Self-contained, biomolecular motor-driven protein sorting and concentrating in an ultrasensitive microfluidic chip

We developed a molecular sorter that operates without external power or control by integrating the microtubule-based, biological motor kinesin into a microfluidic channel network to sort, transport, and concentrate molecules. In our devices, functionalized microtubules that capture analyte molecules are steered along kinesin-coated microchannel tracks toward a collector structure, concentrated, and trapped. Using fluorescent analyte molecules and nanoliter sample volumes, we demonstrated 14 fM sensitivity, even in the presence of high concentrations of other proteins.     

Lensfree sensing on a microfluidic chip using plasmonic nanoapertures

We demonstrate lensfree on-chip sensing within a microfluidic channel using plasmonic nanoapertures that are illuminated by a partially coherent quasimonochromatic source. In this approach, lensfree diffraction patterns of metallic nanoapertures located at the bottom of a microfluidic channel are recorded using an optoelectronic sensor-array. These lensfree diffraction patterns can then be rapidly processed, using phase recovery techniques, to back propagate the optical fields to an arbitrary depth, creating digitally focused complex transmission patterns. Cross correlation of these patterns enables lensfree on-chip sensing of the local refractive index surrounding the near-field of the plasmonic nanoapertures. Based on this principle, we experimentally demonstrate lensfree sensing of refractive index changes as small as ～2×10(-3). This on-chip sensing approach could be quite useful for development of label-free microarray technologies by multiplexing thousands of plasmonic structures on the same microfluidic chip, which can significantly increase the throughput of sensing.     

Cytometry and velocimetry on a microfluidic chip using polyelectrolytic salt bridges

This paper reports a polyelectrolytic salt bridge-based electrode (PSBE), which is a key embedded unit in a microchip device that can size-selectively count microparticles and measure their velocities. The construction of salt bridges at specific locations within a microfluidic chip enables dc-driven electrical detection to be performed successfully. This is expected to be a competitive alternative to the optical methods currently used in conventional cell sorters. The PSBEs were fabricated by irradiating ultraviolet light over a patterned mask on the parts of interest, which were filled with an aqueous monomer solution containing diallyldimethylammonium chloride. A pair of such PSBEs was easily formed at the two lateral branches perpendicular to the main microchannel and was found to be very useful for dc impedometry. The human blood cells as well as the fluorescent microbeads passing between the two PSBEs produced impedance signals in proportional to their size. The information about the velocity of a microparticle was extracted from a doublet of the dc impedance signals, which were generated when cells or microbeads sequentially passed through two PSBE pairs separated from each other by a fixed distance. The plot of peak amplitude versus velocity of the moving microbeads and cells indicated only a slight correlation between the size and the velocity, which means that the peak amplitude of the dc impedance signals alone can provide information about the size of the cells in a mixture. The experimental results showed a screening rate of over 1000 cells s(-1) and a velocity of the cells of over 100 mm s(-1). Compared with the previously suggested electrical detection system based on metal electrodes, the sensitivity and selectivity in cell detection were remarkably improved. In addition, the detection unit including the operating circuit became innovatively simple and the whole device could be miniaturized.     

Detection of viable Cryptosporidium parvum using DNA-modified liposomes in a microfluidic chip

This paper describes a microfluidic chip that enables the detection of viable Cryptosporidium parvum by detecting RNA amplified by nucleic-acid-sequence-based amplification (NASBA). The mRNA serving as the template for NASBA is produced by viable C. parvum as a response to heat shock. The chip utilizes sandwich hybridization by hybridizing the NASBA-generated amplicon between capture probes and reporter probes in a microfluidic channel. The reporter probes are tagged with carboxyfluorescein-filled liposomes. These liposomes, which generate fluorescence intensities not obtainable from single fluorophores, allow the detection of very low concentrations of targets. The limit of detection of the chip is 5 fmol of amplicon in 12.5 microL of sample solution. Samples of C. parvum that underwent heat shock, extraction, and amplification by NASBA were successfully detected and clearly distinguishable from controls. This was accomplished without having to separate the amplified RNA from the NASBA mixture. The microfluidic chip can easily be modified to detect other pathogens. We envision its use in mu-total analysis systems (mu-TAS) and in DNA-array chips utilized for environmental monitoring of pathogens.     

Quantitative polymerase chain reaction using infrared heating on a microfluidic chip

The IR-mediated polymerase chain reaction (IR-PCR) in microdevices is an established technique for rapid amplification of nucleic acids. In this report, we have expanded the applicability of the IR-PCR to quantitative determination of starting copy number by integrating fluorescence detection during the amplification process. Placing the microfluidic device between an IR long-pass filter and a hot mirror reduced the background to a level that enabled fluorescence measurements to be made throughout the thermal cycling process. The average fluorescence intensity during the extension step showed the expected trend of an exponential increase followed by a plateau phase in successive cycles. PUC19 templates at different starting copy numbers were amplified, and the threshold cycle showed an increase for decreasing amounts of starting DNA. The amplification efficiency was 80%, and the gel separation indicated no detectable nonspecific product. A melting curve was generated using IR heating, and this indicated a melting temperature of 85 °C for the 304 bp amplicon, which compared well to the melting temperature obtained using a conventional PCR system. This methodology will be applicable in other types of IR-mediated amplification systems, such as isothermal amplification, and in highly integrated systems that combine pre- and post-PCR processes.     

High throughput assay of diffusion through Cx43 gap junction channels with a microfluidic chip

This paper describes a microfluidic-based assay capable of measuring gap-junction mediated dye diffusion in cultured cells. The technique exploits multistream laminar flow to selectively expose cells to different environments, enabling continuous loading of cells in one compartment while monitoring, in real time, dye diffusion into cells of a neighboring compartment. A simple one-dimensional diffusion model fit to the data extracted the diffusion coefficient of four different dyes, 5-(6)-carboxyfluorescein, 5-chloromethylfluorescein, Oregon green 488 carboxylic acid, and calcein. Different inhibitors were assayed for their ability to reduce dye coupling. The chip can screen multiple inhibitors in parallel in the same cell preparation, demonstrating its potential for high throughput. The technique provides a convenient method to measure gap junction mediated diffusion and a screen for drugs that affect gap junction communication.     

Cytotoxicity of cadmium-containing quantum dots based on a study using a microfluidic chip

There is a lack of reliable nanotoxicity assays available for monitoring and quantifying multiple cellular events in cultured cells. In this study, we used a microfluidic chip to systematically investigate the cytotoxicity of three kinds of well-characterized cadmium-containing quantum dots (QDs) with the same core but different shell structures, including CdTe core QDs, CdTe/CdS core–shell QDs, and CdTe/CdS/ZnS core-shell-shell QDs, in HEK293 cells. Using the microfluidic chip combined with fluorescence microscopy, multiple QD-induced cellular events including cell morphology, viability, proliferation, and QD uptake were simultaneously analysed. The three kinds of QDs showed significantly different cytotoxicities. The CdTe QDs, which are highly toxic to HEK293 cells, resulted in remarkable cellular and nuclear morphological changes, a dose-dependent decrease in cell viability, and strong inhibition of cell proliferation; the CdTe/CdS QDs were moderately toxic but did not significantly affect the proliferation of HEK293 cells; while the CdTe/CdS/ZnS QDs had no detectable influence on cytotoxicity with respect to cell morphology, viability, and proliferation. Our data indicated that QD cytotoxicity was closely related to their surface structures and specific physicochemical properties. This study also demonstrated that the microfluidic chip could serve as a powerful tool to systematically evaluate the cytotoxicity of nanoparticles in multiple cellular events.     

环烯烃聚合物(COP)微流控芯片的制备及其与聚甲基丙烯酸甲酯(PMMA)微流控芯片的性能对比

采用热压方法制备了环烯烃聚合物(COP)微流控芯片.考虑到温度对微结构热压成形的质量影响最大,基于材料的粘弹性特性,通过变温准蠕变实验获得了热压参考温度Tr.实验证明,在该温度下热压成形,宽度和深度方向的复制精度分别达到了97.6%和94.3%.为了研究制备的COP微流控芯片的性能,将其和同一模具制备的PMMA微流控芯片进行了性能对比实验.通过背景荧光实验、电泳实验和DNA分析实验三方面的研究表明,与PMMA芯片相比,COP芯片背景荧光低,电泳效率高,检测重现性相对标准偏差小于2.5%,适用于生化分析.     

Multistage isoelectric focusing in a polymeric microfluidic chip

This paper reports a protocol that improves the resolving power of isoelectric focusing (IEF) in a polymeric microfluidic chip. This method couples several stages of IEF in series by first focusing proteins in a straight channel using broad-range ampholytes and then refocusing segments of the first channel into secondary channels that branch from the first one at T-junctions. Experiments demonstrate that several fluorescent proteins that had focused within a segment of the straight channel in the first stage were refocused at significantly higher resolution due to the shallower pH gradient and higher electrical field gradient. Two variants of green fluorescent protein from the second-stage IEF fractionation were further separated in a third stage. Three stages of IEF were completed in less than 25 min at electric field strengths ranging from 50 to 214 V/cm.     

微流控LAMP芯片构建及在MRSA快速检测中的应用研究

构建一种基于环介导等温扩增(loop-mediated isothermal amplification,LAMP),集细菌在线裂解、核酸提取、目标基因扩增和产物检测一体化的用于病原菌快速检测的集成式微流控芯片。以耐甲氧西林金黄色葡萄球菌(methicillin-resistant staphylococcus,MRSA)为模式菌,以mec A为靶基因,在优化条件下用芯片实现对病原菌的在线检测,完成对101~106cfu MRSA的在线裂解、LAMP扩增和产物测定,采用荧光原位检测可得101~105cfu的检测范围和101cfu的检出限。该微流控LAMP芯片结构简单,操作便捷,可在1 h内实现对MRSA mec A基因的快速检测,具有较高的灵敏度和特异性,为下一步临床生物样本病原菌快速检测微流控芯片系统的构建奠定前期研究基础。     

Integration of dialysis membranes into a poly(dimethylsiloxane) microfluidic chip for isoelectric focusing of proteins using whole-channel imaging detection

A poly(dimethylsiloxane) microfluidic chip-based cartridge is developed and reported here for protein analysis using isoelectic focusing (IEF)-whole-channel imaging detection (WCID) technology. In this design, commercial dialysis membranes are integrated to separate electrolytes and samples and to reduce undesired pressure-driven flow. Fused-silica capillaries are also incorporated in this design for sample injection and channel surface preconditioning. This structure is equivalent to that of a commercial fused-silica capillary-based cartridge for adapting to an IEF analyzer (iCE280 analyzer) to perform IEF-WCID. The successful integration of dialysis membranes into a microfluidic chip significantly improves IEF repeatability by eliminating undesired pressure-driven hydrodynamics and also makes sample injection much easier than that using the first-generation chip as reported recently. In this study, two microfluidic chips with a 100-microm-high, 100-microm-wide and a 200-microm-high, 50-microm-wide microchannel, respectively, were applied for qualitative and quantitative analysis of proteins. The mixture containing six pI markers with a pH range of 3-10 was successfully separated using IEF-WCID. The pH gradient exhibited a good linearity by plotting the pI value versus peak position, and the correlation coefficient reached 0.9994 and 0.9995 separately for the two chips. The separation of more complicated human hemoglobin control sample containing HbA, HbF, HbS, and HbC was also achieved. Additionally, for the quantitative analysis, a good linearity of IEF peak value versus myoglobin concentration in the range of 20-100 microg/mL was obtained.     

Adsorption-resistant acrylic copolymer for prototyping of microfluidic devices for proteins and peptides

A poly(ethylene glycol)-functionalized acrylic copolymer was developed for fabrication of microfluidic devices that are resistant to protein and peptide adsorption. Planar microcapillary electrophoresis (microCE) devices were fabricated from this copolymer with the typical cross pattern to facilitate sample introduction. In contrast to most methods used to fabricate polymeric microchips, the photopolymerization-based method used with the copolymer reported in this work was of the soft lithography type, and both patterning and bonding could be completed within 10 min. In a finished microdevice, the cover plate and patterned substrate were bonded together through strong covalent bonds. Additionally, because of the resistance of the copolymer to adsorption, fabricated microfluidic devices could be used without surface modification to separate proteins and peptides. Separations of fluorescein isothiocyanate-labeled protein and peptide samples were accomplished using these new polymeric microCE microchips. Separation efficiencies as high as 4.7 x 10(4) plates were obtained in less than 40 s with a 3.5-cm separation channel, yielding peptide and protein peaks that were symmetrical.     

pH-controlled diffusion of proteins with different pI values across a nanochannel on a chip

Electrostatic interactions dominate the diffusion of proteins in a nanochannel, which was measured and modeled for pH values above and below the isoelectric points of three lectin proteins. Maximal diffusion coefficients were obtained when the proteins were neutral at their pI. This pH-controlled transport led to a separation of biomolecules across the nanochannel. A pI shift was observed, due to reversible adsorption of proteins on the walls, which affects the pH in the nanochannel.     

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.     

Electrokinetic protein preconcentration using a simple glass/poly(dimethylsiloxane) microfluidic chip

We discovered that a protein concentration device can be constructed using a simple one-layer fabrication process. Microfluidic half-channels are molded using standard procedures in PDMS; the PDMS layer is reversibly bonded to a glass base such as a microscope slide. The microfluidic channels are chevron-shaped, in mirror image orientation, with their apexes designed to pass within approximately 20 microm of each other, forming a thin-walled section between the channels. When an electric field is applied across this thin-walled section, negatively charged proteins are observed to concentrate on the anode side of it. About 10(3)-10(6)-fold protein concentration was achieved in 30 min. Subsequent separation of two different concentrated proteins is easily achieved by switching the direction of the electric field in the direction parallel to the thin-walled section. We hypothesize that a nanoscale channel forms between the PDMS and the glass due to the weak, reversible bonding method. This hypothesis is supported by the observation that, when the PDMS and glass are irreversibly bonded, this phenomenon is not observed until a very high E-field was applied and dielectric breakdown of the PDMS is observed. We therefore suspect that the ion exclusion-enrichment effect caused by electrical double layer overlapping induces cationic selectivity of this nanochannel. This simple on-chip protein preconcentration and separation device could be a useful component in practically any PDMS-on-glass microfluidic device used for protein assays.     

Isoelectric focusing in a poly(dimethylsiloxane) microfluidic chip

This paper reports the application of ampholyte-based isoelectric focusing in poly(dimethylsiloxane) (PDMS) using methylcellulose (MC) to reduce electroosmosis and peak drift. Although the characteristics of PDMS make it possible to fabricate microfluidic chips using soft lithography, unstable electroosmotic flow (EOF) and cathodic drift are significant problems when this medium is used. This paper demonstrates that EOF is greatly reduced in PDMS by applying a dynamic coat of MC to the channel walls and that higher concentrations of MC can be used to increase the viscosity of the electrode solutions in order to suppress pH gradient drift and reduce "compression"of the pH gradient. To illustrate the effect of MC on performance, several fluorescent proteins were focused in microchip channels 5 microm deep by 300 microm wide by 2 cm long in 3-10 min using broad-range ampholytes at electric field strengths ranging from 25 to 100 V/cm.     

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.     

微流控芯片制作及电特性研究

采用热压和键合的方法制作玻璃和有机聚合物(PMMA)芯片,对玻璃和PMMA芯片在高压直流电场作用下的伏安特性进行了研究和分析。实验表明,玻璃芯片的伏安线性区域为1100V,PMMA芯片为700V,由于玻璃的导热性能优于PMMA,所以玻璃芯片的伏安线性区域大于PMMA芯片。在此线性段内,根据基尔霍夫电流定律将芯片简化为等效电阻模型,研究了分离电压以及分离焦耳热对芯片分离效果的影响因素,为微流控芯片的优化设计提供了理论依据。     

Automated electric valve for electrokinetic separation in a networked microfluidic chip

This paper describes an automated electric valve system designed to reduce dispersion and sample loss into a side channel when an electrokinetically mobilized concentration zone passes a T-junction in a networked microfluidic chip. One way to reduce dispersion is to control current streamlines since charged species are driven along them in the absence of electroosmotic flow. Computer simulations demonstrate that dispersion and sample loss can be reduced by applying a constant additional electric field in the side channel to straighten current streamlines in linear electrokinetic flow (zone electrophoresis). This additional electric field was provided by a pair of platinum microelectrodes integrated into the chip in the vicinity of the T-junction. Both simulations and experiments of this electric valve with constant valve voltages were shown to provide unsatisfactory valve performance during nonlinear electrophoresis (isotachophoresis). On the basis of these results, however, an automated electric valve system was developed with improved valve performance. Experiments conducted with this system showed decreased dispersion and increased reproducibility as protein zones isotachophoretically passed the T-junction. Simulations of the automated electric valve offer further support that the desired shape of current streamlines was maintained at the T-junction during isotachophoresis. Valve performance was evaluated at different valve currents based on statistical variance due to dispersion. With the automated control system, two integrated microelectrodes provide an effective way to manipulate current streamlines, thus acting as an electric valve for charged species in electrokinetic separations.