Single-cell Trapping Research Articles

Target trapping and in situ single-cell genetic marker detection with a focused optical beam

Optical trapping of single particles or cells with the capability of in situ bio-sensing or genetic profiling opens the possibility of rapid screening of biological specimens. However, common optical tweezers suffer from the lack of long-range forces. Consequently, their application areas are predominantly limited to target manipulation instead of biological diagnostics. To solve this problem, we herein report an all-in-one approach by combining optical forces and convective drag forces generated through localized optothermal effect for long-range target manipulation. The device consists of a 2D array of gold coated polydimethylsiloxane (PDMS) micro-wells, which are immersed by colloidal particles or cell solution. Upon excitation of a 785-nm laser, the hydrodynamic convective force and optical forces will drag the targets of interest into their designated micro-wells. Moreover, the plasmonic thermal dissipation provides a constant temperature environment for following cell analysis procedures of cell isolation, lysis and isothermal nucleic acid amplification for the detection of genetic markers. With the merits of fabrication simplicity, short sample-to-answer cycle time and the compatibility with optical microscopes, the reported technique offers an attractive and highly versatile approach for on-site single cell analysis systems.

Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing

A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.

A Microfluidic Micropipette Aspiration Device to Study Single-Cell Mechanics Inspired by the Principle of Wheatstone Bridge.

The biomechanical properties of single cells show great potential for early disease diagnosis and effective treatments. In this study, a microfluidic device was developed for quantifying the mechanical properties of a single cell. Micropipette aspiration was integrated into a microfluidic device that mimics a classical Wheatstone bridge circuit. This technique allows us not only to effectively alter the flow direction for single-cell trapping, but also to precisely control the pressure exerted on the aspirated cells, analogous to the feature of the Wheatstone bridge that can precisely control bridge voltage and current. By combining the micropipette aspiration technique into the microfluidic device, we can effectively trap the microparticles and Hela cells as well as measure the deformability of cells. The Young’s modulus of Hela cells was evaluated to be 387 ± 77 Pa, which is consistent with previous micropipette aspiration studies. The simplicity, precision, and usability of our device show good potential for biomechanical trials in clinical diagnosis and cell biology research.

Dielectrophoretic trapping of single leukemic cells using the conventional and compact optical measurement systems.

Here, we report a microfluidic same-single-cell analysis to study the inhibition of multidrug resistance due to drug efflux on single leukemic cells. Drug efflux inhibition was investigated in the microfluidic chip using two different fluorescence detection systems, namely, a compact single-cell bioanalyzer and the conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, a compact signal generator was used to conduct dielectrophoretic cell trapping together with the compact SCB. By using the DEP force, a single acute myeloid leukemia cell was trapped in the cell retention structure of the chip. This allowed us to detect dye accumulation in the MDR leukemic cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 were used as the P-glycoprotein inhibitor and fluorescent dye, respectively. The result showed that the Rh123 fluorescence signal in a single-cell increased dramatically over its same-cell control on both fluorescence detection systems due to the inhibition by CsA.

Bio-O-Pump: a novel portable microfluidic device driven by osmotic pressure

The concepts of lab on a chip, miniaturised fluidic systems, and biochips entail the use of a fluid to perform analogue or digital operations. The temporal and spatial fluidic drive and control in microfluidic systems usually involves a complex pump, tubing, and connection system. Reducing the number of external components is crucial for use by scientists without an engineering background. In this paper, we present a novel pump-free device that uses an osmotic-pressure-driven flow to control and modulate fluid flow in microfluidic networks. The flow rate was regulated by controlling the osmotic area and the concentration of the draw solution, which comprised an easily accessible solution of electrolytes such as commercial sterile saline buffer and sodium chloride solution. The Bio-O-Pump can continuously generate liquid movement up to a flow rate of 4.88 mL h−1 over 1 h. This research also presents a single cell trapping application for biomedical uses.

Time Sequential Single-Cell Patterning with High Efficiency and High Density.

Single-cell capture plays an important role in single-cell manipulation and analysis. This paper presents a microfluidic device for deterministic single-cell trapping based on the hydrodynamic trapping mechanism. The device is composed of an S-shaped loop channel and thousands of aligned trap units. This arrayed structure enables each row of the device to be treated equally and independently, as it has row periodicity. A theoretical model was established and a simulation was conducted to optimize the key geometric parameters, and the performance was evaluated by conducting experiments on MCF-7 and Jurkat cells. The results showed improvements in single-cell trapping ability, including loading efficiency, capture speed, and the density of the patterned cells. The optimized device can achieve a capture efficiency of up to 100% and single-cell capture efficiency of up to 95%. This device offers 200 trap units in an area of 1 mm2, which enables 100 single cells to be observed simultaneously using a microscope with a 20× objective lens. One thousand cells can be trapped sequentially within 2 min; this is faster than the values obtained with previously reported devices. Furthermore, the cells can also be recovered by reversely infusing solutions. The structure can be easily extended to a large scale, and a patterned array with 32,000 trap sites was accomplished on a single chip. This device can be a powerful tool for high-throughput single-cell analysis, cell heterogeneity investigation, and drug screening.

A microfluidic platform with pneumatically switchable single-cell traps for selective intracellular signals probing

To investigate rapid suspension cell signaling, a microfluidic platform was urgently needed for flexibly manipulation of single cells and simultaneous generation of controllable chemical signals to stimulate single cells. In this paper, a microfluidic biosensor was developed to monitor intracellular calcium signal, integrated with single-cell trapping, chemical stimulation and releasing. Selective entrapment and discharge of individual cell were achieved by controlling the deformable membrane with pneumatic traps. The activation of intracellular calcium signal was qualitatively and quantitatively investigated by high-controllable chemical single-cell stimulation based on flexible hydrodynamic gating. And performing chemical stimulation and control assay in the same channel would improve the experimental robustness and effectiveness. Further investigation of the cellular responses to ATP pulses of varying concentrations and durations indicated that 20 μM ATP pulses with duration as short as 200 ms resulted in the same level of Ca2+ response induced by sustained stimulations. Washing with buffer for 30 s was sufficient for single cell to recover from receptor desensitization caused by ATP stimulation. In addition, the responses of cells to ATP stimulation were heterogeneous. The developed microfluidic method opens up a new avenue for intracellular signaling studies and drug screening.

High-Throughput Separation, Trapping, and Manipulation of Single Cells and Particles by Combined Dielectrophoresis at a Bipolar Electrode Array.

Microfluidic systems have been developed widely in scaled-down processes of laboratory techniques, but they are usually limited in achieving stand-alone functionalities. It is highly desirable to exploit an integrated microfluidic device with multiple capabilities such as cell separation, single-cell trapping, and cell manipulation. Herein, we reported a microfluidic platform integrated with actuation electrodes, for separating cells and microbeads, and bipolar electrodes, for trapping, rotating, and propelling single cells and microbeads. The separation of cells and microbeads can be first achieved by deflective dielectrophoresis (DEP) barriers. Trapping experiments with yeast cells and polystyrene (PS) microbeads suspended in aqueous solutions with different conductivities were then conducted, showing that both cells and particles can be trapped at the center of wireless electrodes by negative DEP force. Upon application of a rotating electric field, yeast cells exhibit translational movement along the electrode edges, and self-rotation is seen at an array of bipolar electrodes when electrorotational torque and traveling wave DEP force are applied on the cells. The current approach allows us to switch the propulsion and rotation direction of cells by varying the frequency of the applied electric field. Beyond the achievements of single-cell manipulation, this system permits effective control of several particles or cells simultaneously. The integration of parallel sorting and single trapping stages within a microfluidic chip enables the prospect of high-throughput cell separation, single trapping, and large-scale cell locomotion and rotation in a noninvasive and disposable format, showing great potential in single-cell analysis, targeted drug delivery, and surgery.

An integrated microfluidic platform for size-selective single-cell trapping of monocytes from blood.

Reliable separation and isolation of target single cells from bodily fluids with high purity is of great significance for an accurate and quantitative understanding of the cellular heterogeneity. Here, we describe a fully integrated single-blood-cell analysis platform capable of size-selective cell separation from a population containing a wide distribution of sizes such as diluted blood sample and highly efficient entrapment of single monocytes. The spiked single U937 cells (human monocyte cell line) are separated in sequence by two different-sized microfilters for removing large cell clumps, white blood cells, and red blood cells and then discriminated by dielectrophoretic force and isolated individually by downstream single-cell trapping arrays. When 2% hematocrit blood cells with a final ratio of 1:1000 U937 cells were introduced under the flow rate of 0.2 ml/h, 400 U937 cells were trapped sequentially and deterministically within 40 s with single-cell occupancy of up to 85%. As a proof-of-concept, we also demonstrated single monocyte isolation from diluted blood using the integrated microfluidic device. This size-selective, label-free, and live-cell enrichment microfluidic single blood-cell isolation platform for the processing of cancer and blood cells has a myriad of applications in areas such as single-cell genetic analysis, stem cell biology, point-of-care diagnostics, and cancer diagnostics.

Inkjet-Printing Patterned Chip on Sticky Superhydrophobic Surface for High-Efficiency Single-Cell Array Trapping and Real-Time Observation of Cellular Apoptosis.

Single-cell assays have broad applications in cellular studies, tissue engineering, fundamental studies of cell-cell interactions, and understanding of cell-to-cell variations. Most existing methods for micron-sized cell patterning are still based on lithography-based microfabrication process. Thus, exploiting new mask-free strategies while maintaining high-precision single-cell patterning is still a great challenge. Here, we presented a facile, low-cost, and mask-free approach for constructing high-resolution patterning on sticky superhydrophobic (SH) substrates based on inkjet printing with ordinary precision. In this work, the SH surface with both high contact angle and relatively high contact angle hysteresis can not only obtain high-resolution spots but also avoid droplets bouncing behavior. We improved the feature size of printed protein spots as small as 4 μm, which is much smaller than protein spots used for single-cell trapping. Moreover, with the assistance of a narrow microchannel, the inkjet-printing patterned chip with fibronectin ink allows for fast and high-efficiency trapping of multiple single-cell arrays. Using this method, single-cell occupancy could reach approximately 81% within 30 min on subcellular-sized patterning chip, and there was no significant effect on cell viability. As a proof of concept, this chip has been applied to study the real-time apoptosis of single cells and demonstrated the potential in cells' heterogeneity analysis.

Pipette Petri Dish Single-Cell Trapping (PP-SCT) in Microfluidic Platforms: A Passive Hydrodynamic Technique

Microfluidics-based biochips play a vital role in single-cell research applications. Handling and positioning of single cells at the microscale level are an essential need for various applications, including genomics, proteomics, secretomics, and lysis-analysis. In this article, the pipette Petri dish single-cell trapping (PP-SCT) technique is demonstrated. PP-SCT is a simple and cost-effective technique with ease of implementation for single cell analysis applications. In this paper a wide operation at different fluid flow rates of the novel PP-SCT technique is demonstrated. The effects of the microfluidic channel shape (straight, branched, and serpent) on the efficiency of single-cell trapping are studied. This article exhibited passive microfluidic-based biochips capable of vertical cell trapping with the hexagonally-positioned array of microwells. Microwells were 35 μm in diameter, a size sufficient to allow the attachment of captured cells for short-term study. Single-cell capture (SCC) capabilities of the microfluidic-biochips were found to be improving from the straight channel, branched channel, and serpent channel, accordingly. Multiple cell capture (MCC) was on the order of decreasing from the straight channel, branch channel, and serpent channel. Among the three designs investigated, the serpent channel biochip offers high SCC percentage with reduced MCC and NC (no capture) percentage. SCC was around 52%, 42%, and 35% for the serpent, branched, and straight channel biochips, respectively, for the tilt angle, θ values were between 10–15°. Human lung cancer cells (A549) were used for characterization. Using the PP-SCT technique, flow rate variations can be precisely achieved with a flow velocity range of 0.25–4 m/s (fluid channel of 2 mm width and 100 µm height). The upper dish (UD) can be used for low flow rate applications and the lower dish (LD) for high flow rate applications. Passive single-cell analysis applications will be facilitated using this method.

Development of micro‐inclined well array for trapping single cells

AbstractWe investigated the shape of a well to trap single cells for the purpose of improving the cell trapping rate after cell seeding and liquid exchange in a cell microarray. The wells having vertical and inclined tapered side‐wall were made using three‐dimensional UV photolithography. Changes in cell trapping rate by the shape of the wells were evaluated by trapping tests with fluorescent microbeads and living cells. As a result of trapping test with living cells, bead residual rate of the microarray with inclined tapered wells is up to 2.9 times higher than that with vertical wells.

Liquid Metal-Based Multifunctional Micropipette for 4D Single Cell Manipulation.

A novel manufacturing approach to fabricate liquid metal‐based, multifunctional microcapillary pipettes able to provide electrodes with high electrical conductivity for high‐frequency electrical stimulation and measurement is proposed. 4D single cell manipulation is realized by applying multifrequency, multiamplitude, and multiphase electrical signals to the microelectrodes near the pipette tip to create 3D dielectrophoretic trap and 1D electrorotation, simultaneously. Functions such as single cell trapping, patterning, transfer, and rotation are accomplished. Cell viability and multiday proliferation characterization has confirmed the biocompatibility of this approach. This is a simple, low‐cost, and fast fabrication process that requires no cleanroom and photolithography step to manufacture 3D microelectrodes and microchannels for easy access to a wide user base for broad applications.

A novel dual-well array chip for efficiently trapping single-cell in large isolated micro-well without complicated accessory equipment.

Conventional cell-sized well arrays have advantages of high occupancy, simple operation, and low cost for capturing single-cells. However, they have insufficient space for including reagents required for cell treatment or analysis, which restricts the wide application of cell-sized well arrays as a single-cell research tool alone. Here, we present a novel dual-well array chip, which integrates capture-wells (20 μm in diameter) with reaction-wells (100 μm in diameter) and describe a flow method for convenient single-cell analysis requiring neither complicated infra-structure nor high expenditure, while enabling highly efficient single cell trapping (75.8%) with only 11.3% multi-cells. Briefly, the cells are first loaded into the dual-wells by gravity and then multi-cells in the reaction-wells are washed out by phosphate buffer saline. Next, biochemical reagents are loaded into reaction-wells using the scraping method and the chip is packed as a sandwich structure. We thereby successfully measured intracellular β-galactosidase activity of K562 cells at the single-cell level. We also used computational simulations to illustrate the working principle of dual-well structure and found out a relationship between the wall shear stress distribution and the aspect ratio of the dual-well array chip which provides theoretical guidance for designing multi-wells chip for convenient single-cell analysis. Our work produced the first dual-well chip that can simultaneously provide a high occupancy rate for single cells and sufficient space for reagents, as well as being low in cost and simple to operate. We believe that the feasibility and convenience of our method will enhance its use as a practical single-cell research tool.

Single cell trapping by capillary pumping using NOA81 replica moulded stencils

In this contribution, we demonstrate that the optical adhesive NOA81 (Norland Products Inc.) can be used to replicate optically transparent single cell microsieve structures with exquisite resolution, enabling the fabrication of cheap stencils for single cell trapping applications by the combination of replica moulding and laser micromachining. In addition, we demonstrate an interesting capillary pumping mechanism for gently loading single neuronal cells which eliminates the need for equipment such as pumps and syringes. We demonstrate that capillary pumping through a microsieve generates gentle cell trapping velocities (<13.3 μm/s), enabling reproducible cell trapping efficiencies of 80% with high cell survival rates (90% over 1 week of culture) and facilitating the formation of spatially standardized neuronal networks.

Finite Element Simulation of Microfluidic Biochip for High Throughput Hydrodynamic Single Cell Trapping

In this paper, a microfluidic device capable of trapping a single cell in a high throughput manner and at high trapping efficiency is designed simply through a concept of hydrodynamic manipulation. The microfluidic device is designed with a series of trap and bypass microchannel structures for trapping individual cells without the need for microwell, robotic equipment, external electric force or surface modification. In order to investigate the single cell trapping efficiency, a finite element model of the proposed design has been developed using ABAQUS-FEA software. Based on the simulation, the geometrical parameters and fluid velocity which affect the single cell trapping are extensively optimized. After optimization of the trap and bypass microchannel structures via simulations, a single cell can be trapped at a desired location efficiently.

Tunable Confinement for Bridging Single-Cell Manipulation and Single-Molecule DNA Linearization.

DNA linearization by nanoconfinement has offered a new avenue toward large-scale genome mapping. The ability to smoothly interface the widely different length scales from cell manipulation to DNA linearization is critical to the development of single-cell genomic mapping or sequencing technologies. Conventional nanochannel technologies for DNA analysis suffer from complex fabrication procedures, DNA stacking at the nanochannel entrance, and inefficient solution exchange. In this work, a dynamic and tunable confinement strategy is developed to manipulate and linearize genomic-length DNA molecules from a single cell. By leveraging pneumatic microvalve control and elastomeric collapse, an array of nanochannels with confining dimension down to 20 nm and length up to sub-millimeter is created and can be dynamically tuned in size. The curved edges of the microvalve form gradual transitions from microscale to nanoscale confinement, smoothly facilitating DNA entry into the nanochannels. A unified micro/nanofluidic device that integrates single-cell trapping and lysis, DNA extraction, purification, labeling, and linearization is developed based on dynamically controllable nanochannels. Mbp-long DNA molecules are extracted directly from a single cell and in situ linearized in the nanochannels. The device provides a facile and promising platform to achieve the ultimate goal of single-cell, single-genome analysis.

Parallel trapping of single motile cells based on vibration-induced flow

We propose an on-chip cell manipulation method for trapping single motile cells in parallel. The proposed method traps large ( $$\gtrsim \,50\,\upmu \hbox {m}$$ ) motile cells in parallel, which is difficult to achieve by conventional cell manipulation methods based on optical, acoustic, electric, or magnetic forces. The trapping method exploits the flow induced by applying a vibration to a microfluidic chip with microstructures on its surface. By applying a rectilinear vibration to a chip with pairs of micropillars, we can trap single motile cells within the local flow generated between the micropillars. Using the proposed method, we trapped single Euglena gracilis cells (of size 50–100 $$\upmu \hbox {m}$$ ) in parallel. Moreover, we evaluate the trapping performance for various micropillar array design parameters and the controllability of the trapping-flow velocity by varying the amplitude of the vibration. The proposed method was then demonstrated in a motility evaluation of motile cells. The demonstration confirms the potential of the proposed method in realizing high-throughput motility evaluations of single motile cells.

High-Dimensional mRNA and Protein Content Measurements in Single Cells with Single-Molecule Sensitivity

Although there are well established methodologies to measure and model the relationship between mRNA and protein content in bulk, new techniques are necessary to measure the fundamental expression of these compounds at the single-cell level with single-molecule sensitivity. Examining the heterogeneity of mRNA and protein expression at the single-cell level can lead to fundamental information about the cellular response to external stimuli, including the sensitivity, timing, and regulatory interactions of these genes. Our initial results demonstrate the ability to simultaneously quantify up to five genes of interest in single cells using conventional wide-field imaging and labeling methods. Single-molecule mRNA content is measured by single-molecule fluorescence in-situ hybridization (smFISH) while protein content is quantified though the use of antibody probes. Full system automation of the 3D microscope scan and custom image analysis routines allows hundreds of individual cells to be automatically segmented and the mRNA-protein content to be digitally counted. To mimic immune response to bacterial infection, THP-1 cells are stimulated with lipopolysaccharide (LPS) and up to five genes of interest (e.g. IL1β, TNF-α, TLR2) are simultaneously monitored to examine the distribution of single-cell response to this pathogenic stimulus. Despite these initial successes, it is highly desirable to increase the number of genes simultaneously monitored. To this end we are designing a new microfluidic platform to precisely trap and lyse single cells within an analysis chamber for high-dimensional gene analysis. Within this diffusion limited chamber, the mRNA and protein probes are spatially printed (using ink jet and dip-pen technology) to create specific capture and fluorescent detection regions. Spatially arraying the detection regions, combined with super-resolution fluorescent read-out, should enable multi-parameter analysis beyond the ∼5 color fluorescent limit, allowing tens-to-hundreds of genes to be simultaneously analyzed in each single cell.

A microfluidic microwell device for immunomagnetic single-cell trapping

Single-cell analysis has been widely applied in various biomedical applications, such as cancer diagnostics, immune status monitoring, and drug screening. To perform an accurate and rapid cellular analysis, various magnetic-activated cell sorting techniques are available in the markets. However, large sample requirement and uneven magnetic field distribution limit its application in single-cell trapping and following analysis. To address these problems, we developed a microfluidic microwell device for immunomagnetic single-cell trapping. By adding a microwell layer between the microchannel and magnet, the magnetic field along the device becomes more uniform. Besides, magnetic beads can be retained in the array of microwell after the high-speed washing step, whereas untrapped beads would be flushed away, resulting in high single-particle trapping efficiency (62%) and purity (99.6%). To achieve large-area single-cell trapping, we introduced a “sweeping” loading protocol to further expand the single-particle trapping range. In the microwell region near to the edge of the magnet, over 3000 single magnetic beads were trapped in a 10 mm2 area. Finally, we demonstrated immunomagnetic-labeled THP-1 cells can successfully be trapped at single-cell level in the microwell. The cell trapping process can be done in 10 min. We believe the platform with an accurate and efficient single-cell trapping functionality could potentially be used for various cellular analyses at the single-cell level.