High Throughput‐Per‐Footprint Inertial Focusing

Matching the scale of microfluidic flow systems with that of microelectronic chips for realizing monolithically integrated systems still needs to be accomplished. However, this is appealing only if such re‐scaling does not compromise the fluidic throughput. This is related to the fact that the cost of microelectronic circuits primarily depends on the layout footprint, while the performance of many microfluidic systems, like flow cytometers, is measured by the throughput. The simple operation of inertial particle focusing makes it a promising technique for use in such integrated flow cytometer applications, however, microfluidic footprints demonstrated so far preclude monolithic integration. Here, the scaling limits of throughput‐per‐footprint (TPFP) in using inertial focusing are explored by studying the interplay between theory, the effect of channel Reynolds numbers up to 1500 on focusing, the entry length for the laminar flow to develop, and pressure resistance of the microchannels. Inertial particle focusing is demonstrated with a TPFP up to 0.3 L/(min cm²) in high aspect‐ratio rectangular microfluidic channels that are readily fabricated with a post‐CMOS integratable process, suggesting at least a 100‐fold improvement compared to previously demonstrated techniques. Not only can this be an enabling technology for realizing cost‐effective monolithically integrated flow cytometry devices, but the methodology represented here can also open perspectives for miniaturization of many biomedical microfluidic applications requiring monolithic integration with microelectronics without compromising the throughput.     

Microfluidic-based cell sorting of Francisella tularensis infected macrophages using optical forces

We have extended the principle of optical tweezers as a noninvasive technique to actively sort hydrodynamically focused cells based on their fluorescence signal in a microfluidic device. This micro fluorescence-activated cell sorter (microFACS) uses an infrared laser to laterally deflect cells into a collection channel. Green-labeled macrophages were sorted from a 40/60 ratio mixture at a throughput of 22 cells/s over 30 min achieving a 93% sorting purity and a 60% recovery yield. To rule out potential photoinduced cell damage during optical deflection, we investigated the response of mouse macrophage to brief exposures (<4 ms) of focused 1064-nm laser light (9.6 W at the sample). We found no significant difference in viability, cell proliferation, activation state, and functionality between infrared-exposed and unexposed cells. Activation state was measured by the phosphorylation of ERK and nuclear translocation of NF-kappaB, while functionality was assessed in a similar manner, but after a lipopolysaccharide challenge. To demonstrate the selective nature of optical sorting, we isolated a subpopulation of macrophages highly infected with the fluorescently labeled pathogen Francisella tularensis subsp. novicida. A total of 10,738 infected cells were sorted at a throughput of 11 cells/s with 93% purity and 39% recovery.     

Critical buckling load of paper honeycomb under out‐of‐plane pressure

Two out‐of‐plane buckling criteria for paper honeycomb are proposed by analysing the structure properties and the collapse mechanism of paper honeycomb: these are based on the peeling strength and ring crush strength of the chipboard wall. Taking into account the orthotropic, initial deflection and large deflection properties of the chipboard wall, the two new mechanical models and the calculation methods are developed to represent the out‐of‐plane critical load of paper honeycomb. Theoretical calculations and test results show that the models are suitable for describing the collapse mechanism of paper honeycomb. The peeling strength and ring crush strength determine the critical buckling load of paper honeycomb in different stretch phases. The out‐of‐plane critical buckling load can be predicted when the two models are integrated. Copyright © 2005 John Wiley & Sons, Ltd.     

Multinode acoustic focusing for parallel flow cytometry

Flow cytometry can simultaneously measure and analyze multiple properties of single cells or particles with high sensitivity and precision. Yet, conventional flow cytometers have fundamental limitations with regards to analyzing particles larger than about 70 μm, analyzing at flow rates greater than a few hundred microliters per minute, and providing analysis rates greater than 50,000 per second. To overcome these limits, we have developed multinode acoustic focusing flow cells that can position particles (as small as a red blood cell and as large as 107 μm in diameter) into as many as 37 parallel flow streams. We demonstrate the potential of such flow cells for the development of high throughput, parallel flow cytometers by precision focusing of flow cytometry alignment microspheres, red blood cells, and the analysis of a CD4+ cellular immunophenotyping assay. This approach will have significant impact toward the creation of high throughput flow cytometers for rare cell detection applications (e.g., circulating tumor cells), applications requiring large particle analysis, and high volume flow cytometry.     

Microfluidic‐Based Approaches in Targeted Cell/Particle Separation Based on Physical Properties: Fundamentals and Applications

Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high‐efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label‐free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor‐intensive steps of labeling molecular signatures of cells. In general, microfluidic‐based cell sorting approaches can separate cells using “intrinsic” (e.g., fluid dynamic forces) versus “extrinsic” external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label‐free microfluidic‐based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic‐based cell separation methods are discussed.     

On‐Demand Droplet Collection for Capturing Single Cells

Droplet‐based microfluidic techniques are extensively used in efficient manipulation and genome‐wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single‐droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on‐demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on‐demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell‐to‐cell variations.     

An equilibrium method for continuous-flow cell sorting using dielectrophoresis

Separations represent a fundamental unit operation in biology and biotechnology. Commensurate with their importance is the diversity of methods that have been developed for performing them. One important class of separations are equilibrium gradient methods, wherein a medium with some type of spatial nonuniformity is combined with a force field to focus particles to equilibrium positions related to those particles' intrinsic properties. A second class of techniques that is nonequilibrium exploits labels to sort particles based upon their extrinsic properties. While equilibrium techniques such as iso-electric focusing (IEF) have become instrumental within analytical chemistry and proteomics, cell separations predominantly rely upon the second, label-based class of techniques, exemplified by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). To extend the equilibrium techniques available for separating cells, we demonstrate the first implementation of a new microfluidic equilibrium separation method, which we call isodielectric separation (IDS), for sorting cells based upon electrically distinguishable phenotypes. IDS is analogous to isoelectric focusing, except instead of separating amphoteric molecules in a pH gradient using electrophoresis, we separate cells and particles in an electrical conductivity gradient using dielectrophoresis. IDS leverages many of the advantages of microfluidics and equilibrium gradient separation methods to create a device that is continuous-flow, capable of parallel separations of multiple (>2) subpopulations from a heterogeneous background, and label-free. We demonstrate the separation of polystyrene beads based upon surface conductance as well as sorting nonviable from viable cells of the budding yeast Saccharomyces cerevisiae.     

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer‐related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time‐consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h^?1 in contrast to a flow rate of 1 mL h^?1 standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.     

Continuous sorting of microparticles using dielectrophoresis

Sorting of particles such as cells is a critical process for many biomedical applications, and it is challenging to integrate it into an analytical microdevice. We report an effective and flexible dielectrophoresis (DEP)-based microfluidic device for continuous sorting of multiple particles in a microchannel. The particle sorter is composed of two components-a DEP focusing unit and a Movable DEP Trap (MDT). The trap is formed by an array of microelectrodes at the bottom of the channel and a transparent electrode plate placed at the top. The location of the trap is dependent on the configuration of voltages on the array and therefore is addressable. Flowing particles are first directed and focused into a single particle stream by the focusing unit. The streamed particles are then sorted into different fractions using the movable trap by rapidly switching the applied voltage. The performance of the sorter is demonstrated by successfully sorting microparticles in a continuous flow. The proposed DEP-based microfluidic sorter can be implemented in applications such as sample preparation and cell sorting for subsequent analytical processing, where sorting of particles is needed.     

基元控制的方式优化自聚焦换能器聚焦声场的研究

根据HY2900聚焦超声治疗系统聚焦换能器采用6基元自聚焦方式的特点,通过对6基元聚焦换能器排序及数量的控制,达到了避让特定部位的目的。采用亥姆霍兹-基尔霍夫积分计算并用HY2900声场测量软件测量换能器在脱气水中声场的分布。对6基元换能器排序进行定义,结合水听器控制基元数及排序。在对换能器基元全驱动,关闭1、6基元,关闭1、2、6基元,关闭1、3、5基元的四种状况下测量聚焦换能器途径声场及焦平面声场分布。研究发现焦点上10 mm声场分布平面直接反映换能器基元数及随排序的改变换能器聚焦声途径的变化,可通过对换能器基元排序及数量的控制,优化其途径声场分布。     

An integrated microfluidic system for reaction, high-sensitivity detection, and sorting of fluorescent cells and particles

Presented is a novel approach for an integrated micro total analysis system (microTAS) based on a microfluidic on-chip device that supports ultrasensitive confocal detection of fluorescent cells and particles and subsequently allows for their precise sorting in the fluid phase with respect to spectroscopic properties, such as brightness and color. The hybrid silicone elastomer/glass chip first comprises a branched channel system to initiate fluid mixing and to hydrodynamically focus the sample solution down to a thin flow layer, matching the size of the confocal detection volume placed at that position and, thus, providing a high detection efficiency. In the subsequent on-chip module, the dispersed cells or particles can be sorted into two different output channels. The sorting process is realized by a perpendicular deflection stream that can be switched electrokinetically. The performance of the automated sorting routine is demonstrated by precise partition of a mixture of differently colored fluorescent beads. Moreover, the specifically branched channel geometry allows for direct implementation of reaction steps prior to detection and sorting, which is demonstrated by inducing a selective recognition reaction between the fluorescent protein R-phycoerythrin and a mixture of live bacterial cells exhibiting or lacking the respective surface antigens.     

Lab‐on‐a‐Chip‐Based High‐Throughput Screening of the Genotoxicity of Engineered Nanomaterials

The continuous increasing of engineered nanomaterials (ENMs) in our environment, their combinatorial diversity, and the associated genotoxic risks, highlight the urgent need to better define the possible toxicological effects of ENMs. In this context, we present a new high‐throughput screening (HTS) platform based on the cytokinesis‐block micronucleus (CBMN) assay, lab‐on‐chip cell sorting, and automated image analysis. This HTS platform has been successfully applied to the evaluation of the cytotoxic and genotoxic effects of silver nanoparticles (AgNPs) and silica nanoparticles (SiO₂NPs). In particular, our results demonstrate the high cyto‐ and genotoxicity induced by AgNPs and the biocompatibility of SiO₂NPs, in primary human lymphocytes. Moreover, our data reveal that the toxic effects are also dependent on size, surface coating, and surface charge. Most importantly, our HTS platform shows that AgNP‐induced genotoxicity is lymphocyte sub‐type dependent and is particularly pronounced in CD2+ and CD4+ cells.     

Discontinuous Nanoporous Membranes Reduce Non‐Specific Fouling for Immunoaffinity Cell Capture

The microfluidic isolation of target cells using adhesion‐based surface capture has been widely explored for biology and medicine. However, high‐throughput processing can be challenging due to interfacial limitations such as transport, reaction, and non‐specific fouling. Here, it is shown that antibody‐functionalized capture surfaces with discontinuous permeability enable efficient target cell capture at high flow rates by decreasing fouling. Experimental characterization and theoretical modeling reveal that “wall effects” affect cell–surface interactions and promote excess surface accumulation. These issues are partially circumvented by reducing the transport and deposition of cells near the channel walls. Optimized microfluidic devices can be operated at higher cell concentrations with significant improvements in throughput.     

3D‐Structured Stretchable Strain Sensors for Out‐of‐Plane Force Detection

Stretchable strain sensors, as the soft mechanical interface, provide the key mechanical information of the systems for healthcare monitoring, rehabilitation assistance, soft exoskeletal devices, and soft robotics. Stretchable strain sensors based on 2D flat film have been widely developed to monitor the in‐plane force applied within the plane where the sensor is placed. However, to comprehensively obtain the mechanical feedback, the capability to detect the out‐of‐plane force, caused by the interaction outside of the plane where the senor is located, is needed. Herein, a 3D‐structured stretchable strain sensor is reported to monitor the out‐of‐plane force by employing 3D printing in conjunction with out‐of‐plane capillary force‐assisted self‐pinning of carbon nanotubes. The 3D‐structured sensor possesses large stretchability, multistrain detection, and strain‐direction recognition by one single sensor. It is demonstrated that out‐of‐plane forces induced by the air/fluid flow are reliably monitored and intricate flow details are clearly recorded. The development opens up for the exploration of next‐generation 3D stretchable sensors for electronic skin and soft robotics.     

Magnetic Tweezers‐Based 3D Microchannel Electroporation for High‐Throughput Gene Transfection in Living Cells

A novel high‐throughput magnetic tweezers‐based 3D microchannel electroporation system capable of transfecting 40 000 cells/cm² on a single chip for gene therapy, regenerative medicine, and intracellular detection of target mRNA for screening cellular heterogeneity is reported. A single cell or an ordered array of individual cells are remotely guided by programmable magnetic fields to poration sites with high (>90%) cell alignment efficiency to enable various transfection reagents to be delivered simultaneously into the cells. The present technique, in contrast to the conventional vacuum‐based approach, is significantly gentler on the cellular membrane yielding >90% cell viability and, moreover, allows transfected cells to be transported for further analysis. Illustrating the versatility of the system, the GATA2 molecular beacon is delivered into leukemia cells to detect the regulation level of the GATA2 gene that is associated with the initiation of leukemia. The uniform delivery and a sharp contrast of fluorescence intensity between GATA2 positive and negative cells demonstrate key aspects of the platform for gene transfer, screening and detection of targeted intracellular markers in living cells.     

Position‐dependent defocus processing for acoustic holography images

Acoustic holography is a transmission‐based ultrasound imaging method that uses optical image reconstruction and provides a larger field of view than pulse‐echo ultrasound imaging. A focus parameter controls the position of the focal plane along the optical axis, and the images obtained contain defocused content from objects not near the focal plane. Moreover, it is not always possible to bring all objects of interest into simultaneous focus. In this article, digital image processing techniques are presented to (1) identify a “best focused” image from a sequence of images taken with different focus settings and (2) simultaneously focus every pixel in the image through fusion of pixels from different frames in the sequence. Experiments show that the three‐dimensional image information provided by acoustic holography requires position‐dependent filtering for the enhancement step. It is found that filtering in the spatial domain is more computationally efficient than in the frequency domain. In addition, spatial domain processing gives the best performance. © 2002 Wiley Periodicals, Inc. Int J Imaging Syst Technol 12, 101–111, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ima.10017     

Smart Nanovehicles Based on pH‐Triggered Disassembly of Supramolecular Peptide‐Amphiphiles for Efficient Intracellular Drug Delivery

A novel type of nanovehicle (NV) based on stimuli‐responsive supramolecular peptide‐amphiphiles (SPAs, dendritic poly (L‐lysine) non‐covalently linked poly (L‐leucine)) is developed for intracellular drug delivery. To determine the pH‐dependent mechanism, the supramolecular peptide‐amphiphile system (SPAS) is investigated at different pH conditions using a variety of physical and chemical approaches. The pH‐triggered disassembly of SPAS can be attributed to the disappearance of non‐covalent interactions within SPAs around the isoelectric point of poly (L‐leucine). SPAS is found to encapsulate guest molecules at pH 7.4 but release them at pH 6.2. In this way, SPAS is able to act as a smart NV to deliver its target to tumor cells using intracellular pH as a trigger. The DOX‐loaded NVs are approximately 150 nm in size. In vitro release profiles and confocal laser scanning microscopy (CLSM) images of HepG2 cells confirm that lower pH conditions can trigger the disassembly of NVs and so achieve pH‐dependent intracellular DOX delivery. In vitro cytotoxicity of the DOX‐loaded NVs to HepG2 cells demonstrate that the smart NVs enhance the efficacy of hydrophobic DOX. Fluorescence‐activated cell sorting (FACS) and CLSM results show that the NVs can enhance the endocytosis of DOX into HepG2 cells considerably and deliver DOX to the nuclei.     

Solution‐Processing of High‐Purity Semiconducting Single‐Walled Carbon Nanotubes for Electronics Devices

High‐purity semiconducting single‐walled carbon nanotubes (s‐SWCNTs) are of paramount significance for the construction of next‐generation electronics. Until now, a number of elaborate sorting and purification techniques for s‐SWCNTs have been developed, among which solution‐based sorting methods show unique merits in the scale production, high purity, and large‐area film formation. Here, the recent progress in the solution processing of s‐SWCNTs and their application in electronic devices is systematically reviewed. First, the solution‐based sorting and purification of s‐SWCNTs are described, and particular attention is paid to the recent advance in the conjugated polymer‐based sorting strategy. Subsequently, the solution‐based deposition and morphology control of a s‐SWCNT thin film on a surface are introduced, which focus on the strategies for network formation and alignment of SWCNTs. Then, the recent advances in electronic devices based on s‐SWCNTs are reviewed with emphasis on nanoscale s‐SWCNTs' high‐performance integrated circuits and s‐SWCNT‐based thin‐film transistors (TFT) array and circuits. Lastly, the existing challenges and development trends for the s‐SWCNTs and electronic devices are briefly discussed. The aim is to provide some useful information and inspiration for the sorting and purification of s‐SWCNTs, as well as the construction of electronic devices with s‐SWCNTs.     

Intuitive, image-based cell sorting using optofluidic cell sorting

We present a microfluidic cell-sorting device which augments microscopy with the capability to perform facile image-based cell sorting. This combination enables intuitive, complex phenotype sorting based on spatio-temporal fluorescence or cell morphology. The microfluidic device contains a microwell array that can be passively loaded with mammalian cells via sedimentation and can be subsequently inspected with microscopy. After inspection, we use the scattering force from a focused infrared laser to levitate cells of interest from their wells into a flow field for collection. First, we demonstrate image-based sorting predicated on whole-cell fluorescence, which could enable sorting based on temporal whole-cell fluorescence behavior. Second, we demonstrate image-based sorting predicated on fluorescence localization (nuclear vs whole-cell fluorescence), highlighting the capability of our approach to sort based on imaged subcellular events, such as localized protein expression or translocation events. We achieve postsort purities up to 89% and up to 155-fold enrichment of target cells. Optical manipulation literature and a direct cell viability assay suggest that cells remain viable after using our technique. The architecture is highly scalable and supports over 10 000 individually addressable trap sites. Our approach enables sorting of significant populations based on subcellular spatio-temporal information, which is difficult or impossible with existing widespread sorting technologies.