An integrated microfluidic cell array for apoptosis and proliferation analysis induction of breast cancer cells

In vitro sensitivity testing of tumor cells could rationalize and improve the choice of chemotherapy and hormone therapy. In this report, a microfluidic device made from poly(dimethylsiloxane) and glass was developed for an assay of drug induced cytotoxicity. We evaluated the apoptotic and proliferation-inhibitory effects of anticancer drugs mitomycin C (MMC) and tamoxifen (TAM) using MCF-7 breast cancer cells. MMC and TAM both induced apoptosis and inhibited proliferation of MCF-7 cells in a concentration-dependent manner. MMC caused the expression of antiapoptotic protein Bcl-2 a dose-dependent reduction in MCF-7 cells. The expression of Bcl-2 did not change significantly in MCF-7 cells treated by TAM. The results in the microfluidic device were correlated well with the data obtained from the parallel experiments carried out in the conventional culture plates. The developed microfluidic device could be a potential useful tool for high content screening and high throughput screening research.     

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.     

Long-term microfluidic glucose and lactate monitoring in hepatic cell culture

Monitoring cellular bioenergetic pathways provides the basis for a detailed understanding of the physiological state of a cell culture. Therefore, it is widely used as a tool amongst others in the field of in vitro toxicology. The resulting metabolic information allows for performing in vitro toxicology assays for assessing drug-induced toxicity. In this study, we demonstrate the value of a microsystem for the fully automated detection of drug-induced changes in cellular viability by continuous monitoring of the metabolic activity over several days. To this end, glucose consumption and lactate secretion of a hepatic tumor cell line were continuously measured using microfluidically addressed electrochemical sensors. Adapting enzyme-based electrochemical flat-plate sensors, originally designed for human whole-blood samples, to their use with cell culture medium supersedes the common manual and laborious colorimetric assays and off-line operated external measurement systems. The cells were exposed to different concentrations of the mitochondrial inhibitor rotenone and the cellular response was analyzed by detecting changes in the rates of the glucose and lactate metabolism. Thus, the system provides real-time information on drug-induced liver injury in vitro.     

Transport and shear in a microfluidic membrane bilayer device for cell culture

Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a "membrane bilayer" microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.     

Controlled electroporation of the plasma membrane in microfluidic devices for single cell analysis

Chemical cytometry on a single cell level is of interest to various biological fields ranging from cancer to stem cell research. The impact chemical cytometry can exert in these fields depends on the dimensionality of the retrievable analytes content. To this point, the number of different analytes identifiable and additionally their subcellular localization is of interest. To address this, we present an electroporation based approach for selective lysis of only the plasma membrane, which permits analysis of the dissolved cytoplasm, while reducing contributions from the nucleus and membrane bound fractions of the cell analytes. The use of 100 μs long pulse and a well defined DC electric field gradient of ∼4.5 kV·cm⁻¹ generated by 3D electrodes initiates release of a cytoplasm marker in ≪1 s, while retaining nuclear fluorescence markers.     

Analyzing shear stress-induced alignment of actin filaments in endothelial cells with a microfluidic assay

The physiology of vascular endothelial cells is strongly affected by fluid shear stress on their surface. In this study, a microfluidic assay was employed to analyze the alignment of actin filaments in endothelial cells in response to shear stress. When cells were cultured in microfluidic channels and subjected to shear stress, the alignment of filaments in the channel direction was significantly higher than in static cultures. By adding inhibitory drugs, the roles of several signaling proteins in the process of alignment were determined. Thus, it is shown how microfluidic technology can be employed to provide a mechanistic insight into cell physiology.     

Label-free isolation of a prostate cancer cell among blood cells and the single-cell measurement of drug accumulation using an integrated microfluidic chip

Circulating tumor cells (CTCs) are found in the blood of patients with cancer. Although these cells are rare, they can provide useful information for chemotherapy. However, isolation of these rare cells from blood is technically challenging because they are small in numbers. An integrated microfluidic chip, dubbed CTC chip, was designed and fabricated for conducting tumor cell isolation. As CTCs usually show multidrug resistance (MDR), the effect of MDR inhibitors on chemotherapeutic drug accumulation in the isolated single tumor cell is measured. As a model of CTC isolation, human prostate cancer cells were mixed with mouse blood cells and the label-free isolation of the tumor cells was conducted based on cell size difference. The major advantages of the CTC chip are the ability for fast cell isolation, followed by multiple rounds of single-cell measurements, suggesting a potential assay for detecting the drug responses based on the liquid biopsy of cancer patients.     

Release monitoring of single cells on a microfluidic device coupled with fluorescence microscopy and electrochemistry

A method for monitoring the biological exocytotic phenomena on a microfluidic system was proposed. A microfluidic device coupled with functionalities of fluorescence imaging and amperometric detection has been developed to enable the real-time monitoring of the exocytotic events. Exocytotic release of single SH-SY5Y neuroblastoma cells was studied. By staining the cells located on integrated microelectrodes with naphthalene-2,3-dicarboxaldehyde, punctuate fluorescence consistent with localization of neurotransmitters stored in vesicles was obtained. The stimulated exocytotic release was successfully observed at the surface of SH-SY5Y cells without refitting the commercial inverted fluorescence microscope. Spatially and temporally resolved exocytotic events from single cells on a microfluidic device were visualized in real time using fluorescence microscopy and were amperometrically recorded by the electrochemical system simultaneously. This coupled technique is simple and is hoped to provide new insights into the mechanisms responsible for the kinetics of exocytosis.     

Design and optimization of a double-enzyme glucose assay in microfluidic lab-on-a-chip

An electrokinetic driven microfluidic lab-on-a-chip was developed for glucose quantification using double-enzyme assay. The enzymatic glucose assay involves the two-step oxidation of glucose, which was catalyzed by hexokinase and glucose-6-phosphate dehydrogenase, with the concomitant reduction of NADP⁺ to NADPH. A fluorescence microscopy setup was used to monitor the different processes (fluid flow and enzymatic reaction) in the microfluidic chip. A two-dimensional finite element model was applied to understand the different aspects of design and to improve the performance of the device without extensive prototyping. To our knowledge this is the first work to exploit numerical simulation for understanding a multisubstrate double-enzyme on-chip assay. The assay is very complex to implement in electrokinetically driven continuous system due to the involvement of many species, which has different transport velocity. With the help of numerical simulation, the design parameters, flow rate, enzyme concentration, and reactor length, were optimized. The results from the simulation were in close agreement with the experimental results. A linear relation exists for glucose concentrations from 0.01 to 0.10 g l⁻¹. The reaction time and the amount of enzymes required were drastically reduced compared to off-chip microplate analysis.     

Generation of nitric oxide gradients in microfluidic devices for cell culture using spatially controlled chemical reactions

In this paper, we develop a microfluidic device capable of generating nitric oxide (NO) gradients for cell culture using spatially controlled chemical reactions. NO plays an essential role in various biological activities, including nervous, immune, and cardiovascular systems. The device developed in this paper can control NO gradients without utilizing expensive and hazardous high purity NO gas sources or direct addition of NO donors. Consequently, the device provides an efficient, cost-effective, robust, and stable platform to generate NO gradients for cell culture studies. In the experiments, NO gradients are first characterized using a NO-sensitive fluorescence dye, and cell experiments using aortic smooth muscle cells are conducted. The results demonstrate that the device can alter the intracellular NO concentrations and further affect the Ca²⁺ concentration oscillation for the cells. The device developed in this paper provides a powerful platform for researchers better study the biological roles of NO and its spatial distribution using in vitro cell models with minimal instrumentation.     

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.     

Diffusion phenomena of cells and biomolecules in microfluidic devices

Biomicrofluidics is an emerging field at the cross roads of microfluidics and life sciences which requires intensive research efforts in terms of introducing appropriate designs, production techniques, and analysis. The ultimate goal is to deliver innovative and cost-effective microfluidic devices to biotech, biomedical, and pharmaceutical industries. Therefore, creating an in-depth understanding of the transport phenomena of cells and biomolecules becomes vital and concurrently poses significant challenges. The present article outlines the recent advancements in diffusion phenomena of cells and biomolecules by highlighting transport principles from an engineering perspective, cell responses in microfluidic devices with emphases on diffusion- and flow-based microfluidic gradient platforms, macroscopic and microscopic approaches for investigating the diffusion phenomena of biomolecules, microfluidic platforms for the delivery of these molecules, as well as the state of the art in biological applications of mammalian cell responses and diffusion of biomolecules.     

Study of flow behaviors on single-cell manipulation and shear stress reduction in microfluidic chips using computational fluid dynamics simulations

Various single-cell retention structures (SCRSs) were reported for analysis of single cells within microfluidic devices. Undesirable flow behaviors within micro-environments not only influence single-cell manipulation and retention significantly but also lead to cell damage, biochemical heterogeneity among different individual cells (e.g., different cell signaling pathways induced by shear stress). However, the fundamentals in flow behaviors for single-cell manipulation and shear stress reduction, especially comparison of these behaviors in different microstructures, were not fully investigated in previous reports. Herein, flow distribution and induced shear stress in two different single-cell retention structures (SCRS I and SCRS II) were investigated in detail to study their effects on single-cell trapping using computational fluid dynamics (CFD) methods. The results were successfully verified by experimental results. Comparison between these two SCRS shows that the wasp-waisted configuration of SCRS II has a better performance in trapping and manipulating long cylinder-shaped cardiac myocytes and provides a safer “harbor” for fragile cells to prevent cell damage due to the shear stress induced from strong flows. The simulation results have not only explained flow phenomena observed in experiments but also predict new flow phenomena, providing guidelines for new chip design and optimization, and a better understanding of the cell micro-environment and fundamentals of microfluidic flows in single-cell manipulation and analysis.     

Characterization of microfluidic shear-dependent epithelial cell adhesion molecule immunocapture and enrichment of pancreatic cancer cells from blood cells with dielectrophoresis

Current microfluidic techniques for isolating circulating tumor cells (CTCs) from cancer patient blood are limited by low capture purity, and dielectrophoresis (DEP) has the potential to complement existing immunocapture techniques to improve capture performance. We present a hybrid DEP and immunocapture Hele-Shaw flow cell to characterize DEP''s effects on immunocapture of pancreatic cancer cells (Capan-1, PANC-1, and BxPC-3) and peripheral blood mononuclear cells (PBMCs) with an anti-EpCAM (epithelial cell adhesion molecule) antibody. By carefully specifying the applied electric field frequency, we demonstrate that pancreatic cancer cells are attracted to immunocapture surfaces by positive DEP whereas PBMCs are repelled by negative DEP. Using an exponential capture model to interpret our capture data, we show that immunocapture performance is dependent on the applied DEP force sign and magnitude, cell surface EpCAM expression level, and shear stress experienced by cells flowing in the capture device. Our work suggests that DEP can not only repel contaminating blood cells but also enhance capture of cancer cell populations that are less likely to be captured by traditional immunocapture methods. This combination of DEP and immunocapture techniques to potentially increase CTC capture purity can facilitate subsequent biological analyses of captured CTCs and research on cancer metastasis and drug therapies.     

Prostate specific antigen in patients of benign prostate hypertrophy and carcinoma prostate

Prostate Specific Antigen (PSA) has emerged as the most applicable and important tumor marker for carcinoma prostate. In the present study PSA was determined in serum of healthy subjects, patients of benign prostate hypertrophy (BPH) and Carcinoma Prostate (Ca−P) to evaluate its diagnostic efficiency in day to day management of prostate cancer patients and in differentiating patients of early prostate cancer from those with BPH. Receiver operating characteristic curve (ROC) revealed 2 ng/ml and 10 ng/ml cut off serum PSA level for BPH and untreated carcinoma prostate patients (Ca−P). An extremely significant increase (P<0.0001) was observed in mean PSA concentration in BPH patients and adenocarcinoma prostate patients when compared to healthy males. Clinical relevance of PSA was highlighted by a case study of cancer patient prior to any therapy till death.     

The microfluidic system for studies of carcinoma and normal cells interactions after photodynamic therapy (PDT) procedures

This study reports on the use of a microsystem for evaluation of photodynamic therapy (PDT) procedures on the "mixed" (carcinoma-normal) cultures. Balb/3T3 (normal mouse embryo) and A549 (human lung carcinoma) cells were tested in separated and "mixed" cultures. Interactions and migration of cells cultured together were observed. The PDT procedures were examined in the hybrid (PDMS/glass) microsystem which contains cell culture microchambers integrated with network of microchannels. We investigated that the number of dead cells after PDT procedures is dependent on the kind of cell culture. Moreover, the influence of the carcinoma cells on the viability of normal cells in the "mixed" culture was observed.     

Discrimination between the human prostate normal cell and cancer cell by using a novel electrical impedance spectroscopy controlling the cross-sectional area of a microfluidic channel

The prostate biopsy method shows a high false negative result because the suspicious tissue considered as cancer is not confirmed during tissue sampling. Thus, repeated biopsy procedures and diagnostic errors in relation to prostate cancer frequently occur. The purpose of this research is to enhance the prostate cancer detection rate by using microfluidic electrical impedance spectroscopy (μEIS), which allows real-time measurement of the electrical impedance of a single human prostate normal cell and cancer cell. The μEIS was equipped with a movable flexible membrane, which is operated by pneumatic pressure to capture the single cell on the surface of sensing electrodes. The forced tight contact between the cell and electrodes makes it possible to measure the electrical characteristics of the cell with a high sensitivity. The μEIS discriminates well between normal human prostate cells (RWPE-1) and cancer cells (PC-3) at 8.7 kHz based on the electrical signal responses of the cells. The average difference rates of admittance magnitude and susceptance are 54.55% and 54.59%, respectively. The developed μEIS also shows high repeatability, which was verified by a deionized water test conducted before and after each cell assay; the maximum variance of both the impedance and admittance at 8.7 kHz was as small as 9.48%.     

Integrated electrical concentration and lysis of cells in a microfluidic chip

Lysing cells is an important step in the analysis of intracellular contents. Concentrating cells is often required in order to acquire adequate cells for lysis. This work presents an integrated concentration and lysis of mammalian cells in a constriction microchannel using dc-biased ac electric fields. By adjusting the dc component, the electrokinetic cell motion can be precisely controlled, leading to an easy switch between concentration and lysis of red blood cells in the channel constriction. These two operations are also used in conjunction to demonstrate a continuous concentration and separation of leukemia cells from red blood cells in the same microchannel. The observed cell behaviors agree reasonably with the simulation results.     

Fabrication of a gel particle array in a microfluidic device for bioassays of protein and glucose in human urine samples

This paper describes a simple method for fabricating a series of poly(ethylene glycol) diacrylate (PEG-DA) hydrogel microstructures inside microfluidic channels as probe for proteins and glucose. In order to demonstrate the feasibility of this newly developed system, bovine serum albumin (BSA) was chosen as a model protein. PEG microcolumns were used for the parallel detection of multiple components. Using tetrabromophenol blue (TBPB) and the horseradish peroxidase/glucose oxidase reaction system, bovine serum albumin (BSA) and glucose in human urine were detected by color changes. The color changes for BSA within a concentration range of 1-150 μM, and glucose within a range of 50 mM-2 M could be directly distinguished by eyes or precisely identified by optical microscope. To show the practicability of the gel particle array, protein and glucose concentrations of real human urine samples were determined, resulting in a good correlation with hospital analysis. Notably, only a 5 μL sample was needed for a parallel measurement of both analytes. Conveniently, no special readout equipment or power source was required during the diagnosis process, which is promising for an application in rapid point-of-care diagnosis.     

One step antibody-mediated isolation and patterning of multiple cell types in microfluidic devices

Cell-cell interactions play a key role in regeneration, differentiation, and basic tissue function taking place under physiological shear forces. However, current solutions to mimic such interactions by micro-patterning cells within microfluidic devices have low resolution, high fabrication complexity, and are limited to one or two cell types. Here, we present a microfluidic platform capable of laminar patterning of any biotin-labeled peptide using streptavidin-based surface chemistry. The design permits the generation of arbitrary cell patterns from heterogeneous mixtures in microfluidic devices. We demonstrate the robust co-patterning of α-CD24, α-ASGPR-1, and α-Tie2 antibodies for rapid isolation and co-patterning of mixtures of hepatocytes and endothelial cells. In addition to one-step isolation and patterning, our design permits step-wise patterning of multiple cell types and empty spaces to create complex cellular geometries in vitro. In conclusion, we developed a microfluidic device that permits the generation of perfusable tissue-like patterns in microfluidic devices by directly injecting complex cell mixtures such as differentiated stem cells or tissue digests with minimal sample preparation.