Controlled viable release of selectively captured label-free cells in microchannels

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.     

The effects of flow type on aptamer capture in differential mobility cytometry cell separations

In this work, differential mobility cytometry (DMC) was used to monitor cell separation based on aptamer recognition for target cells. In this device, open-tubular capillaries coated with Sgc8 aptamers were used as affinity chromatography columns for separation. After cells were injected into the columns, oscillating flow was generated to allow for long-term cell adhesion studies. This process was monitored by optical microscopy, and differential imaging was used to analyze the cells as they adhered to the affinity surface. We investigated the capture time, capture efficiency, purity of target and control cells, as well as the reusability of the affinity columns. Capture time for both CCRF-CEM cells and Jurkat T cells was 0.4 ± 0.2 s, which demonstrated the high separation affinity between aptamers and target cells. The capture efficiency for CCRF-CEM cells was 95% and purity was 99% in a cell mixture. With the advantage of both high cell capture efficiency and purity, DMC combined with aptamer-based separation emerges as a powerful tool for rare cell enrichment. In addition, aptamer-based DMC channels were found to be more robust than antibody based channels with respect to reuse of the separation device.     

A micropillar-integrated smart microfluidic device for specific capture and sorting of cells

An integrated smart microfluidic device consisting of nickel micropillars, microvalves, and microchannels was developed for specific capture and sorting of cells. A regular hexagonal array of nickel micropillars was integrated on the bottom of a microchannel by standard photolithography, which can generate strong induced magnetic field gradients under an external magnetic field to efficiently trap superparamagnetic beads (SPMBs) in a flowing stream, forming a bed with sufficient magnetic beads as a capture zone. Fluids could be manipulated by programmed controlling the integrated air-pressure-actuated microvalves, based on which in situ bio-functionalization of SPMBs trapped in the capture zone was realized by covalent attachment of specific proteins directly to their surface on the integrated microfluidic device. In this case, only small volumes of protein solutions (62.5 nL in the capture zone; 375 nL in total volume needed to fill the device from inlet A to the intersection of outlet channels F and G) can meet the need for protein! The newly designed microfluidic device reduced greatly chemical and biological reagent consumption and simplified drastically tedious manual handling. Based on the specific interaction between wheat germ agglutinin (WGA) and N-acetylglucosamine on the cell membrane, A549 cancer cells were effectively captured and sorted on the microfluidic device. Capture efficiency ranged from 62 to 74%. The integrated microfluidic device provides a reliable technique for cell sorting.     

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.     

Integration of Lateral Filter Arrays with Immunoaffinity for Circulating‐Tumor‐Cell Isolation

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size‐based separation with immunoaffinity‐based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two‐dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.     

Switchable surface traps for injectable bead-based chromatography in PDMS microfluidic channels

We report here a reversible microchannel surface capture system for stimuli-responsive grafted bioanalytical beads. Poly(N-isopropylacrylamide) (PNIPAAm) was grafted onto polydimethylsiloxane (PDMS) surfaces by a UV-mediated graft polymerization from a photoinitiator that was preadsorbed in the channel wall. The surface grafting density and resulting switchable hydrophilic/hydrophobic properties were controlled by varying the photo-illumination times and/or the initiator concentration. At limiting PNIPAAm-graft densities, the surfaces demonstrated minimal contact angles of 35 degrees below the lower critical solution temperature (LCST) and maximal contact angles of 82 degrees above it. These contact angles could be varied depending on the graft density. The surface grafts are spatially limited to the photo-illuminated region to define where the trap is constructed. The surface traps capture PNIPAAm-grafted nanobeads uniformly above the LCST and facilitate their rapid release as the temperature is reversed to below the LCST. This dual surface trap and injectable chromatography system could be useful in many applications, such as affinity separations, immunoassays, and enzyme bioprocesses, by providing for the controlled capture and release of chromatography beads.     

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single‐cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 μm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 μm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP‐release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single‐cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP‐release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single‐cell analysis.     

Blood cell capture in a sawtooth dielectrophoretic microchannel

Biological fluids can be considered to contain information-rich mixtures of biochemicals and particles that enable clinicians to accurately diagnose a wide range of pathologies. Rapid and inexpensive analysis of blood and other bodily fluids is a topic gaining substantial attention in both science and medicine. One line of development involves microfluidic approaches that provide unique advantages over entrenched technologies, including rapid analysis times, microliter sample and reagent volumes, potentially low cost, and practical portability. The present study focuses on the isolation and concentration of human blood cells from small-volume samples of diluted whole blood. Separation of cells from the matrix of whole blood was accomplished using constant potential insulator-based gradient dielectrophoresis in a converging, sawtooth-patterned microchannel. The channel design enabled the isolation and concentration of specific cell types by exploiting variations in their characteristic physical properties. The technique can operate with isotonic buffers, allowing capture of whole cells, and reproducible capture occurred at specific locales within the channel over a global applied voltage range of 200–700 V.     

Design and fabrication of an integrated cell processor for single embryo cell manipulation

This paper presents an integrated cell processor for the automatic handling of individual embryo cells. The integrated processor can perform various functions such as cell transport, isolation, orientation, and immobilization. These functions are indispensable and frequently used for the manipulation of single cells, but can only be carried out by a skillful operator. The purpose of this study was the integration and automation of these functions for effective cell manipulation, using a MEMS approach. The isolation of a cell was performed using polypyrrole (PPy) valves in a microchannel into which cells were transported. The orientation of cells was controlled by electrorotation (ER), and the target cell was immobilized by suction from a microhole. All of these functions were seamlessly realized on a single chip. Excellent experimental results with mouse (B6CBA) embryo cells showed that this device could substitute for routine and cumbersome manual work. It is expected that the integrated chip will contribute significantly to faster and more reliable manipulation of cells.     

Microfluidic array cytometer based on refractive optical tweezers for parallel trapping, imaging and sorting of individual cells

Analysis of genetic and functional variability in populations of living cells requires experimental techniques capable of monitoring cellular processes such as cell signaling of many single cells in parallel while offering the possibility to sort interesting cell phenotypes for further investigations. Although flow cytometry is able to sequentially probe and sort thousands of cells per second, dynamic processes cannot be experimentally accessed on single cells due to the sub-second sampling time. Cellular dynamics can be measured by image cytometry of surface-immobilized cells, however, cell sorting is complicated under these conditions due to cell attachment. We here developed a cytometric tool based on refractive multiple optical tweezers combined with microfluidics and optical microscopy. We demonstrate contact-free immobilization of more than 200 yeast cells into a high-density array of optical traps in a microfluidic chip. The cell array could be moved to specific locations of the chip enabling us to expose in a controlled manner the cells to reagents and to analyze the responses of individual cells in a highly parallel format using fluorescence microscopy. We further established a method to sort single cells within the microfluidic device using an additional steerable optical trap. Ratiometric fluorescence imaging of intracellular pH of trapped yeast cells allowed us on the one hand to measure the effect of the trapping laser on the cells' viability and on the other hand to probe the dynamic response of the cells upon glucose sensing.     

Artificial Cytoskeleton Assembly for Synthetic Cell Motility

Imitation of cellular processes in cell-like compartments is a current research focus in synthetic biology. Here, a method is introduced for assembling an artificial cytoskeleton in a synthetic cell model system based on a poly(N-isopropyl acrylamide) (PNIPAM) composite material. Toward this end, a PNIPAM-based composite material inside water-in-oil droplets that are stabilized with PNIPAM-functionalized and commercial fluorosurfactants is introduced. The temperature-mediated contraction/release behavior of the PNIPAM-based cytoskeleton is investigated. The reversibility of the PNIPAM transition is further examined in bulk and in droplets and it could be shown that hydrogel induced deformation could be used to controllably manipulate droplet-based synthetic cell motility upon temperature changes. It is envisioned that a combination of the presented artificial cytoskeleton with naturally occurring components might expand the bandwidth of the bottom-up synthetic biology.     

Synthesis of hydrophilic boronate affinity monolithic capillary for specific capture of glycoproteins by capillary liquid chromatography

Boronate affinity chromatography is an important tool for specific isolation of cis-diol-containing compounds such as glycoproteins, RNA and carbohydrates. Boronate functionalized monolithic capillaries have been recently developed for specific capture of cis-diol-containing small biomolecules, but the apparent hydrophobicity of the columns prevents them from specific capture of glycoproteins. In this paper, a hydrophilic boronate affinity monolithic capillary was prepared by in situ free radical polymerization, using 4-vinylphenylboronic acid (VPBA) and N, N′-methylenebisacrylamide (MBAA) as functional monomer and cross-linker, respectively. The prepared poly(VPBA-co-MBAA) monolithic capillary exhibited uniform open channel network and high density of accessible boronic acid. Due to the utilization of hydrophilic cross-linker, the prepared column was hydrophilic, allowing for specific capture of glycoproteins.     

Evaluation of a surface-sampling probe electrospray mass spectrometry system for the analysis of surface-deposited and affinity-captured proteins

A combined self-aspirating electrospray emitter/surfacing-sampling probe coupled with an ion trap mass spectrometer was used to sample and mass analyze proteins from surfaces. The sampling probe mass spectrometer system was used to sample and detect lysozyme that had been deposited onto a glass slide using a piezoelectric spotter or murine gamma-interferon affinity captured on a glass slide using surface-immobilized anti-gamma-interferon antibody. The detection level for surface-deposited lysozyme (spot size < or =200 microm) was approximately 1.0 fmol (approximately 100 fmol/mm2) as determined from the ability to measure accurately the protein molecular mass from the mass spectrum acquired by sampling the deposit. These detection limits may be sufficient for certain applications in which protein fractions from a separation method are collected onto a surface. Radiolabeled proteins were used to quantify the surface density of immobilized antibody and the efficiency of capture of the gamma-interferon on glass and higher surface area ceramic supports. The capture density of gamma-interferon at surface saturation ranged from about 23 to 50 fmol/mm2 depending on the capture surface. Nonetheless, mass spectrometric detection of affinity capture protein was successful in some cases, but the results were not reproducible. Thus, improvement of the sampling system, ionization efficiency and/or capture density will be necessary for practical sampling of affinity-captured proteins. The means to accomplish improved sampling system detection limits and to increase the absolute amounts of protein captured per unit area are discussed.     

Trapping and releasing of single microparticles and cells in a microfluidic chip

A microfluidic device was designed and fabricated to capture single microparticles and cells by using hydrodynamic force and selectively release the microparticles and cells of interest via negative dielectrophoresis by activating selected individual microelectrodes. The trap microstructure was optimized based on numerical simulation of the electric field as well as the flow field. The capture and selective release functions of the device were verified by multi-types microparticles with different diameters and K562 cells. The capture efficiencies/release efficiencies were 95.55% ± 0.43%/96.41% ± 1.08% and 91.34% ± 0.01%/93.67% ± 0.36% for microparticles and cells, respectively. By including more traps and microelectrodes, the device can achieve high throughput and realize the visual separation of microparticles/cells of interest in a large number of particle/cell groups.     

Electrochemical real-time polymerase chain reaction

In this work, we report the first electrochemistry-based real-time polymerase chain reaction technique for sequence-specific nucleic acid detection. This new technique builds upon the advantages of the well-established fluorescence-based counterpart, such as short assay time (simultaneous target DNA amplification and detection). In addition, this electrochemical approach could employ simple and miniaturizable instrumentation compared to the bulky and expensive optics required in the fluorescence-based schemes. We have demonstrated a proof-of-concept experiment showing that the utilization of solid-phase extension of the electrode surface-immobilized capture probe with Fc-dUTP during PCR resulted in the accumulation of the redox marker on the transducer surface. This new technique can be applied to a microfabricated PCR electrochemical device for point-of-care diagnostics as well as on-site environmental monitoring and biowarfare agent detection.     

Manipulating microparticles with single surface-immobilized nanoparticles

This experimental study explores the capture and manipulation of micrometer-scale particles by single surface-immobilized nanoparticles. The nanoparticles, approximately 10 nm in diameter, are cationic and therefore attract the micrometer-scale silica particles in an analyte suspension. The supporting surface on which the nanoparticles reside is negative (also silica) and repulsive toward approaching microparticles. In the limit where there are as few as 9 nanoparticles per square micrometer of collector, it becomes possible to capture and hold micrometer-scale silica particles with single nanoparticles. The strong nanoparticle-microparticle attractions, their nanometer-scale protrusion forward of the supporting surface, and their controlled density on the supporting surface facilitate microparticle-surface contact occurring through a single nanoelement. This behavior differs from most particle-particle, cell-cell, or particle (or cell)-surface interactions that involve multiple ligand-receptor bonds or much larger contact areas. Despite the limited contact of microparticles with surface-immobilized nanoparticles, microparticles resist shear forces of 9 pN or more but can be released through an increase in the ionic strength. The ability of nanoparticles to reversibly trap and hold much larger targets has implications in materials self-assembly, cell capture, and sorting applications, whereas the single point of contact affords precision in particle manipulation.     

Immunomagnetic T cell capture from blood for PCR analysis using microfluidic systems

A one-step immunomagnetic separation technique was performed on a microfluidic platform for the isolation of specific cells from blood samples. The cell isolation and purification studies targeted T cells, as a model for low abundance cells (about 1:10,000 cells), with more dilute cells as the ultimate goal. T cells were successfully separated on-chip from human blood and from reconstituted blood samples. Quantitative polymerase chain reaction analysis of the captured cells was used to characterize the efficiency of T cell capture in a variety of flow path designs. Employing many (4-8), 50 microm deep narrow channels, with the same overall cross section as a single, 3 mm wide channel, was much more effective in structuring dense enough magnetic bead beds to trap cells in a flowing stream. The use of 8-multiple bifurcated flow paths increased capture efficiencies from approximately 20 up to 37%, when compared to a straight 8-way split design, indicating the value of ensuring uniform flow distribution into each channel in a flow manifold for effective cell capture. Sample flow rates of up to 3 microL min(-1) were evaluated in these capture beds.     

Ultra-thin temperature controllable microwell array chip for continuous real-time high-resolution imaging of living single cells

Single-cell imaging, a powerful analytical method to study single-cell behavior, such as gene expression and protein profiling, provides an essential basis for modern medical diagnosis. The coding and localization function of microfluidic chips has been developed and applied in living single-cell imaging in recent years. Simultaneously, chip-based living single-cell imaging is also limited by complicated trapping steps, low cell utilization, and difficult high-resolution imaging. To solve these problems, an ultra-thin temperature-controllable microwell array chip (UTCMA chip) was designed to develop a living single-cell workstation in this study for continuous on-chip culture and real-time high-resolution imaging of living single cells. The chip-based on ultra-thin ITO glass is highly matched with an inverted microscope (or confocal microscope) with a high magnification objective (100 × oil lens), and the temperature of the chip can be controlled by combining it with a home-made temperature control device. High-throughput single-cell patterning is realized in one step when the microwell array on the chip uses hydrophilic glass as the substrate and hydrophobic SU-8 photoresist as the wall. The cell utilization rate, single-cell capture rate, and microwell occupancy rate are all close to 100% in the microwell array. This method will be useful in rare single-cell research, extending its application in the biological and medical-related fields, such as early diagnosis of disease, personalized therapy, and research-based on single-cell analysis.     

Screening of cardioprotective diseases bioactive components from Danshen extracts and LC–MS analysis

A rapid and efficient analysis and screening method is adopted for cell affinity capture coupled with HPLC–MS (CAC–HPLC–MS) analysis of bioactive components that have possible efficiency against cardiovascular diseases. This method involves affinity capture, concentration, and separation of bioactive components from Danshen library using oxidatively damaged endothelial cells induced by H₂O₂, as well as analysis and identification of targeted compounds with HPLC and MS. It combines the specific interaction between cell membrane receptors and bioactive components with the powerful analysis and identification function of HPLC–MS. The CAC–HPLC–MS method was also used for analysis and screening of bioactive components from crude extracts of Danshen. A total of 19 components were found to be bound to oxidatively damaged endothelial cells with seven of these identified. Existing literature confirms that these seven components have many activities related to cardioprotective diseases. Therefore, the combination of biological affinity capture with HPLC–MS should be regarded as an attractive method with great potential for rapid and efficient screening of bioactive components related to anti-cardiovascular diseases from natural product libraries.     

Membrane protein isolation by in situ solubilization, partitioning and affinity adsorption in aqueous two-phase systems. Purification of the human type 1 11beta-hydroxysteroid dehydrogenase

Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11beta-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg(-1) min(-1) of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg(-1) min(-1) was achieved.