Comparison of inlet geometry in microfluidic cell affinity chromatography

Cell separation based on microfluidic affinity chromatography is a widely used methodology in cell analysis research when rapid separations with high purity are needed. Several successful examples have been reported with high separation efficiency and purity; however, cell capture at the inlet area and inlet design have not been extensively described or studied. The most common inlets-used to connect the microfluidic chip to pumps, tubing, etc.-are vertical (top-loading) inlets and parallel (in-line) inlets. In this work, we investigated the cell capture behavior near the affinity chip inlet area and compared the different performances of vertical inlet devices and parallel inlet devices. Vertical inlet devices showed significant cell capture capability near the inlet area, which led to the formation of cell blockages as the separation progressed. Cell density near the inlet area was much higher than that in the remaining channel, whereas for parallel inlet chips cell density at the inlet area was similar to that in the rest of the channel. In this paper, we discuss the effects of inlet type on chip fabrication, nonspecific binding, cell capture efficiency, and separation purity. We also discuss the possibility of using vertical inlets in negative-selection separations. Our findings show that inlet design is critical and must be considered when fabricating cell affinity microfluidic devices.     

Multitarget dielectrophoresis activated cell sorter

The ability to rapidly and efficiently isolate specific viruses, bacteria, or mammalian cells from complex mixtures lies at the heart of biomedical applications ranging from in vitro diagnostics to cell transplantation therapies. Unfortunately, many current selection methods for cell separation, such as magnetic activated cell sorting (MACS), only allow the binary separation of target cells that have been labeled via a single parameter (e.g., magnetization). This limitation makes it challenging to simultaneously enrich multiple, distinct target cell types from a multicomponent sample. We describe here a novel approach to specifically label multiple cell types with unique synthetic dielectrophoretic tags that modulate the complex permittivities of the labeled cells, allowing them to be sorted with high purity using the multitarget dielectrophoresis activated cell sorter (MT-DACS) chip. Here we describe the underlying physics and design of the MT-DACS microfluidic device and demonstrate approximately 1000-fold enrichment of multiple bacterial target cell types in a single-pass separation.     

Open-tubular capillary cell affinity chromatography: single and tandem blood cell separation

In this paper, an open-tubular capillary cell affinity chromatography (OT-CAC) method to enrich and separate target cells is described. Open tubular capillaries coated with anti-CD4, anti-CD14, or anti-CD19 antibodies were used as affinity chromatography columns to separate target blood cells. Cells were eluted using either shear force or bubbles. Bubbles were used to elute the captured cells without diluting the captured cells appreciably, while maintaining viability (the viability of the recovered cells was 85.83 +/- 7.34%; the viability of the cells was 90.41 +/- 3.49% before separation). Several aspects of the OT-CAC method were studied, such as the affinity of one antibody between two different cell lines, the effect of shear force, and the recovery of captured cells. Single- and multicell type separations were demonstrated by isolating CD4+ cells with antiCD4 coated capillary and isolating CD4+ and CD19+ cells with two capillaries in tandem from blood samples. In the one cell type isolation test, an average of 87.7% of the recovered cells from antiCD4 capillary were lymphocytes and an average of 97.7% of those lymphocytes were CD4+ cells. In the original blood sample, only 14.2% of the leukocytes were CD4+ cells. Two capillary columns were also run in tandem, separating two blood cell types from a single sample with high purity. The use of different elution shear forces was demonstrated to selectively elute one cell type. This method is an inexpensive, rapid, and effective method to separate target cells from blood samples.     

Integrative Magneto-Microfluidic Separation of Immune Cells Facilitates Clinical Functional Assays

Accurately analyzing the functional activities of natural killer (NK) cells in clinical diagnosis remains challenging due to their coupling with other immune effectors. To address this, an integrated immune cell separator is required, which necessitates a streamlined sample preparation workflow including immunological cell isolation, removal of excess red blood cells (RBCs), and buffer exchange for downstream analysis. Here, a self-powered integrated magneto-microfluidic cell separation (SMS) chip is presented, which outputs high-purity target immune cells by simply inputting whole blood. The SMS chip intensifies the magnetic field gradient using an iron sphere-filled inlet reservoir for high-performance immuno-magnetic cell selection and separates target cells size-selectively using a microfluidic lattice for RBC removal and buffer exchange. In addition, the chip incorporates self-powered microfluidic pumping through a degassed polydimethylsiloxane chip, enabling the rapid isolation of NK cells at the place of blood collection within 40 min. This chip is used to isolate NK cells from whole blood samples of hepatocellular cancer patients and healthy volunteers and examined their functional activities to identify potential abnormalities in NK cell function. The SMS chip is simple to use, rapid to sort, and requires small blood volumes, thus facilitating the use of immune cell subtypes for cell-based diagnosis.     

Microfluidic platform for liquid chromatography-tandem mass spectrometry analyses of complex peptide mixtures

A microfluidic chip that integrates all the fluidic components of a gradient liquid chromatography (LC) system is described. These chips were batch-fabricated on a silicon wafer using photolithographic processes and with Parylene as the main structural material. The fabricated chip includes three electrolysis-based electrochemical pumps, one for loading the sample and the other two for delivering the solvent gradient; platinum electrodes for delivering current to the pumps and establishing the electrospray potential; a low-volume static mixer; a column packed with silica-based reversed-phase support; integrated frits for bead capture; and an electrospray nozzle. The fabricated structures were able to withstand pressures in excess of 250 psi. The device was used to perform a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a mixture of peptides from the trypsin digestion of bovine serum albumen (BSA). Gradient elution through the 1.2-cm column was performed at a flow rate of 80 nL/min. Compared to the analysis of the same sample using a commercial nanoflow LC system, the chromatographic resolution was nearly as good, and the total cycle time was significantly reduced because of the minimal volume between the pumps and the column. Results demonstrate the potential of mass-produced, low-cost microfluidic systems capable of performing LC separations for proteomics applications.     

Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip

Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient.     

Gradient elution moving boundary electrophoresis for high-throughput multiplexed microfluidic devices

A novel method for performing electrophoretic separations is described-gradient elution moving boundary electrophoresis (GEMBE). The technique utilizes the electrophoretic migration of chemical species in combination with variable hydrodynamic bulk counterflow of the solution through a separation capillary or microfluidic channel. Continuous sample introduction is used, eliminating the need for a sample injection mechanism. Only analytes with an electrophoretic velocity greater than the counterflow velocity enter the separation channel. The counterflow velocity is varied over time so that each analyte is brought into the separation column at different times, allowing for high-resolution separations in very short channels. The new variable of bulk flow acceleration affords a new selectivity parameter to electrophoresis analogous to gradient elution compositions in chromatography. Because it does not require extra channels or access ports to form an injection zone and because separations can be performed in very short channels, GEMBE separations can be implemented in much smaller areas on a micro-fluidic chip as compared to conventional capillary electrophoresis. Demonstrations of GEMBE separations of small dye molecules, amino acids, DNA, and immunoassay products are presented. A low-cost, polymeric, eight-channel multiplexed microfluidic device was fabricated to demonstrate the reduced area requirements of GEMBE; the device was less than 1 in.2 in area and required only n + 1 fluidic access ports per n analyses (in this instance, nine ports for eight analyses). Parallel separations of fluorescein and carboxyfluorescein yielded less than 3% relative standard deviation (RSD) in interchannel migration times and less than 5% RSD in both peak and height measurements. The device was also used to generate a calibration curve for a homogeneous insulin immunoassay using each of the eight channels as a calibration point with less than 5% RSD at each point with replicate analyses.     

Microfluidic-Assisted CTC Isolation and In Situ Monitoring Using Smart Magnetic Microgels

Capturing rare disease-associated biomarkers from body fluids can offer an early-stage diagnosis of different cancers. Circulating tumor cells (CTCs) are one of the major cancer biomarkers that provide insightful information about the cancer metastasis prognosis and disease progression. The most common clinical solutions for quantifying CTCs rely on the immunomagnetic separation of cells in whole blood. Microfluidic systems that perform magnetic particle separation have reported promising outcomes in this context, however, most of them suffer from limited efficiency due to the low magnetic force generated which is insufficient to trap cells in a defined position within microchannels. In this work, a novel method for making soft micromagnet patterns with optimized geometry and magnetic material is introduced. This technology is integrated into a bilayer microfluidic chip to localize an external magnetic field, consequently enhancing the capture efficiency (CE) of cancer cells labeled with the magnetic nano/hybrid microgels that are developed in the previous work. A combined numerical-experimental strategy is implemented to design the microfluidic device and optimize the capturing efficiency and to maximize the throughput. The proposed design enables high CE and purity of target cells and real-time time on-chip monitoring of their behavior. The strategy introduced in this paper offers a simple and low-cost yet robust opportunity for early-stage diagnosis and monitoring of cancer-associated biomarkers.     

Tag-free microfluidic separation of cells against multiple markers

Conventional cell separation against multiple markers generally requires the attachment of antibody tags, typically fluorescent or magnetic, to selected cell types in a heterogeneous suspension. This work describes how such separation can be accomplished in a series of microfluidic systems without the need for such tags. Two capture stages containing antibody-functionalized alginate hydrogels are utilized for the isolation of CD34+ and Flk1+ cells from untreated, whole human blood. The capture-release capability of these degradable coatings is harnessed by a mixing chamber and a simple valving system such that the suspension emerging from the first capture stage is prepared for the second capture stage for further enrichment. With this configuration, we demonstrate the isolation of CD34+/Flk1+ endothelial progenitor cells from blood enabled by the depletion of CD34+/Flk1-hematopoietic stem cells population. This ability to achieve isolation of cells against multiple markers in an untagged separation method is of particular significance in applications involving cell implantation-based therapeutics including tissue engineering and molecular analysis.     

Gradient elution isotachophoresis for enrichment and separation of biomolecules

A novel format for performing capillary isotachophoresis (ITP) is described -- gradient elution ITP (GEITP). GEITP merges the recently described electrophoretic separation technique of gradient elution moving boundary electrophoresis (GEMBE) with an ITP enrichment step. GEMBE utilizes a combination of continuous sample injection with a pressure-controlled counterflow; as the counterflow is reduced, analytes are sequentially eluted onto the separation column and detected as boundary interfaces. By incorporating leading electrolytes into the counterflow and terminating electrolytes into the sample matrix, an ionic interface can be formed near the capillary inlet. The discontinuous buffer system forms highly enriched analyte zones outside of the capillary, which are then eluted onto the separation capillary as the counterflow is reduced. Separation of fluorescent analytes was achieved either through discrete electrolyte spacers added to the sample or by using ampholyte mixtures to form a continuum of spacers. As the ITP process occurs off-column, extremely short length separations can be achieved, as demonstrated by a separation in 30 microm. The effects of various parameters on the GEITP enrichment process are investigated, including initial counterflow rates, electric field, leading electrolyte concentration, and counterflow acceleration, which is an adjustable parameter allowing for highly flexible separations. Typical enhancements in limits of detection and sensitivity were greater than 10,000-fold and were achieved in less than 2 min, yielding low-picomolar detection limits using arc lamp illumination and low-cost CCD detection. An optimized system afforded greater than 100,000-fold improvement in detection of carboxyfluorescein in 8 min. Specific examples of enrichment and separation demonstrated include the following: small dye molecules, DNA, amino acid mixtures, and protein mixtures.     

Pressure injection on a valved microdevice for electrophoretic analysis of submicroliter samples

A recent report describes a reversible valve that can be used in series to achieve diaphragm pumping on chip (Grover, W. H.; Skelley, A. M.; Liu, C. N.; Lagally, E. T.; and Mathies, R. A. Sens. Actuators, B 2003, 89, 315-323). Here, the functionality of an integrated diaphragm pump on a hybrid PDMS-glass microchip to perform pressure injections for electrophoretic separations is demonstrated. A chip design that can perform both pressure and electrokinetic (EK) injection is described, and a mixture of fluorescein and ROX dyes in borate buffer is utilized as a model sample system. Multiple electrophoretic separations of sample injected with pressure and voltage are compared. Over multiple EK injections, an electrophoretic bias is observed and the injected analytes are not representative of the sample, with the peak area ratio changing 20% after 20 runs. Over multiple pressure injections, however, the sample composition is maintained, with a 3.6% CV over 20 runs. The data presented show the ability to alternate between injection types and pressure-inject a representative sample volume after a bias has already been observed with multiple EK injections. Multiple pressure injections have been performed on sample volumes as low as 500 nL while maintaining sample composition, supporting its use in integrated systems for small-volume sampling.     

A high-throughput continuous sample introduction interface for microfluidic chip-based capillary electrophoresis systems

The development of efficient sample introduction and pretreatment systems for microfluidic chip-based analytical systems is important for their application to real-life samples. In this work, world-to-chip interfacing was achieved by a novel flow-through sampling reservoir featuring a guided overflow design. The flow-through reservoir was fabricated on a 30 x 60 x 3 mm planar glass chip of crossed-channel design used for capillary electrophoresis separations. The 20-microL sample reservoir was produced from a section of plastic pipet tip and fixed at one end of the sampling channel. Sample change was performed by pumping 80-microL samples sandwiched between air segments at approximately 0.48 mL/min flow rate through the flow-through reservoir, introduced from an access hole on the bottom side of the chip. A filter paper collar wrapped tightly around the reservoir guided the overflowing sample solution into a plastic trough surrounding the reservoir and then to waste. The performance of the system was demonstrated in the separation and determination of FITC-labeled arginine, glycine, phenylalanine, and glutamic acid with LIF detection, by continuously introducing a train of different samples through the system without electrical interruption. Employing a separation channel of 4 cm (2-cm effective separation length) and 1.4-kV separation voltage, maximum throughputs of 80/h were achieved with <4.1% carryover and precisions ranging from 1.5% for arginine to 2.6% RSD (n = 11) for glycine. The sampling system was tested in the continuous monitoring of the derivatizing process of amino acids by FITC over a period of 4 h, involving 166 analytical cycles. An outstanding overall precision of 4.8% RSD (n = 166) was achieved for the fluorescein internal standard.     

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.     

Atmospheric pressure photoionization-mass spectrometry with a microchip heated nebulizer

A novel, microfabricated heated nebulizer chip for atmospheric pressure photoionization-mass spectrometry (APPI-MS) is presented. The chip consists of fluidic and gas inlets, a mixer, and a nozzle etched onto silicon wafer that is anodically bonded to a Pyrex glass wafer, on which an aluminum heater is sputtered. A krypton discharge lamp is used as the source for 10-eV photons to initiate the photoionization process. Dopant, delivered as part of the sample solution, is used to achieve efficient ionization. The use of the microfabricated heated nebulizer with APPI in the analysis of four analytes is demonstrated, and the spectra are compared to those obtained with a conventional APPI source. Ionization in positive and negative ion modes was successfully achieved and the spectra were mainly similar to those obtained with conventional APPI, indicating that the ionization in microfabricated and conventional APPI sources takes place by the same mechanisms. The flow rates with conventional APPI are approximately 100 muL/min, whereas the microchip heated nebulizer allows the use of flow rates 0.05-5 muL/min, thus being compatible with microfluidic separation systems or micro- and nano-LC. A stable signal was demonstrated throughout a 5-h measurement, which proved the excellent stability of the micro-APPI. The same heated nebulizer chip can be used for weeks.     

Microfabricated system for parallel single-cell capillary electrophoresis

Performing single-cell electrophoresis separations using multiple parallel microchannels offers the possibility of both increasing throughput and eliminating cross-contamination between different separations. The instrumentation for such a system requires spatial and temporal control of both single-cell selection and lysis. To address these problems, a compact platform is presented for single-cell capillary electrophoresis in parallel microchannels that combines optical tweezers for cell selection and electromechanical lysis. Calcein-labeled acute myloid leukemia (AML) cells were selected from an on-chip reservoir and transported by optical tweezers to one of four parallel microfluidic channels. Each channel entrance was manufactured by F2-laser ablation to form a 20- to 10-microm tapered lysis reservoir, creating an injector geometry effective in confining the cellular contents during mechanical shearing of the cell at the 10-microm capillary entrance. The contents of individual cells were simultaneously injected into parallel channels resulting in electrophoretic separation as recorded by laser-induced fluorescence of the labeled cellular contents.     

Electrokinetic injection techniques in microfluidic chips

The separation efficiency of a microfluidic chip is influenced to a significant degree by the flow field conditions within the injection microchannel. Therefore, an understanding of the physics of the flow within this channel is beneficial in the design and operation of such a system. The configuration of an injection system is determined by the volume of the sample plug that is to be delivered to the separation process. Accordingly, this paper addresses the design and testing of injection systems with a variety of configurations, including a simple cross, a double-T, and a triple-T configuration. This paper also presents the design of a unique multi-T injection configuration. Each injection system cycles through a predetermined series of steps, in which the electric field magnitude and distribution within the various channels is strictly manipulated, to effectuate a virtual valve. The uniquemulti-T configuration injection system presented within this paper has the ability to simulate the functions of the cross, double-T, and triple-T systems through appropriate manipulations of the electric field within its various channels. In other words, the proposed design successfully combines several conventional injection systems within a single microfluidic chip.     

A titanium dioxide nanorod array as a high-affinity nano-bio interface of a microfluidic device for efficient capture of circulating tumor cells

Nanomaterials show promising opportunities to address clinical problems (such as insufficient capture of circulating tumor cells;CTCs) via the high surface area-to-volume ratio and high affinity for biological cells.However,how to apply these nanomaterials as a nano-bio interface in a microfluidic device for efficient CTC capture with high specificity remains a challenge.In the present work,we first found that a titanium dioxide (TiO2) nanorod array that can be conveniently prepared on multiple kinds of substrates has high affinity for tumor cells.Then,the TiO2 nanorod array was vertically grown on the surface of a microchannel with hexagonally patterned Si micropillars via a hydrothermal reaction,forming a new kind of a micro-nano 3D hierarchically structured microfluidic device.The vertically grown TiO2 nanorod array was used as a sensitive nano-bio interface of this 3D hierarchically structured microfluidic device,which showed high efficiency of CTC capture (76.7％ ± 7.1％) in an artificial whole-blood sample.     

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

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

Dielectrophoretic cell separation and gene expression profiling on microelectronic chip arrays

Cell membrane dielectric properties of five different cultivated cell lines and human peripheral blood mononuclear cells (PBMC) were determined from dielectrophoretic crossover frequency measurements on a 5 x 5 microelectronic chip array. Based on distinct dielectric property differences between individual cell types, efficient cell separations were achieved by dielectrophoresis on this 5 x 5 array, which included separation of monocytic cells (U937) or human T cell leukemia virus type 1 (HTLV-1) tax-transformed cells (Ind-2) from PBMC, as well as separation of neuroblastoma cells (SH-SY5Y) from glioma cells (HTB). The purity of dielectrophoretically separated cells can be greater than 95%. Expression profiles of IL-1, TNF-alpha, and TGF-beta genes for U937 cells mixed with PBMC before and after the separation were determined by a means of electric field-facilitated hybridization on a 10 x 10 microelectronic chip array. By using the expression levels of pure U937 cells as a control, it was shown that the gene expression profiles of the postseparation cells were significantly different from those of the preseparation cell mixtures. The increase in gene expression levels for U937 cells upon lipopolysaccharide induction could be accurately determined only in the postseparation cells, while the preseparation samples masked these changes. Furthermore, by cultivating the separated HTB and SH-SY5Y cells and measuring expression of the stress-related gene c-fos, dielectrophoretic forces were shown to have little effect on cell survival and stress. The presented approach of using microelectronic chip arrays for both cell separation and gene expression profiling provides a great potential for accurate genetic analysis of specific cell subpopulations in heterogeneous samples.     

Nanocapillary array interconnects for gated analyte injections and electrophoretic separations in multilayer microfluidic architectures

An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyses. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyses.