heights were 4% in vitro, and detection limits for the excitatory amino acids were approximately 60 nM. For in vivo measurements, push-pull probes were implanted in the striatum of anesthetized rats and amino acid concentrations were monitored while sampling at 50 nL/min. o-Phosphorylethanolamine, glutamate, aspartate, taurine, glutamine, serine, and glycine were all detected with stable peak heights observed for over 4 h with relative standard deviations of 10% in vivo. Basal concentrations of glutamate were 1.9 +/- 0.6 microM (n = 4) in good agreement with similar methods. Monitoring of dynamic changes of neurotransmitters resulting from 10-min applications of 70 mM K(+) through the push channel of the pump was demonstrated. The combined system allows temporal resolution for multianalyte monitoring of approximately 45 s with spatial resolution 65-fold better than conventional microdialysis probe with 4-mm length. The system demonstrates the feasibility of sampling from a complex microenvironment with transfer to a microfluidic device for on-line analysis.     

Velocity measurement of particles flowing in a microfluidic chip using Shah convolution Fourier transform detection

A noninvasive radiative technique, based on Shah convolution Fourier transform detection, for velocity measurement of particles in fluid flows in a microfluidic chip, is presented. It boasts a simpler instrumental setup and optical alignment than existing measurement methods and a wide dynamic range of velocities measurable. A glass-PDMS microchip with a layer of patterned Cr to provide multiple detection windows which are 40 microns wide and 70 microns apart is employed. The velocities of fluorescent microspheres, which were electrokinetically driven in the channel of the microfluidic chip, were determined. The effects of increasing the number of detection windows and sampling period were investigated. This technique could have wide applications, ranging from the determination of the velocity of particles in pressure-driven flow to the measurement of electrophoretic mobilities of single biological cells.     

集成一次性微流控芯片的便携式荧光检测系统

本文研究了一种新型的生物化学分析系统,该系统包括便携式荧光检测仪和带光纤的微流控芯片．采用基于MEMS技术的微泵将待测物与荧光试剂的混合物导入微流控芯片,采用PMT检测受激发产生的荧光,荧光强度与待测物浓度成一定比例．激发光则通过光纤将光源LED光信号导入微沟道中．随着液体在微沟道中的流动,可连续分析和检测不同的样品．该系统检测1～1000μg／L浓度的荧光素具有0．966的相关系数．基于荧光猝灭原理,该系统还可检测浓度为5ng／μL的硝基化合物．该生化分析系统除具有便携式和一次性微流控芯片优点外,还具有成本低．试剂、样品消耗量少,且分析时间短等优点该系统能实现现场检测,可应用于临床诊断、环境检测及生物战剂检测等领域     

Effect of Hydroxyapatite Nanorods on the Fate of Human Adipose‐Derived Stem Cells Assessed In Situ at the Single Cell Level with a High‐Throughput,Real‐Time Microfluidic Chip

The fate of stem cells at the single cell level with limited communication with other cells is still unknown due to the lack of an efficient tool for highly accurate molecular detection. Moreover, the conditional sensitivity of biological experiments requires a sufficient number of parallel experiments to support a conclusion. In this work, a microfluidic single cell chip is designed for use with a protein chip to investigate the effect of hydroxyapatite (HAp) on the osteogenic differentiation of human adipose‐derived stem cells (hADSCs) in situ at the single cell level. By successfully detecting secretory proteins in situ, it is found that the HAp nanorods enhance osteogenic differentiation at the single cell level. In the chip, the single cell seeding approach confirms the osteogenic differentiation of the hADSCs, which endocytoses HAp, by reducing the influence of the factors secreted by neighboring differentiating cells. Most importantly, more than 7000 microchambers provide a sufficient number of parallel experiments for statistical analysis, which ensure a high level of repeatability of the HAp nanorod‐induced osteogenic differentiation. The microfluidic chip comprising single cell culture microchambers with in situ detection capability is a promising tool for research on cell behavior or cell fate at the single cell level.     

Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing

Rapid detection and identification of influenza virus is becoming increasingly important in the face of concerns over an influenza pandemic. A fully integrated and self-contained microfluidic device has been developed to rapidly identify influenza A hemagglutinin and neuraminidase subtypes and sequence portions of both genes. The device consists of a DNA microarray with 12 000 features and a microfluidic cartridge that automates the fluidic handling steps required to carry out a genotyping assay for pathogen identification and sequencing. The fully integrated microfluidic device consists of microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross talk of the stored reagents. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows the detection and identification of influenza virus in a rapid and automated fashion.     

Interactions of human osteoprogenitors with porous ceramic following diffusion chamber implantation in a xenogeneic host

Porous calcium phosphate ceramics are useful bone graft substitutes on account of their osteoconductive properties and lack of toxicity, but they lack osteogenicity and are brittle in nature. Osteogenic properties, and increased biomechanical properties, could be induced by combining them with human bone-forming cell populations. Progress has been hampered both by the lack of a suitable experimental assay of in vivo human bone formation and a suitable in vivo test system with which to study such cells in association with biomaterials. Here, trabecular bone-derived cells and marrow stromal fibroblastic cells from four human donors aged between 14 and 27 y have been cultured in vitro then combined with a porous ceramic within diffusion chambers and implanted into athymic mice. Bone and cartilage formation was found within the chambers primed with cells cultured in the continuous presence of dexamethasone and ascorbate. These tissues were found in close apposition to the ceramic, confirming that the material is biocompatible and bioactive. These findings demonstrate both that appropriately primed human-cell populations can express the fully differentiated osteoblastic phenotype in the diffusion-chamber model, and also that this is a useful system in which to test the interactions of such cell populations with putative biomaterials.     

Microfluidic chips for the study of cell migration under the effect of chemicals

Numerical simulation of the formation of a chemoattractant gradient in reaction chambers of a chip having different geometries enabled the determination of a structure suitable for the study of cell migration, in accordance with which hybrid polymer–glass microfluidic devices were manufactured. Verification of the procedures of alignment of cells in the reaction chamber of the chip by centrifugal force and subsequent culturing of the cells showed that microfluidic chips can be used to study cell migration under the effect of the chemoattractant gradient in vitro.     

A novel honeycomb cell assay kit designed for evaluating horizontal cell migration in response to functionalized self-assembling peptide hydrogels

A clear understanding on cell migration behaviors contributes to designing novel biomaterials in tissue engineering and elucidating related tissue regeneration processes. Many traditional evaluation methods on cell migration including scratch assay and transwell migration assay possess all kinds of limitations. In this study, a novel honeycomb cell assay kit was designed and made of photosensitive resin by 3D printing. This kit has seven hexagonal culture chambers so that it can evaluate the horizontal cell migration behavior in response to six surrounding environments simultaneously, eliminating the effect of gravity on cells. Here this cell assay kit was successfully applied to evaluate endothelial cell migration cultured on self-assembling peptide (SAP) RADA (AcN-RADARADARADARADA-CONH₂) nanofiber hydrogel toward different functionalized SAP hydrogels. Our results indicated that the functionalized RADA hydrogels with different concentration of bioactive motifs of KLT or PRG could induce cell migration in a dose-dependent manner. The total number and migration distance of endothelial cells on functionalized SAP hydrogels significantly increased with increasing concentration of bioactive motif PRG or KLT. Therefore, the honeycomb cell assay kit provides a simple, efficient and convenient tool to investigate cell migration behavior in response to multi-environments simultaneously.     

合成远红外陶瓷功能聚酯的研究

本论述以苯二甲酸二甲酯（ＤＭＴ）、乙二醇（ＥＧ）为原料，远红外陶瓷粉改性剂，采用间歇式聚合方法合成远红外功能聚酯。对陶瓷粉的研磨分散，陶瓷粉用量对聚酯切片性能和聚酯的热性能、远红外发射性能、保温性能的影响进行了研究。同时对改性聚酯的保健功能和毒性也进行了分析。研究结果表明：添加１％的陶瓷粉可以制得具有远红外功能的聚酯。     

Nanodevices: Location of Biomarkers and Reagents within Agarose Beads of a Programmable Nano‐bio‐chip (Small 5/2011)

The slow development of cost‐effective medical microdevices with strong analytical performance characteristics is due to a lack of selective and efficient analyte capture and signaling. The recently developed programmable nano‐bio‐chip (PNBC) is a flexible detection device with analytical behavior rivaling established macroscopic methods. The PNBC system employs ≈300 μm‐diameter bead sensors composed of agarose “nanonets” that populate a microelectromechanical support structure with integrated microfluidic elements. The beads are an efficient and selective protein‐capture medium suitable for the analysis of complex fluid samples. Microscopy and computational studies probe the 3D interior of the beads. The relative contributions that the capture and detection of moieties, analyte size, and bead porosity make to signal distribution and intensity are reported. Agarose pore sizes ranging from 45 to 620 nm are examined and those near 140 nm provide optimal transport characteristics for rapid (<15 min) tests. The system exhibits efficient (99.5%) detection of bead‐bound analyte along with low (≈2%) nonspecific immobilization of the detection probe for carcinoembryonic antigen assay. Furthermore, the role analyte dimensions play in signal distribution is explored, and enhanced methods for assay building that consider the unique features of biomarker size are offered.     

On-chip transformation of bacteria

On-chip transformation of Escherichia coli cells was accomplished for the first time using a microbial array chip. The continuous E. coli transformation procedures were performed on a chip in which the microcompartment was composed of PDMS microfluidic channels and a silicon substrate predeposited with different plasmid DNAs. The PDMS microfluidic device enabled the parallel transformation of E. coli cells with various plasmid DNAs by separating each transformation area. The phenotypic differences reflecting different plasmid DNAs were identified by various approaches such as colorimetry, fluorometry, and electrochemical methods. This microbial array chip could become a versatile tool for many cell biological applications.     

Self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and DNA microarray detection

A fully integrated biochip device that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and DNA microarray sensors was developed to perform DNA analysis of complex biological sample solutions. Sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis), polymerase chain reaction, DNA hybridization, and electrochemical detection were performed in this fully automated and miniature device. Cavitation microstreaming was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads and accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from approximately milliliters of whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.     

Toward integrated detection and graphene-based removal of contaminants in a lab-on-a-chip platform

A novel miniaturized microfluidic platform was developed for the simultaneous detection and removal of polybrominated diphenyl ethers (PBDEs).The platform consists of a polydimethylsiloxane (PDMS) microfluidic chip for an immunoreaction step,a PDMS chip with an integrated screen-printed electrode (SPCE) for detection,and a PDMS-reduced graphene oxide (rGO) chip for physical adsorption and subsequent removal of PBDE residues.The detection was based on competitive immunoassay-linked binding between PBDE and PBDE modified with horseradish peroxidase (HRP-PBDE) followed by the monitoring of enzymatic oxidation of o-aminophenol (o-AP) using square wave anodic stripping voltammetry (SW-ASV).PBDE was detected with good sensitivity and a limit of detection similar to that obtained with a commercial colorimetric test (0.018 ppb),but with the advantage of using lower reagent volumes and a reduced analysis time.The use of microfluidic chips also provides improved linearity and a better reproducibility in comparison to those obtained with batch-based measurements using screen-printed electrodes.In order to design a detection system suitable for toxic compounds such as PBDEs,a reduced graphene oxide-PDMS composite was developed and optimized to obtain increased adsorption (based on both the hydrophobicity and π-π stacking between rGO and PBDE molecules) compared to those of non-modified PDMS.To the best of our knowledge,this is the first demonstration of electrochemical detection of flame retardants and a novel application of the rGO-PDMS composite in a biosensing system.This system can be easily applied to detect any analyte using the appropriate immunoassay and it supports operation in complex matrices such as seawater.     

Bone-like apatite coating on Mg-PSZ/Al2O3 composites using bioactive systems

A biomimetic method was used to promote bioactivity on zirconia/alumina composites. The composites were composed of 80 vol% Mg-PSZ and 20 vol% Al₂O₃. Samples of these bioinert materials were immersed in simulated body fluid (SBF) for 7 days on either a bed of wollastonite ceramics or bioactive glass. After those 7 days, the samples were immersed in a more concentrated solution (1.4 SBF) for 14 days. Experiments were also performed without using a bioactive system during the first stage of immersion. A bone-like apatite layer was formed on the surface of all the materials tested, using wollastonite the bioactive layer was thicker and its morphology was close to that observed on the existing bioactive systems. A thinner apatite layer consisting of small agglomerates was obtained using bioactive glass. The thickness of the ceramic layers was within the range of 15 to 30 μm.     

Effect of glass–ceramic microstructure on its in vitro bioactivity

Two routes were used to obtain a glass–ceramic composed of 43.5 wt % SiO₂ – 43.5 wt % CaO – 13 wt % ZrO₂. Heat treatment of a glass monolith produced a glass–ceramic (WZ1) containing wollastonite-2M and tetragonal zirconia as crystalline phases. The WZ1 did not display bioactivity in vitro. Ceramizing the glass via powder technology routes formed a bioactive glass–ceramic (WZ2). The two glass–ceramics, WZ1 and WZ2, were composed of the same crystalline phases, but differed in microstructure. The in vitro studies carried out on WZ2 showed the formation of an apatite-like layer on its surface during exposure to a simulated body fluid. This paper examined the influence of both chemical and morphological factors on the in vitro bioactivitity. The interfacial reaction product was examined by scanning and transmission electron microscopy. Both instruments were fitted with energy-dispersive X-ray analyzers. Measurements of the pH made directly at the interface of the two glass–ceramics were important in understanding their different behavior during exposure to the same physiological environment.     

Computer-controlled microcirculatory support system for endothelial cell culture and shearing

Endothelial cells (ECs) lining the inner lumen of blood vessels are continuously subjected to hemodynamic shear stress, which is known to modify EC morphology and biological activity. This paper describes a self-contained microcirculatory EC culture system that efficiently studies such effects of shear stress on EC alignment and elongation in vitro. The culture system is composed of elastomeric microfluidic cell shearing chambers interfaced with computer-controlled movement of piezoelectric pins on a refreshable Braille display. The flow rate is varied by design of channels that allow for movement of different volumes of fluid per variable-speed pump stroke. The integrated microfluidic valving and pumping system allowed primary EC seeding and differential shearing in multiple compartments to be performed on a single chip. The microfluidic flows caused ECs to align and elongate significantly in the direction of flow according to their exposed levels of shear stress. This microfluidic system overcomes the small flow rates and the inefficiencies of previously described microfluidic and macroscopic systems respectively to conveniently perform parallel studies of EC response to shear stress.     

Developments toward a microfluidic system for long-term monitoring of dynamic cellular events in immobilized human cells

A microfluidic system for long-term real-time monitoring of dynamic cellular events of immobilized human cells was investigated. The luciferase reporter gene activity in the reporter cell line HFF11, based on HeLa cells, was used as the model system. The cells were immobilized on silicon flow-through microchips and continuously supplied with a cell medium at 2 microL/min while maintaining the chip at 37 degrees C. The HFF11 cell line was designed for high-throughput screening of ligands for seven-transmembrane receptors. When a ligand binds, the receptor is activated and a cascade of intracellular reactions starts, ending with the synthesis of the reporter protein Photinus luciferase. The major goal was to develop a microfluidic system for continuous long-term assaying of the intracellular reporter gene activity in real time and determine the conditions, which could minimize cells stress and hence unspecific expression of the reporter gene. In the resulting microfluidic system and assay protocol, the cell microchip could be kept and assayed for a period up to 30 h. The developed system and data outcome was compared with a corresponding microtiter plate performed with the same cell line to highlight the advantages obtained in the microfluidic format.