Effect of surface nanotopography on immunoaffinity cell capture in microfluidic devices

Immunoaffinity microfluidic devices have recently become a popular choice to isolate specific cells for many applications. To increase cell capture efficiency, several groups have employed capture beds with nanotopography. However, no systematic study has been performed to quantitatively correlate surface nanopatterns with immunoaffinity cell immobilization. In this work, we controlled substrate topography by depositing close-packed arrays of silica nanobeads with uniform diameters ranging from 100 to 1150 nm onto flat glass. These surfaces were functionalized with a specific antibody and assembled as the base in microfluidic channels, which were then used to capture CD4+ T cells under continuous flow. It is observed that capture efficiency generally increases with nanoparticle size under low flow rate. At higher flow rates, cell capture efficiency becomes increasingly complex; it initially increases with the bead size then gradually decreases. Surprisingly, capture yield plummets atop depositions of some particle diameters. These dips likely stem from dynamic interactions between nanostructures on the substrate and cell membrane as indicated by roughness-insensitive cell capture after glutaraldehyde fixing. This systematic study of surface nanotopography and cell capture efficiency will help optimize the physical properties of microfluidic capture beds for cell isolation from biological fluids.     

An integrated microfluidic culture device to regulate endothelial cell differentiation from embryonic stem cells

We developed an integrated microfluidic culture device to regulate embryonic stem (ES) cell fate. The integrated microfluidic culture device consists of an air control channel and a fluidic channel with 4×4 micropillar arrays. We hypothesized that the microscale posts within the micropillar arrays would enable the control of uniform cell docking and shear stress profiles. We demonstrated that ES cells cultured for 6 days in the integrated microfluidic culture device differentiated into endothelial cells. Therefore, our integrated microfluidic culture device is a potentially powerful tool for directing ES cell fate.     

Fabrication and performance of a microfluidic traveling-wave electrophoresis system

A microfluidic traveling-wave electrophoresis (TWE) system is reported that uses a locally defined traveling electric field wave within a microfluidic channel to achieve band transport and separation. Low voltages, over a range of -0.5 to +0.5 V, are used to avoid electrolysis and other detrimental redox reactions while the short distance between electrodes, ～25 μm, provides high electric fields of ～200 V cm(-1). It is expected that the low voltage requirements will simplify the future development of smaller portable devices. The TWE device uses four interdigitated electrode arrays: one interdigitated electrode array pair is on the top of the microchannel and the other interdigitated electrode array pair is on the microchannel bottom. The top and bottom substrates are joined by a PDMS spacer that has a nominal height of 15 μm. A pinched injection scheme is used to define a narrow sample band within an injection cross either electrokinetically or hydrodynamically. Separation of two dyes, fluorescein and FLCA, with baseline resolution is achieved in less than 3 min and separation of two proteins, insulin and casein is demonstrated. Investigation of band broadening with fluorescein reveals that sample band widths equivalent to the diffusion limit can be achieved within the microfluidic channel, yielding highly efficient separations. This low level of band broadening can be achieved with capillary electrophoresis, but is not routinely observed in microchannel electrophoresis. Sample enrichment can be achieved very easily with TWE using a device with converging electric field waves controlled by two sets of independently controlled interdigitated electrodes arrays positioned serially along the microchannel. Sample enrichment of 40-fold is achieved without heterogeneous buffer/solvent systems, sorptive, or permselective materials. While there is much room for improvement in device fabrication, and many capabilities are yet to be demonstrated, it is anticipated that the capabilities and performance demonstrated herein will enable new lab-on-a-chip processes and systems.     

Construction of Tumor Tissue Array on An Open-Access Microfluidic Chip

An open-access microfluidic chip which enabled automatic cell distribution and complex multi-step operations was developed. The microfluidic chip featured a key structure in which a nanoporous membrane was sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach took advantage of the characteristics of nanoporous membrane. On one side, this membrane permitted the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution. On the other side, it allowed diffusion-based media exchange and thus, mimicked the endothelial layer. In synergy with a liquid transferring platform, the open-access microfluidic system enabled complex multi-step operations involving medium exchange, drug treatment, and cell viability testing. By using this microfluidic protocol, a 10 × 10 tissue arrays was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays results indicated that the resultant tumor tissue was faithful to that in vivo. Drug testing assays showed that the microfluidic tissue array promised multi-step cell assays under biomimetic microenvironment, thus providing an advantageous tool for cell research.     

Dip-coating crystallization on a superhydrophobic surface: a million mounted crystals in a 1 cm2 array

Silicon wafers (silicon dioxide surfaces) were patterned by photolithograpy to contain 3 μm (width) × 6 μm (length) × 40 μm (height) staggered rhombus posts in a square array (20 μm center-to-center spacing). These surfaces were hydrophobized using a vapor phase reaction with tridecafluorooctyldimethylchlorosilane and exhibit "superhydrophobicity" (water contact angles of θ(A)/θ(R) = 169°/156°). When a section of a wafer is submerged in and withdrawn from water, the superhydrophobic surface emerges, apparently completely dry. If the same procedure is performed using aqueous sodium chloride as the liquid bath, individual crystals of the salt can be observed on the top of each of the posts. "Dip-coating crystallization" using an aqueous sodium chloride solution of 4.3 M produces crystals with ～1 μm dimensions. A less concentrated solution, 1 M NaCl, renders crystals with ～500 nm dimensions. These experiments suggest that superhydrophobic surfaces that emerge from water and are "apparently completely dry" are, in fact, decorated with micrometer-size (several femtoliters) sessile water drops that rapidly evaporate. This simple technique is useful for preparation of very small liquid drops or puddles (of controlled composition) and for preparation of arrays of controlled size, crystalline substances (dip-coating crystallization).     

Mechanical disruption of mammalian cells in a microfluidic system and its numerical analysis based on computational fluid dynamics

The lysis of mammalian cells is an essential part of different lab-on-a-chip sample preparation methods, which aim at the release, separation, and subsequent analysis of DNA, proteins, or metabolites. Particularly for the analysis of compartmented in vivo metabolism of mammalian cells, such a method must be very fast compared to the metabolic turnover-rates, it should not affect the native metabolite concentrations, and should ideally leave cell organelles undamaged. So far, no such a method is available. We have developed a microfluidic system for the effective rapid mechanical cell disruption and established a mathematical model to describe the efficiency of the system. Chinese hamster ovary (CHO) cells were disrupted with high efficiency by passing through two consecutive micronozzle arrays. Simultaneous cell compression and shearing led to a disruption rate of ≥90% at a sample flow rate of Q = 120 μL min(-1) per nozzle passage, which corresponds to a mean fluid velocity of 13.3 m s(-1) and a mean Reynolds number of 22.6 in the nozzle gap. We discussed the problem of channel clogging by cellular debris and the resulting flow instability at the micronozzle arrays. The experimental results were compared to predictions from Computational Fluid Dynamics (CFD) simulations and the critical energy dissipation rate for the disruption of the CHO cell population with known size distribution was determined to be 4.7 × 10(8) W m(-3). Our model for the calculation of cell disruption on the basis of CFD-data could be applied to other microgeometries to predict intended disruption or undesired cell damage.     

Micro-impedance cytometry for detection and analysis of micron-sized particles and bacteria

The sensitivity of a microfluidic impedance flow cytometer is governed by the dimensions of the sample analysis volume. A small volume gives a high sensitivity, but this can lead to practical problems including fabrication and clogging of the device. We describe a microfluidic impedance cytometer which uses an insulating fluid to hydrodynamically focus a sample stream of particles suspended in electrolyte, through a large sensing volume. The detection region consists of two pairs of electrodes fabricated within a channel 200 μm wide and 30 μm high. The focussing technique increases the sensitivity of the system without reducing the dimensions of the microfluidic channel. We demonstrate detection and discrimination of 1 μm and 2 μm diameter polystyrene beads and also Escherichia coli. Impedance data from single particles are correlated with fluorescence emission measured simultaneously. Data are also compared with conventional flow cytometry and dynamic light scattering: the coefficient of variation (CV) of size is found to be comparable between the systems.     

A vertical microfluidic probe

Performing localized chemical events on surfaces is critical for numerous applications. We earlier invented the microfluidic probe (MFP), which circumvented the need to process samples in closed microchannels by hydrodynamically confining liquids that performed chemistries on surfaces (Juncker et al. Nat. Mater. 2005, 4, 622-628). Here we present a new and versatile probe, the vertical MFP (vMFP), which operates in the scanning mode while overcoming earlier challenges that limited the practical implementation of the MFP technology. The key component of the vMFP is the head, a microfluidic device (～1 cm(2) in area) consisting of glass and Si and having microfluidic features fabricated in-plane in the Si layer. The base configuration of the head has two micrometer-size channels that inject/aspirate liquids and terminate at the apex which is ～1 mm(2). In scanning mode, the head is oriented vertically with the apex parallel to the surface with typical spacing of 1-30 μm. Such length scales and using flow rates from nanoliters/second to microliters/second allow chemical events to be performed on surfaces with tens of picoliter quantities of reagents. Before scanning, the head is clipped on a holder for leak-free, low dead volume interface assembly, providing a simple world-to-chip interface. Surfaces are scanned by mounting the holder on a computer-controlled stage having ～0.1 μm resolution in positioning. We present detailed steps to fabricate vMFP heads having channels with dimensions from 1 μm × 1 μm to 50 μm × 50 μm for liquid localization over areas of 10-10,000 μm(2). Additionally, advanced design strategies are described to achieve high yield in fabrication and to support a broad range of applications. These include particulate filters, redundant aperture architectures, inclined flow-paths that service apertures, and multiple channels to enable symmetric flow confinement. We also present a method to characterize flow confinement and estimate the distance between the head and the surface by monitoring the evolution of a solution of fluorescently labeled antibody on an activated glass surface. This flow characterization reveals regimes of operation suitable for different surface topographies. We further integrate the dispensing of immersion liquid to the vMFP head for processing surfaces for extended periods of time (～60 min). The versatility of the vMFP is exemplified by patterning fluorescently labeled proteins, inactivation of cells using sodium hypochlorite, and staining living NIH fibroblasts with Cellomics. These applications are enabled by the compact design of the head, which provides easy access to the surface, simplifies alignment, and enables processing surfaces having dimensions from the micrometer to the centimeter scale and with large topographical variations. We therefore believe that ease-of-operation, reconfigurability, and conservative use of chemicals by the vMFP will lead to its widespread use by microtechnologists and the chemical and biomedical communities.     

Ratchetlike slip angle anisotropy on printed superhydrophobic surfaces

The fabrication and properties of superhydrophobic surfaces that exhibit ratchet-like anisotropic slip angle behavior is described. The surface is composed of arrays of poly(dimethylsiloxane) (PDMS) posts fabricated by a type of 3D printing. By controlling the dispense parameters, regular arrays of asymmetric posts were deposited such that the slope of the posts was varied from 0 to 50 relative to the surface normal. Advancing and receding contact angles as well as slip angles were measured as a function of the post slope and droplet volume. Ratchetlike slip angle anisotropy was observed on surfaces composed of sloped features. The maximum slip angle difference (for a 180° tilt angle variation) was 32° for 20 μL droplets on surfaces with posts fabricated with a slope of 50°. This slip angle anisotropy is attributed to an increase in the triple contact line (TCL) length as the droplet is tilted in a direction against the post slope whereas the TCL decreases continuously when the drop travels in a direction parallel to the post slope. The increasing length of the TCL creates an increased energy barrier that accounts for the higher slip angles in this direction.     

Solid phase extraction of DNA from biological samples in a post-based, high surface area poly(methyl methacrylate) (PMMA) microdevice

This work describes the performance of poly(methyl methacrylate) (PMMA) microfluidic DNA purification devices with embedded microfabricated posts, functionalized with chitosan. PMMA is attractive as a substrate for creating high surface area (SA) posts for DNA capture because X-ray lithography can be exploited for extremely reproducible fabrication of high SA structures. However, this advantage is offset by the delicate nature of the posts when attempting bonding to create a closed system, and by the challenge of functionalizing the PMMA surface with a group that invokes DNA binding. Methods are described for covalent functionalization of the post surfaces with chitosan that binds DNA in a pH-dependent manner, as well as for bonding methods that avoid damaging the underlying post structure. A number of geometric posts designs are explored, with the goal of identifying post structures that provide the requisite surface area without a concurrent rise in fluidic resistance that promotes device failure. Initial proof-of-principle is shown by recovery of prepurified human genomic DNA (hgDNA), with real-world utility illustrated by purifying hgDNA from whole blood and demonstrating it to be PCR-amplifiable.     

Continuous protein production in nanoporous, picolitre volume containers

The synthetic manufacture of functional proteins enables a bottom-up understanding of the workings of biological systems and opens new opportunities for the treatment of disease. Cell-free protein synthesis is a practical approach for enabling such manufacturing, however, it is typically carried out in fairly large volumes, when compared to a natural cell, leading to increases in cost and loss of efficiency. Here we demonstrate continuous cell free protein synthesis in arrays of cellular scale containers that continuously exchange energy and materials with their environment. A multiscale fabrication process allows the monolithic integration of nanoporous silicon containers within an addressable microfluidic network. Synthesis of enhanced green fluorescent protein (eGFP) in the containers continues beyond 24 h and yields more than twice the amount of protein, on a per volume basis, than conventional scale batch reactions. By mimicking the physical volume and controlled flux of a natural cell, the resulting "cell mimic" devices can enable fundamental studies of biological systems as well as serve applications related to the functional screening of proteins and the on-demand production of biologics.     

Membrane assisted passive sampler for triazine compounds in water bodies--characterization of environmental conditions and field performance

This article reports the integration of the fiber optic-particle plasmon resonance (FO-PPR) biosensor with a microfluidic chip to reduce response time and improve detection limit. The microfluidic chip made of poly(methyl methacrylate) had a flow-channel of dimensions 4.0 cm × 900 μm × 900 μm. A partially unclad optical fiber with gold or silver nanoparticles on the core surface was placed within the flow-channel, where the volume of the flow space was about 14 μL. Results using sucrose solutions of various refractive indexes show that the refractive index resolution improves by 2.4-fold in the microfluidic system. The microfluidic chip is capable of delivering a precise amount of biological samples to the detection area without sample dilution. Several receptor/analyte pairs were chosen to examine the biosensing capability of the integrated platform: biotin/streptavidin, biotin/anti-biotin, DNP/anti-DNP, OVA/anti-OVA, and anti-MMP-3/MMP-3. Results show that the response time to achieve equilibrium can be shortened from several thousand seconds in a conventional liquid cell to several hundred seconds in a microfluidic flow-cell. In addition, the detection limit also improves by about one order of magnitude. Furthermore, the normalization by using the relative change of transmission response as the sensor output alleviate the demand on precise optical alignment, resulting in reasonably good chip-to-chip measurement reproducibility.     

Motion of single long DNA molecules through arrays of magnetic columns

We present a videomicroscopy study of T4 DNA (169 kbp) in microfluidic arrays of posts formed by the self-assembly of magnetic beads. We observe DNA moving through an area of 10 000 microm(2), typically containing 100-600 posts. We determine the distribution of the contact times with the posts and the distribution of passage times across the field of view for hundreds of DNA per experiment. The contact time is well approximated by a Poisson process, scaling like the inverse of the field strength, independent of the density of the array. The distribution of passage times allows us to estimate the mean velocity and dispersivity of the DNA during its motion over distances long compared to our field of view. We compare these values with those computed from a lattice Monte Carlo model and geometration theory. We find reasonable quantitative agreement between the lattice Monte Carlo model and experiment, with the error increasing with increasing post density. The deviation between theory and experiment is attributed to the high mobility of DNA after disengaging from the posts, which leads to a difference between the contact time and the total time lost by colliding. Classical geometration theory furnishes surprisingly good agreement for the dispersivity, while geometration theory with a mean free path significantly overestimates the dispersivity.     

Characterization of a hybrid dielectrophoresis and immunocapture microfluidic system for cancer cell capture

The capture of circulating tumor cells (CTCs) from cancer patient blood enables early clinical assessment as well as genetic and pharmacological evaluation of cancer and metastasis. Although there have been many microfluidic immunocapture and electrokinetic techniques developed for isolating rare cancer cells, these techniques are often limited by a capture performance tradeoff between high efficiency and high purity. We present the characterization of shear‐dependent cancer cell capture in a novel hybrid DEP–immunocapture system consisting of interdigitated electrodes fabricated in a Hele‐Shaw flow cell that was functionalized with a monoclonal antibody, J591, which is highly specific to prostate‐specific membrane antigen expressing prostate cancer cells. We measured the positive and negative DEP response of a prostate cancer cell line, LNCaP, as a function of applied electric field frequency, and showed that DEP can control capture performance by promoting or preventing cell interactions with immunocapture surfaces, depending on the sign and magnitude of the applied DEP force, as well as on the local shear stress experienced by cells flowing in the device. This work demonstrates that DEP and immunocapture techniques can work synergistically to improve cell capture performance, and it will aid in the design of future hybrid DEP–immunocapture systems for high‐efficiency CTC capture with enhanced purity.     

Interconnected ordered nanoporous networks of colloidal crystals integrated on a microfluidic chip for highly efficient protein concentration

We report a controllable method to fabricate silica colloidal crystals at defined position in microchannel of microuidic devices using simple surface modification. The formed PCs (photonic crystals) in microfluidic channels were stabilized by chemical cross-linking of Si-O-Si bond between neighboring silica beads. The voids among colloids in PCs integrated on microfluidic devices form interconnected nanoporous networks, which show special electroosmotic properties. Due to the "surface-charge induced ion depletion effect" mechanism, FITC-labeled proteins can be efficiently and selectively concentrated in the anodic boundary of the ion depletion zone. Using this device, about 10(3) - to 10(5)-fold protein concentration was achieved within 10 min. The present simple on chip protein concentration device could be a potential sample preparation component in microfluidic systems for practical biochemical assays.     

多功能芯片对合成样本中肝癌细胞HepG2的测试研究

设计并制作了一种集多孔流分离(Multi-orifice flow fractionation,MOFF)技术与磁捕获技术于一体的用于特异性分离和捕获合成样本中肝癌细胞HepG2的多功能微流控细胞芯片.此芯片由玻璃基片和PDMS微通道盖片组成,PDMS盖片上含有3条进样通道、MOFF分离区和六边形腔体的细胞富集检测区.其中,MOFF分离区总长20 mm,由80组长度为0.18 mm、深度为50μm、收缩区域宽度为0.06 mm、扩张区域宽度为0.20 mm的半菱形收缩/扩张重复单元组成,每组收缩/扩张重复单元间的夹角为103.0°.实验以肝癌细胞HepG2-血细胞混悬液为样本;根据磁珠表面修饰c-Met抗体能与肝癌细胞HepG2特异性结合的原理,通过表面羧基化的磁珠、EDC(1 mg/mL)、NHS(1 mg/mL)和c-Met抗体制备了浓度为50μg/mL的免疫磁珠(Anti-MNCs)悬浮液.在样本流速为50μL/min条件下,利用外加磁场实现了血细胞合成样本中微量肝癌细胞HepG2的有效捕获;采用微波加热法以柠檬酸、硫脲为原料制备了用于荧光标记HepG2的碳量子点,在芯片上实现了血液中肝癌细胞HepG2的原位荧光可视化观测.对芯片检测区捕获到的HepG2进行了显微计数分析,对500μL血细胞(107 cell/mL)中含10个HepG2细胞的合成样本,捕获效率达到88.5%±6.7%(n=20).结果表明,所设计的多模式多功能的微流控芯片具有良好的肿瘤细胞分离和检测功能.     

Laser-induced thermal bubbles for microfluidic applications

We present a unique bubble generation technique in microfluidic chips using continuous-wave laser-induced heat and demonstrate its application by creating micro-valves and micro-pumps. In this work, efficient generation of thermal bubbles of controllable sizes has been achieved using different geometries of chromium pads immersed in various types of fluid. Effective blocking of microfluidic channels (cross-section 500 × 40 μm(2)) and direct pumping of fluid at a flow rate of 7.2-28.8 μl h(-1) with selectable direction have also been demonstrated. A particular advantage of this technique is that it allows the generation of bubbles at almost any location in the microchannel and thus enables microfluidic control at any point of interest. It can be readily integrated into lab-on-a-chip systems to improve functionality.     

Integration of a nanoporous platinum thin film into a microfluidic system for non-enzymatic electrochemical glucose sensing.

In this paper, we describe an amperometric-type enzymeless glucose sensing system based on a nanoporous platinum (Pt) electrode embedded in a microfluidic chip. This microchip system is comprised of a microfluidic transport channel network and a miniaturized electrochemical cell for nonenzymatic glucose sensing. Sample and buffer solutions were transferred to the cell by programmed electroosmotic flow (EOF). A nanoporous Pt electrode with the roughness factor of 200.6 was utilized to determine glucose concentrations in phosphate buffered saline (PBS) by the direct oxidation of glucose, without any separation process. The sensitivity of the developed system is 1.65 microA cm-2 mM-1 in the glucose concentration range from 1-10 mM in PBS.     

Integration of large-area polymer nanopillar arrays into microfluidic devices using in situ polymerization cast molding

Presented here is a simple and robust approach for the integration of mixed-scale (nm-cm) structures into fluidic devices. We report on devices composed of large-area polymer nanopillar arrays of high aspect ratio (33-667) integrated into microfluidic channels fabricated by cast-molding polymerization of methyl methacrylate with mechanically/lithographically patterned, nanoporous aluminium oxide (AAO) templates. The microchannels containing the nanopillar arrays can be chemically functionalized and used for a variety of applications, such as separation beds or solid-phase reactors/extractors.