Insulator-based dielectrophoretic single particle and single cancer cell trapping

Trapping of individual cells at specific locations in a microfluidic lab-on-a-chip platform is essential for single cell studies, especially those requiring individual stimulation followed by downstream analysis. To this aim, we have designed microdevices based on direct current (DC) insulator-based dielectrophoresis (iDEP) acting as individual single cell traps. We present both the design of a negative iDEP trap and a positive iDEP trap using insulating posts integrated at microchannel intersections. We obtained electric field distributions via numerical simulations adapted to the intersection and trap geometry with which we predict single particle pathlines. With polystyrene particles of 10?μm diameter, we demonstrated an effective design for a single particle trap in the case of negative dielectrophoresis. The onset trapping voltage shows an inverse relation to the buffer conductivity, thus indicating the influence of electrokinetic effects on the trapping behavior. Additionally, we demonstrated the proof-of-principle of single MCF-7 breast cancer cell trapping in a positive iDEP trap. Our single particle trapping experiments were further in very good agreement with numerical simulations. To ensure that no significant damage occurred to the cells during the experiment, we further optimized medium conditions to ensure viability of the cells for at least 1?h, more than sufficient for microfluidic trapping experiments. Our results thus indicated the successful design of DC iDEP traps, which can easily be integrated into a variety of microchip operations for single cell analysis.     

Quantitative modeling of dielectrophoretic traps

We present quantitative modeling software for simulating multiple forces acting on a single particle in a microsystem. In this paper, we focus on dielectrophoretic (DEP) trapping of single cells against fluid flow. The software effectively models the trapping behavior for a range of particles including beads, mammalian cells, viruses, and bacteria. In addition, the software can be used to reveal useful information about the DEP traps - such as multipolar DEP force effects, trap size-selectivity, and effects from varying the flow chamber height. Our modeling software thus serves as a predictive tool, enabling the design of novel DEP traps with superior performance over existing trap geometries. In addition, the software can evaluate a range of trap dimensions to determine the effects on trapping behavior, thus optimizing the trap geometry before it is even fabricated. The software is freely available to the scientific community at: .     

Bacterial chromosome extraction and isolation

We have used diffusive mixing and dielectrophoretic trapping to lyse Escherichia coli cells in a microfabricated environment and trap the E. coli chromosome. We characterize the conditions needed for efficient lysis of the cells, and conditions needed for the dielectrophoretic trapping of the chromatin without the trapping of cytoplasmic proteins.     

Dynamic single cell culture array

It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning approximately 1 mm(2) with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.     

Enhanced microfiltration devices configured with hydrodynamic trapping and a rain drop bypass filtering architecture for microbial cells detection

Ultra-fine (<1 microm) microfilters are required to effectively trap microbial cells. We designed microfilters featuring a rain drop bypass architecture, which significantly reduces the likelihood of clogging at the cost of limited cell loss. The new rain drop bypass architecture configuration has a substantially lower pressure drop and allows a better efficiency in trapping protozoan cells (Cryptosporidium parvum and Giardia lamblia) in comparison to our previous generation of a microfilter device. A modified version displaying sub-micron filter gaps was adapted to trap and detect bacterial cells (Escherichia coli), through a method of cells labeling, which aims to amplify the fluorescence signal emission and therefore the sensitivity of detection.     

A microfluidic-based hydrodynamic trap: design and implementation

We report an integrated microfluidic device for fine-scale manipulation and confinement of micro- and nanoscale particles in free-solution. Using this device, single particles are trapped in a stagnation point flow at the junction of two intersecting microchannels. The hydrodynamic trap is based on active flow control at a fluid stagnation point using an integrated on-chip valve in a monolithic PDMS-based microfluidic device. In this work, we characterize device design parameters enabling precise control of stagnation point position for efficient trap performance. The microfluidic-based hydrodynamic trap facilitates particle trapping using the sole action of fluid flow and provides a viable alternative to existing confinement and manipulation techniques based on electric, optical, magnetic or acoustic force fields. Overall, the hydrodynamic trap enables non-contact confinement of fluorescent and non-fluorescent particles for extended times and provides a new platform for fundamental studies in biology, biotechnology and materials science.     

Frequency discretization in dielectrophoretic assisted cell sorting arrays to isolate neural cells

We present an automated dielectrophoretic assisted cell sorting (DACS) device for dielectric characterization and isolation of neural cells. Dielectrophoretic (DEP) principles are often used to develop cell sorting techniques. Here we report the first statistically significant neuronal sorting using DACS to enrich neurons from a heterogeneous population of mouse derived neural stem/progenitor cells (NSPCs) and neurons. We also study the dielectric dispersions within a heterogeneous cell population using a Monte-Carlo (MC) simulation. This simulation model explains the trapping behavior of populations as a function of frequency and predicts sorting efficiencies. The platform consists of a DEP electrode array with three multiplexed trapping regions that can be independently activated at different frequencies. A novel microfluidic manifold enables cell sorting by trapping and collecting cells at discrete frequency bands rather than single frequencies. The device is used to first determine the percentage of cells trapped at these frequency bands. With this characterization and the MC simulation we choose the optimal parameters for neuronal sorting. Cell sorting experiments presented achieve a 1.4-fold neuronal enrichment as predicted by our model.     

Hyphenation of thermogravimetry and soft single photon ionization–ion trap mass spectrometry (TG–SPI–ITMS) for evolved gas analysis

Thermal processes are part of many industrial treatments; therefore, it is of great interest to gain more insight of these processes. Evolved gas analysis (EGA) is the most straightforward way to make chemical reactions in thermal processes accessible for on-line investigations. The sample matrix of evolved off gas e.g., from coffee roasting is a permanently changing and complex mixture of a multitude of substances that have to be analyzed simultaneously for real on-line investigations without any sample trapping or separation device. Therefore, a measurement system as an ion trap mass spectrometer (ITMS) with soft ionization is required with its tandem mass spectrometry capability to provide distinct substance identification unperturbed by the remaining matrix. The presented novel system setup is based on a thermogravimetric device (TG) to simulate the thermal treatment as in industrial processes combined with an ITMS with soft single photon ionization (SPI) to achieve the required substance information. Hence it is possible to gain single mass spectrometric information of expected substances for process control. More comprehensive than that are the two-dimensional MS data which are required for research and process development purposes though. The conducted analyses show that this novel setup is able to provide distinct substance identification in evolved gas of roast and ground coffee powder. To our knowledge, this is the first TG–SPI–ITMS setup with successful application in verifying the identity of different mass traces within a single run.     

Quantitative analysis of the three-dimensional trap stiffness of a dielectrophoretic corral trap

Dielectrophoresis is a robust approach for the manipulation and separation of (bio)particles using microfluidic platforms. We developed a dielectrophoretic corral trap in a microfluidic device that utilizes negative dielectrophoresis to capture single spherical polystyrene particles. Circular-shaped micron-size traps were employed inside the device and the three-dimensional trap stiffness (restoring trapping force from equilibrium trapping location) was analyzed using 4.42 μm particles and 1 MHz of an alternating electric field from 6 V_P-P to 10 V_P-P. The trap stiffness increased exponentially in the x- and y-direction, and linearly in the z-direction. Image analysis of the trapped particle movements revealed that the trap stiffness is increased 608.4, 539.3, and 79.7% by increasing the voltage from 6 V_P-P to 10 V_P-P in the x-, y-, and z-direction, respectively. The trap stiffness calculated from a finite element simulation of the device confirmed the experimental results. This analysis provides important insights to predict the trapping location, strength of the trapping, and optimum geometry for single particle trapping and its applications such as single-molecule analysis and drug discovery.     

Numerical design of electrical-mechanical traps

We present a coupled immersed interface method-boundary element method (IIM-BEM) numerical technique that predicts the behaviour of deformable cells under the effect of both hydrodynamic and electrical forces. This technique is applied to the study of a hybrid electrical-mechanical trap for single-cell trapping. We report on the effect of different combinations of electrode positions and mechanical properties of the trap on the maximum loading and unloading Reynolds numbers. We also report on the effect that cells moving with the flow have on cells which have been already trapped in a cavity.     

3D Insulator‐based dielectrophoresis using DC‐biased,AC electric fields for selective bacterial trapping

Insulator‐based dielectrophoresis (iDEP) is a well‐known technique that harnesses electric fields for separating, moving, and trapping biological particle samples. Recent work has shown that utilizing DC‐biased AC electric fields can enhance the performance of iDEP devices. In this study, an iDEP device with 3D varying insulating structures analyzed in combination with DC biased AC fields is presented for the first time. Using our unique reactive ion etch lag, the mold for the 3D microfluidic chip is created with a photolithographic mask. The 3D iDEP devices, whose largest dimensions are 1 cm long, 0.18 cm wide, and 90 μm deep are then rapidly fabricated by curing a PDMS polymer in the glass mold. The 3D nature of the insulating microstructures allows for high trapping efficiency at potentials as low as 200 Vpp. In this work, separation of Escherichia coli from 1 μm beads and selective trapping of live Staphylococcus aureus cells from dead S. aureus cells is demonstrated. This is the first reported use of DC‐biased AC fields to selectively trap bacteria in 3D iDEP microfluidic device and to efficiently separate particles where selectivity of DC iDEP is limited.     

Dielectrophoresis‐assisted creation of cell aggregates under flow conditions using planar electrodes

We present a microfluidic platform allowing dielectrophoresis‐assisted formation of cell aggregates of controlled size and composition under flow conditions. When specific experimental conditions are met, negative dielectrophoresis allows efficient concentration of cells towards electric field minima and subsequent aggregation. This bottom‐up assembly strategy offers several advantages with respect to the targeted application: first, dielectrophoresis offers precise control of spatial cell organization, which can be adjusted by optimizing electrode design. Then, it could contribute to accelerate the establishment of cell‐cell interactions by favoring close contact between neighboring cells. The trapping geometry of our chip is composed of eight electrodes arranged in a circle. Several parameters have been tested in simulations to find the best configurations for trapping in flow. Those configurations have been tested experimentally with both polystyrene beads and human embryonic kidney cells. The final design and experimental setup have been optimized to trap cells and release the created aggregates on demand.     

Hydrodynamic trapping of Tetrahymena thermophila for the long-term monitoring of cell behaviors

Microfluidic trapping technology has been widely applied for single-cell observation in order to reveal characteristic cell behaviors. However, this strategy has yet to be tested for monitoring highly motile cells, which are often biologically important. In this paper, we seek the conditions that enable effective and long-term trapping of a prominent model ciliate Tetrahymena thermophila within a hydrodynamic microfluidic device. Although motility and flexibility of T. thermophila make it difficult to avoid escaping from the trap, we show that tuning some key parameters in the hydrodynamic circuit was effective to achieve approximately 40 h cell retention, which is long enough to monitor cell behaviors over several generations. Here, we demonstrate the real-time observation of cell division and phagocytic digestion, revealing interesting phenomena such as a wide distribution in doubling time in a poor synthetic medium and heterogeneous time courses in digestion processes. Our results present a strategy for trapping highly motile ciliate cells in order to study the dynamic behaviors of single cells.     

A single-cell drug efflux assay in bacteria by using a directly accessible femtoliter droplet array

Active efflux of drugs, such as antibiotics, from a cell is one of the major mechanisms that cause multi-drug resistance in bacteria. Here we report a method to assess drug efflux activity in individual Escherichia coli cells enclosed and isolated in a directly accessible femtoliter droplet array with a fluorogenic compound. The inhibitory effect of a chemical compound on an exogenously expressed efflux pump system from pathogenic bacteria was easily detected at the single-cell level. We also present a proof-of-principle experiment to screen for the gene encoding a drug efflux pump by collecting individual droplets containing single cells in which the drug efflux activity was restored after introduction of the exogenous gene from pathogenic bacteria. Our approach will be a useful tool to screen novel pump inhibitors and efflux pump genes, and to overcome infectious diseases caused by multi-drug-resistant bacteria.     

Laser trap studies of end-on E. coli adhesion to glass

Rod-shaped Escherichia coli K12:D21 bacteria were previously found to adhere by their ends (poles) [J.F. Jones, J.D. Feick, D. Imoudu, N. Chukwumah, M. Vigeant, D. Velegol, Appl. Environ. Microbiol. 69 (2003) 6515.]. In the current study we used a Nd:YAG 1064 nm laser trap to quantify the fraction of adherent bacteria and the time scale for the adhesion to occur. For the E. coli studied, 15.9+/-3.4% of the bacteria adhered when presented end-on for 15s to a cleaned glass surface that was not treated for specific interactions. These bacteria were found to adhere either instantaneously (approximately <1s) or not at all, and the adhesion was shown to be independent of power (force) of the laser trap. Additionally, for a given bacterium, either 0 or 1 ends were adhesive, never both ends. It is hypothesized that the end-on adhesion of D21 is related to bacterial polarity that dynamically results from the division process. We studied the reattachment of cells after adhesion and subsequent removal, finding that most bacteria reattach, some at least five times. However, a small fraction of D21 did not reattach after the first removal. Bacterial cells with observable division planes were tested for end-on adhesion; none of the 18 cells studied adhered by either end. On the other hand, we examined 50 daughter cells immediately after division, and four of the cells were adhesive. End-on adhesion is shown to be an important initial adhesion strategy for the E. coli strain via a single end with adhesion occurring instantaneously. Knowledge about adherent nanodomains (here, on one end) on bacteria will lead to better predictions of sticking coefficients and bacteria transport through porous media.     

An electrically compensated trap designed to eighth order for FT-ICR mass spectrometry

We present the design, guided by theory to eighth order, and the first evaluation of a Fourier transform ion cyclotron resonance (FT-ICR) compensated trap. The purpose of the new trap is to reduce effects of the nonlinear components of the trapping electric field; those nonliner components introduce variations in the cyclotron frequency of an ion depending on its spatial position (its cyclotron and trapping mode amplitudes). This frequency spread leads to decreased mass resolving power and signal-to-noise. The reduction of the spread of cyclotron frequencies, as explicitly modeled in theory, serves as the basis for our design. The compensated trap shows improved signal-to-noise and at least a threefold increase in mass resolving power compared to the uncompensated trap at the same trapping voltage. Resolving powers (FWHH) as high as 1.7 x 10(7) for the [M + H](+) of vasopressin at m/z 1084.5 in a 7.0-tesla induction can be obtained when using trap compensation.     

Active particle control through silicon using conventional optical trapping techniques

Functional integration of optical trapping techniques with silicon surfaces and environments can be realized with minimal modification of conventional optical trapping instruments offering a method to manipulate, track and position cells or non-biological particles over silicon substrates. This technique supports control and measurement advances including the optical control of silicon-based microfluidic devices and precision single molecule measurement of biological interactions at the semiconductor interface. Using a trapping laser in the near infra-red and a reflective imaging arrangement enables object control and measurement capabilities comparable to trapping through a classical glass substrate. The transmission efficiency of the silicon substrate affords the only reduction in trap stiffness. We implement conventional trap calibration, positioning, and object tracking over silicon surfaces. We demonstrate control of multiple objects including cells and complex non-spherical objects on silicon wafers and fabricated surfaces.     

Quantification of bacterial cells based on autofluorescence on a microfluidic platform

Bacterial counts provide important information during the processes such as pathogen detection and hygiene inspection and these processes are critical for public health and food/pharmaceutical production. In this study, we demonstrate the quantification of the number of bacterial cells based on the autofluorescence from the cell lysate on a microfluidic chip. We tested three model pathogenic bacteria (Listeria monocytogenes F4244, Salmonella Enteritidis PT1 and Escherichia coli O157:H7 EDL 933). In the experiment, a plug of approximately 150 pL containing lysate from 240 to 4100 cells was injected into a microfluidic channel with downstream laser-induced fluorescence detection under electrophoresis conditions. We found that the autofluorescence intensity increased with the number of cells almost linearly for all three bacteria. The autofluorescence remained a single peak when the cell lysate contained a mixture of different bacterial species. We also demonstrate a simple microfluidic device that integrates entrapment and electrical lysis of bacterial cells with fluorescence detection. Such a device can carry out the quantification of bacterial cells based on lysate autofluorescence without off-chip procedures. This study offers a simple and fast solution to on-chip quantification of bacterial cells without labeling. We believe that the method can be extended to other bacterial species.     

Monitoring induced gene expression of single cells in a multilayer microchip

We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods.     

Bubbles no more: in-plane trapping and removal of bubbles in microfluidic devices

Gas bubbles present a frequent challenge to the on-chip investigation and culture of biological cells and small organs. The presence of a single bubble can adversely impair biological function and often viability as it increases the wall shear stress in a liquid-perfused microchannel by at least one order of magnitude. We present a microfluidic strategy for in-plane trapping and removal of gas bubbles with volumes of 0.1-500 nL. The presented bubble trap is compatible with single-layer soft lithography and requires a footprint of less than ten square millimetres. Nitrogen bubbles were consistently removed at a rate of 0.14 μL min(-1). Experiments were complemented with analytical and numerical models to comprehensively characterize bubble removal for liquids with different wetting behaviour. Consistent long-term operation of the bubble trap was demonstrated by removing approximately 4000 bubbles during one day. In a case study, we successfully applied the bubble trap to the on-chip investigation of intact small blood vessels. Scalability of the design was demonstrated by realizing eight parallel traps at a total removal rate of 0.9 μL min(-1) (measured for nitrogen).