Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10–100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.     

ApoStream?, a new dielectrophoretic device for antibody independent isolation and recovery of viable cancer cells from blood

Isolation and enumeration of circulating tumor cells (CTCs) are used to monitor metastatic disease progression and guide cancer therapy. However, currently available technologies are limited to cells expressing specific cell surface markers, such as epithelial cell adhesion molecule (EpCAM) or have limited specificity because they are based on cell size alone. We developed a device, ApoStream^™ that overcomes these limitations by exploiting differences in the biophysical characteristics between cancer cells and normal, healthy blood cells to capture CTCs using dielectrophoretic technology in a microfluidic flow chamber. Further, the system overcomes throughput limitations by operating in continuous mode for efficient isolation and enrichment of CTCs from blood. The performance of the device was optimized using a design of experiment approach for key operating parameters such as frequency, voltage and flow rates, and buffer formulations. Cell spiking studies were conducted using SKOV3 or MDA-MB-231 cell lines that have a high and low expression level of EpCAM, respectively, to demonstrate linearity and precision of recovery independent of EpCAM receptor levels. The average recovery of SKOV3 and MDA-MB-231 cancer cells spiked into approximately 12 × 10⁶ peripheral blood mononuclear cells obtained from 7.5 ml normal human donor blood was 75.4% ± 3.1% (n = 12) and 71.2% ± 1.6% (n = 6), respectively. The intra-day and inter-day precision coefficients of variation of the device were both less than 3%. Linear regression analysis yielded a correlation coefficient (R²) of more than 0.99 for a spiking range of 4–2600 cells. The viability of MDA-MB-231 cancer cells captured with ApoStream was greater than 97.1% and there was no difference in cell growth up to 7 days in culture compared to controls. The ApoStream device demonstrated high precision and linearity of recovery of viable cancer cells independent of their EpCAM expression level. Isolation and enrichment of viable cancer cells from ApoStream enables molecular characterization of CTCs from a wide range of cancer types.     

Bandpass sorting of heterogeneous cells using a single surface acoustic wave transducer pair

Separation and sorting of biological entities (viruses, bacteria, and cells) is a critical step in any microfluidic lab-on-a-chip device. Acoustofluidics platforms have demonstrated their ability to use physical characteristics of cells to perform label-free separation. Bandpass-type sorting methods of medium-sized entities from a mixture have been presented using acoustic techniques; however, they require multiple transducers, lack support for various target populations, can be sensitive to flow variations, or have not been verified for continuous flow sorting of biological cells. To our knowledge, this paper presents the first acoustic bandpass method that overcomes all these limitations and presents an inherently reconfigurable technique with a single transducer pair for stable continuous flow sorting of blood cells. The sorting method is first demonstrated for polystyrene particles of sizes 6, 10, and 14.5 μm in diameter with measured purity and efficiency coefficients above 75 ± 6% and 85 ± 9%, respectively. The sorting strategy was further validated in the separation of red blood cells from white blood cells and 1 μm polystyrene particles with 78 ± 8% efficiency and 74 ± 6% purity, respectively, at a flow rate of at least 1 μl/min, enabling to process finger prick blood samples within minutes.     

Quantification of the specific membrane capacitance of single cells using a microfluidic device and impedance spectroscopy measurement

The specific membrane capacitance (SMC) is an electrical parameter that correlates with both the electrical activity and morphology of the plasma membrane, which are physiological markers for cellular phenotype and health. We have developed a microfluidic device that enables impedance spectroscopy measurements of the SMC of single biological cells. Impedance spectra induced by single cells aspirated into the device are captured over a moderate frequency range (5 kHz–1 MHz). Maximum impedance sensitivity is achieved using a tapered microfluidic channel, which effectively routes electric fields across the cell membranes. The SMC is extracted by curve-fitting impedance spectra to an equivalent circuit model. From our measurement, acute myeloid leukemia (AML) cells are found to exhibit larger SMC values in hypertonic solutions as compared with those in isotonic solutions. In addition, AML cell phenotypes (AML2 and NB4) exhibiting varying metastatic potential yield distinct SMC values (AML2: 16.9 ± 1.9 mF/m² (n = 23); NB4: 22.5 ± 4.7 mF/m² (n = 23)). Three-dimensional finite element simulations of the microfluidic device confirm the feasibility of this approach.     

A low cost design and fabrication method for developing a leak proof paper based microfluidic device with customized test zone

This article describes a fabrication process for the generation of a leak proof paper based microfluidic device and a new design strategy for convenient incorporation of externally prepared test zones. Briefly, a negative photolithographic method was used to prepare the device with a partial photoresist layer on the rear of the device to block the leakage of sample. Microscopy and Field Emission Scanning Electron Microscopy data validated the formation of the photoresist layer. The partial layer of photoresist on the device channel limits sample volume to 7 ± 0.2 μl as compared to devices without the partial photoresist layer which requires a larger sample volume of 10 ± 0.1 μl. The design prototype with a customized external test zone exploits the channel protrusions on the UV exposed photoresist treated paper to bridge the externally applied test zone to the sample and absorbent zones. The partially laminated device with an external test zone has a comparatively low wicking speed of 1.8 ± 0.9 mm/min compared to the completely laminated device with an inbuilt test zone (3.3 ± 1.2 mm/min) which extends the reaction time between the analyte and reagents. The efficacy of the prepared device was studied with colorimetric assays for the non-specific detection of protein by tetrabromophenol blue, acid/base with phenolphthalein indicator, and specific detection of proteins using the HRP-DAB chemistry. The prepared device has the potential for leak proof detection of analyte, requires low sample volume, involves reduced cost of production (∼$0.03, excluding reagent and lamination cost), and enables the integration of customized test zones.     

A chitosan coated monolith for nucleic acid capture in a thermoplastic microfluidic chip

A technique for microfluidic, pH modulated DNA capture and purification using chitosan functionalized glycidyl methacrylate monoliths is presented. Highly porous polymer monoliths are formed and subsequently functionalized off-chip in a batch process before insertion into thermoplastic microchannels prior to solvent bonding, simplifying the overall fabrication process by eliminating the need for on-chip surface modifications. The monolith anchoring method allows for the use of large cross-section monoliths enabling high flowrates and high DNA capture capacity with a minimum of added design complexity. Using monolith capture elements requiring less than 1 mm² of chip surface area, loading levels above 100 ng are demonstrated, with DNA capture and elution efficiency of 54.2% ± 14.2% achieved.     

Continuous flowing micro-reactor for aqueous reaction at temperature higher than 100?°C

Some aqueous reactions in biological or chemical fields are accomplished at a high temperature. When the reaction temperature is higher than 100 °C, an autoclave reactor is usually required to elevate the boiling point of the water by creating a high-pressure environment in a closed system. This work presented an alternative continuous flowing microfluidic solution for aqueous reaction with a reaction temperature higher than 100 °C. The pressure regulating function was successfully fulfilled by a small microchannel based on a delicate hydrodynamic design. Combined with micro heater and temperature sensor that integrated in a single chip by utilizing silicon-based microfabrication techniques, this pressure regulating microchannel generated a high-pressure/high-temperature environment in the upstream reaction zone when the reagents continuously flow through the chip. As a preliminary demonstration, thermal digestion of aqueous total phosphorus sample was achieved in this continuous flowing micro-reactor at a working pressure of 990 kPa (under the working flow rate of 20 nl/s) along with a reaction temperature of 145 °C. This continuous flowing microfluidic solution for high-temperature reaction may find applications in various micro total analysis systems.     

Biomolecule storage on non-modified thermoplastic microfluidic chip by ink-jet printing of ionogels

This paper reports an innovative technique for reagents storage in microfluidic devices by means of a one-step UV-photoprintable ionogel-based microarray on non-modified polymeric substrates. Although the ionogel and the ink-jet printing technology are well published, this is the first study where both are used for long-term reagent storage in lab-on-a-chip devices. This technology for reagent storage is perfectly compatible with mass production fabrication processes since pre-treatment of the device substrate is not necessary and inkjet printing allows for an efficient reagent deposition process. The functionality of this microarray is demonstrated by testing the release of biotin-647 after being stored for 1 month at room temperature. Analysis of the fluorescence of the ionogel-based microarray that contains biotin-647 demonstrated that 90% of the biotin-647 present was released from the ionogel-based microarray after pumping PBS 0.1% Tween at 37 °C. Moreover, the activity of biotin-647 after being released from the ionogel-based microarray was investigated trough the binding capability of this biotin to a microcontact printed chip surface with avidin. These findings pave the way for a novel, one-step, cheap and mass production on-chip reagents storage method applicable to other reagents such as antibodies and proteins and enzymes.     

Polyester μ-assay chip for stem cell studies

The application of microfluidic technologies to stem cell research is of great interest to biologists and bioengineers. This is chiefly due to the intricate ability to control the cellular environment, the reduction of reagent volume, experimentation time and cost, and the high-throughput screening capabilities of microscale devices. Despite this importance, a simple-to-use microfluidic platform for studying the effects of growth factors on stem cell differentiation has not yet emerged. With this consideration, we have designed and characterized a microfluidic device that is easy to fabricate and operate, yet contains several functional elements. Our device is a simple polyester-based microfluidic chip capable of simultaneously screening multiple independent stem cell culture conditions. Generated by laser ablation and stacking of multiple layers of polyester film, this device integrates a 10 × 10 microwell array for cell culture with a continuous perfusion system and a non-linear concentration gradient generator. We performed numerical calculations to predict the gradient formation and calculate the shear stress acting on the cells inside the device. The device operation was validated by culturing murine embryonic stem cells inside the microwells for 5 days. Furthermore, we showed the ability to maintain the pluripotency of stem cell aggregates in response to concentrations of leukemia inhibitory factor ranging from 0 to ∼1000 U/ml. Given its simplicity, fast manufacturing method, scalability, and the cell-compatible nature of the device, it may be a useful platform for long-term stem cell culture and studies.     

Manufacturing and wetting low-cost microfluidic cell separation devices

Deterministic lateral displacement (DLD) is a microfluidic size-based particle separation or filter technology with applications in cell separation and enrichment. Currently, there are no cost-effective manufacturing methods for this promising microfluidic technology. In this fabrication paper, however, we develop a simple, yet robust protocol for thermoplastic DLD devices using regulatory-approved materials and biocompatible methods. The final standalone device allowed for volumetric flow rates of 660 μl min⁻¹ while reducing the manufacturing time to <1 h. Optical profilometry and image analysis were employed to assess manufacturing accuracy and precision; the average replicated post height was 0.48% less than the average post height on the master mold and the average replicated array pitch was 1.1% less than the original design with replicated posts heights of 62.1 ± 5.1 μm (mean ± 6 standard deviations) and replicated array pitches of 35.6 ± 0.31 μm.     

Gold coated optical fibers as three-dimensional electrodes for microfluidic enzymatic biofuel cells: Toward geometrically enhanced performance

For the first time, we report on the preliminary evaluation of gold coated optical fibers (GCOFs) as three-dimensional (3D) electrodes for a membraneless glucose/O₂ enzymatic biofuel cell. Two off-the-shelf 125 μm diameter GCOFs were integrated into a 3D microfluidic chip fabricated via rapid prototyping. Using soluble enzymes and a 10 mM glucose solution flowing at an average velocity of 16 mm s⁻¹ along 3 mm long GCOFs, the maximum power density reached 30.0 ± 0.1 μW cm⁻² at a current density of 160.6 ± 0.3 μA cm⁻². Bundles composed of multiple GCOFs could further enhance these first results while serving as substrates for enzyme immobilization.     

Reconfigurable microfluidics combined with antibody microarrays for enhanced detection of T-cell secreted cytokines

Cytokines are small proteins secreted by leukocytes in blood in response to infections, thus offering valuable diagnostic information. Given that the same cytokines may be produced by different leukocyte subsets in blood, it is beneficial to connect production of cytokines to specific cell types. In this paper, we describe integration of antibody (Ab) microarrays into a microfluidic device to enable enhanced cytokine detection. The Ab arrays contain spots specific to cell-surface antigens as well as anti-cytokine detection spots. Infusion of blood into a microfluidic device results in the capture of specific leukocytes (CD4 T-cells) and is followed by detection of secreted cytokines on the neighboring Ab spots using sandwich immunoassay. The enhancement of cytokine signal comes from leveraging the concept of reconfigurable microfluidics. A three layer polydimethylsiloxane microfluidic device is fabricated so as to contain six microchambers (1 mm × 1 mm × 30 μm) in the ceiling of the device. Once the T-cell capture is complete, the device is reconfigured by withdrawing liquid from the channel, causing the chambers to collapse onto Ab arrays and enclose cell/anti-cytokine spots within a 30 nl volume. In a set of proof-of-concept experiments, we demonstrate that ∼90% pure CD4 T-cells can be captured inside the device and that signals for three important T-cell secreted cytokines, tissue necrosis factor-alpha, interferon-gamma, and interleukin-2, may be enhanced by 2 to 3 folds through the use of reconfigurable microfluidics.     

A microfluidic chip for direct and rapid trapping of white blood cells from whole blood

Blood analysis plays a major role in medical and science applications and white blood cells (WBCs) are an important target of analysis. We proposed an integrated microfluidic chip for direct and rapid trapping WBCs from whole blood. The microfluidic chip consists of two basic functional units: a winding channel to mix and arrays of two-layer trapping structures to trap WBCs. Red blood cells (RBCs) were eliminated through moving the winding channel and then WBCs were trapped by the arrays of trapping structures. We fabricated the PDMS (polydimethylsiloxane) chip using soft lithography and determined the critical flow velocities of tartrazine and brilliant blue water mixing and whole blood and red blood cell lysis buffer mixing in the winding channel. They are 0.25 μl/min and 0.05 μl/min, respectively. The critical flow velocity of the whole blood and red blood cell lysis buffer is lower due to larger volume of the RBCs and higher kinematic viscosity of the whole blood. The time taken for complete lysis of whole blood was about 85 s under the flow velocity 0.05 μl/min. The RBCs were lysed completely by mixing and the WBCs were trapped by the trapping structures. The chip trapped about 2.0 × 10³ from 3.3 × 10³ WBCs.     

Efficient elusion of viable adhesive cells from a microfluidic system by air foam

We developed a new method for releasing viable cells from affinity-based microfluidic devices. The lumen of a microchannel with a U-shape and user-designed microstructures was coated with supported lipid bilayers functionalized by epithelial cell adhesion molecule antibodies to capture circulating epithelial cells of influx solution. After the capturing process, air foam was introduced into channels for releasing target cells and then carrying them to a small area of membrane. The results show that when the air foam is driven at linear velocity of 4.2 mm/s for more than 20 min or at linear velocity of 8.4 mm/s for more than 10 min, the cell releasing efficiency approaches 100%. This flow-induced shear stress is much less than the physiological level (15 dyn/cm²), which is necessary to maintain the intactness of released cells. Combining the design of microstructures of the microfluidic system, the cell recovery on the membrane exceeds 90%. Importantly, we demonstrate that the cells released by air foam are viable and could be cultured in vitro. This novel method for releasing cells could power the microfluidic platform for isolating and identifying circulating tumor cells.     

Parallel generation of uniform fine droplets at hundreds of kilohertz in a flow-focusing module

Droplet-based microfluidic systems enable a variety of biomedical applications from point-of-care diagnostics with third world implications, to targeted therapeutics alongside medical ultrasound, to molecular screening and genetic testing. Though these systems maintain the key advantage of precise control of the size and composition of the droplet as compared to conventional methods of production, the low rates at which droplets are produced limits translation beyond the laboratory setting. As well, previous attempts to scale up shear-based microfluidic systems focused on increasing the volumetric throughput and formed large droplets, negating many practical applications of emulsions such as site-specific therapeutics. We present the operation of a parallel module with eight flow-focusing orifices in the dripping regime of droplet formation for the generation of uniform fine droplets at rates in the hundreds of kilohertz. Elevating the capillary number to access dripping, generation of monodisperse droplets of liquid perfluoropentane in the parallel module exceeded 3.69 × 10⁵ droplets per second, or 1.33 × 10⁹ droplets per hour, at a mean diameter of 9.8 μm. Our microfluidic method offers a novel means to amass uniform fine droplets in practical amounts, for instance, to satisfy clinical needs, with the potential for modification to form massive amounts of more complex droplets.     

Magnetographic array for the capture and enumeration of single cells and cell pairs

We present a simple microchip device consisting of an overlaid pattern of micromagnets and microwells capable of capturing magnetically labeled cells into well-defined compartments (with accuracies >95%). Its flexible design permits the programmable deposition of single cells for their direct enumeration and pairs of cells for the detailed analysis of cell-cell interactions. This cell arraying device requires no external power and can be operated solely with permanent magnets. Large scale image analysis of cells captured in this array can yield valuable information (e.g., regarding various immune parameters such as the CD4:CD8 ratio) in a miniaturized and portable platform.The emergent need for point-of-care devices has spurred development of simplified platforms to organize cells across well-defined templates.¹ These devices employ passive microwells, immunospecific adhesive islands, and electric, optical, and acoustic traps to manipulate cells.^2–6 In contrast, magnetic templating can control the spatial organization of cells through its ability to readily program ferromagnetic memory states.⁷ While it has been applied to control the deposition of magnetic beads,^8–13 it has not been used to direct the deposition of heterogeneous cell pairs, which may help provide critical insight into the function of single cells.^14,15 As such, we developed a simple magnetographic device capable of arraying single cells and pairs of cells with high fidelity. We show this magnetic templating tool can use immunospecific magnetic labels for both the isolation of cells from blood and their organization into spatially defined wells.We used standard photolithographic techniques to fabricate the microchips (see supplementary material¹⁶). Briefly, an array of 10 × 30 μm cobalt micromagnets were patterned by a photolithographic liftoff process and overlaid with a pattern of dumbbell-shaped microwells formed in SU-8 photoresist (Fig. 1(a)). The micromagnets were designed to produce a predominantly vertical field in the microwells by aligning the ends of the micromagnet at the center of each well of the dumbbell. These features were deposited across an area of ≈400 mm² (>50 000 well pairs per microchip) (Fig. 1(b)). Depending on the programmed magnetization state with respect to the external field, magnetic beads or cells were attracted to one pole and repelled by the other pole of each micromagnet, leading to a biased deposition (Fig. 1(c)).¹²Open in a separate windowFIG. 1.Magnetographic array for single cell analysis. (a) SEM image of the dumbbell-shaped well pairs for capturing magnetically labelled cells. (b) Photograph of the finished device. (c) An array of well pairs displaying a pitch of 60 × 120 μm before (top) and 10 min after the deposition of magnetic beads (bottom).To demonstrate the capability of the array to capture cells into a format amenable for rapid image processing, we organized CD3+ lymphocytes using only hand-held permanent magnets. We isolated CD3+ lymphocytes from blood via positive selection using anti-CD3 magnetic nanoparticles (EasySep™, STEMCELL Technologies) with purities confirmed by flow cytometry (97.8%; see supplementary material¹⁶). We then stained 1 × 10⁶ CD3+ cells with anti-CD8 Alexa-488 and anti-CD4 Alexa-647 (5 μl of each antibody in 100 μl for 20 min; BD Bioscience) to determine the CD4:CD8 ratio, a prognostic ratio for assessing the immune system.^17,18Variably spaced neodymium magnets (0.5 in. × 0.5 in. × 1 in.; K&J Magnetics, Inc.) were fixed on either side of the microchip to generate a tunable magnetic field (0–400 G; Fig. 2(a)). Using this setup, fluorescently labeled cells were deposited, and the populations of CD4+ and CD8+ cells were indiscriminately arrayed, imaged, and enumerated using ImageJ. The resulting CD4:CD8 ratio of 1.84 ± 0.18 (Fig. 2(b)) was confirmed by flow cytometry with a high correlation (5.4% difference; Fig. 2(c)), indicating the magnetographic microarray can pattern cells for the rapid and accurate assessment of critical phenotypical parameters without complex equipment (e.g., function generators or flow cytometers).Open in a separate windowFIG. 2.CD8 analysis of CD3+ lymphocytes. (a) Photograph of the magnetographic device activated by permanent magnets (covered with green tape). The CD4:CD8 ratio determined by the (b) magnetographic microarray and (c) and (d) flow cytometry was 1.84 and 1.74, respectively.More complex operations, such as the programmed deposition of cell pairs, can be achieved by leveraging the switchable, bistable magnetization of the micromagnets for the detailed studies of cell-cell interactions (Figs. 3(a)–3(d)).¹² For these studies, a 200 G horizontal field generated from an electromagnetic coil was used to magnetize the micromagnets.¹⁹ We then captured different concentrations of magnetic beads as surrogates for cells (8.4 μm polystyrene, Spherotech, Inc.) and found that higher bead concentrations did not affect the capture accuracy (>95%; see supplementary material¹⁶).Open in a separate windowFIG. 3.Programmed pairing of magnetic beads and CD3+ lymphocytes. (a) Schematic of the magnetographic cell pair isolations. (b) Polarized micromagnets isolate cells of one type to one side in a vertical magnetic field and then cells of a second type to the other side when the field is reversed. (c) Fluorescent image of magnetically trapped green stained (top) and red stained (bottom) cell pairs. (d) SEM image of magnetically labeled cells in the microwells. (e) Capture accuracy of magnetic bead pairs. (Each color (and shape) represents the field strength of the reversed field.) (f) Change in the capture accuracy (loss) of initially captured beads after reversing the magnetic field. The capture accuracy of (g) magnetically labeled cell pairs and (h) the second magnetically labeled cell (for (e)–(h): n = 5; time starts from the deposition of the second set of cells or beads).The opposite side of each micromagnet was then populated with the second (yellow fluorescent) bead by reversing the direction of the applied magnetic field. We tested several field strengths (i.e., 10, 25, 40, or 55 G) to optimize the conditions for isolating the desired bead in the opposite well without ejecting the first bead. If the field strength was too large, the previously deposited beads could be ejected from their wells due to the repulsive magnetic force overcoming gravity.¹² As shown in Figure 3(e), increasing the field strength from 10 to 25 G significantly increased the capture accuracy at 60 min from the deposition of the second bead (p < 0.01), but increases from 25 to 55 G did not affect the capture accuracy (p > 0.10). As shown in Figure 3(f), higher field strengths (i.e., 40 and 55 G) resulted in lower capture accuracies compared to lower field strengths (i.e., 10 and 25 G) (p < 0.01), which was primarily due to ejection of the initially captured beads when the micromagnets reversed their polarity.We then arranged pairs of membrane dyed (calcein AM, Invitrogen; PKH26, Sigma) magnetically labeled CD3+ lymphocytes. First, red stained cells (150 μl of 2 × 10⁴ cells/ml) were deposited on the microchip in the presence of 250 G vertical magnetic field. After 20 min, the field was reversed (i.e., to 40, 55, and 70 G) and green stained cells (150 μl of 2 × 10⁴ cells/ml) were deposited on the microchip with images taken in 10 min intervals. Fluorescence images were overlaid (Fig. 3(c)) and the capture accuracy of cell pairs was determined (ImageJ).As seen in Figure 3(g), the capture accuracy of pairs of CD3+ lymphocytes was lower than that of magnetic beads (Fig. 3(e)). However, as shown in Figure 3(h), the second set of cells (green fluorescent) exhibited an average capture accuracy of 91.8% ± 1.9%. This indicates that the lower capture accuracy of cell pairs was either due to the ejection of initially captured (red fluorescent) cells or the migration of initially captured cells through the connecting channel, resulting from their relatively high deformability compared to magnetic beads.In summary, we developed a simple device capable of organizing magnetic particles, cells, and pairs of cells into well-defined compartments. A major advantage of this system is the use of specific magnetic labels to both isolate cells and program their deposition. While the design of this device does not enable dynamic control of the spacing between captured cell pairs as does some dielectrophoresis-based devices,²⁰ it can easily capture cells with high fidelity using only permanent magnets and has clinical relevance in the assessment of immune parameters. These demonstrations potentiate a relatively simple and robust device where highly organized spatial arrangement of cells facilitates rapid and accurate analyses towards a functional and low-cost point-of-care device.     

A negative-pressure-driven microfluidic chip for the rapid detection of a bladder cancer biomarker in urine using bead-based enzyme-linked immunosorbent assay

This paper describes an integrated microfluidic chip that is capable of rapidly and quantitatively measuring the concentration of a bladder cancer biomarker, apolipoprotein A1, in urine samples. All of the microfluidic components, including the fluid transport system, the micro-valve, and the micro-mixer, were driven by negative pressure, which simplifies the use of the chip and facilitates commercialization. Magnetic beads were used as a solid support for the primary antibody, which captured apolipoprotein A1 in patients'' urine. Because of the three-dimensional structure of the magnetic beads, the concentration range of the target that could be detected was as high as 2000 ng ml⁻¹. Because this concentration is 100 times higher than that quantifiable using a 96-well plate with the same enzyme-linked immunosorbent assay (ELISA) kit, the dilution of the patient''s urine can be avoided or greatly reduced. The limit of detection was determined to be approximately 10 ng ml⁻¹, which is lower than the cutoff value for diagnosing bladder cancer (11.16 ng ml⁻¹). When the values measured using the microfluidic chip were compared with those measured using conventional ELISA using a 96-well plate for five patients, the deviations were 0.9%, 6.8%, 9.4%, 1.8%, and 5.8%. The entire measurement time is 6-fold faster than that of conventional ELISA. This microfluidic device shows significant potential for point-of-care applications.     

Modulating chemotaxis of lung cancer cells by using electric fields in a microfluidic device

We employed direct-current electric fields (dcEFs) to modulate the chemotaxis of lung cancer cells in a microfluidic cell culture device that incorporates both stable concentration gradients and dcEFs. We found that the chemotaxis induced by a 0.5 μM/mm concentration gradient of epidermal growth factor can be nearly compensated by a 360 mV/mm dcEF. When the effect of chemical stimulation was balanced by the electrical drive, the cells migrated randomly, and the path lengths were largely reduced. We also demonstrated electrically modulated chemotaxis of two types of lung cancer cells with opposite directions of electrotaxis in this device.     

Characterizing the dielectric properties of human mesenchymal stem cells and the effects of charged elastin-like polypeptide copolymer treatment

Human mesenchymal stem cells (hMSCs) have three key properties that make them desirable for stem cell therapeutics: differentiation capacity, trophic activity, and ability to self-renew. However, current separation techniques are inefficient, time consuming, expensive, and, in some cases, alter hMSCs cellular function and viability. Dielectrophoresis (DEP) is a technique that uses alternating current electric fields to spatially separate biological cells based on the dielectric properties of their membrane and cytoplasm. This work implements the first steps toward the development of a continuous cell sorting microfluidic device by characterizing native hMSCs dielectric signatures and comparing them to hMSCs morphologically standardized with a polymer. A quadrapole Ti-Au electrode microdevice was used to observe hMSC DEP behaviors, and quantify frequency spectra and cross-over frequency of hMSCs from 0.010–35 MHz in dextrose buffer solutions (0.030 S/m and 0.10 S/m). This combined approach included a systematic parametric study to fit a core-shell model to the DEP spectra over the entire tested frequency range, adding robustness to the analysis technique. The membrane capacitance and permittivity were found to be 2.2 pF and 2.0 in 0.030 S/m and 4.5 pF and 4.1 in 0.10 S/m, respectively. Elastin-like polypeptide (ELP-) polyethyleneimine (PEI) copolymer was used to control hMSCs morphology to spheroidal cells and aggregates. Results demonstrated that ELP-PEI treatment controlled hMSCs morphology, increased experiment reproducibility, and concurrently increased hMSCs membrane permittivity to shift the cross-over frequency above 35 MHz. Therefore, ELP-PEI treatment may serve as a tool for the eventual determination of biosurface marker-dependent DEP signatures and hMSCs purification.     

Rapid microfluidic solid-phase extraction system for hyper-methylated DNA enrichment and epigenetic analysis

Genetic sequence and hyper-methylation profile information from the promoter regions of tumor suppressor genes are important for cancer disease investigation. Since hyper-methylated DNA (hm-DNA) is typically present in ultra-low concentrations in biological samples, such as stool, urine, and saliva, sample enrichment and amplification is typically required before detection. We present a rapid microfluidic solid phase extraction (μSPE) system for the capture and elution of low concentrations of hm-DNA (≤1 ng ml⁻¹), based on a protein-DNA capture surface, into small volumes using a passive microfluidic lab-on-a-chip platform. All assay steps have been qualitatively characterized using a real-time surface plasmon resonance (SPR) biosensor, and quantitatively characterized using fluorescence spectroscopy. The hm-DNA capture/elution process requires less than 5 min with an efficiency of 71% using a 25 μl elution volume and 92% efficiency using a 100 μl elution volume.