Polydimethylsiloxane Microwell Research Articles

A sandwiched photoelectrochemical sensor for high-throughput single-cell analysis based on low-toxic and near-infrared AgInS2 microarray

High-throughput single-cell quantitative analysis is becoming increasingly important to elucidate cellular heterogeneity and further understand some related disease mechanisms. Various techniques including mass spectrometry, fluorescence detection, and electrochemiluminescence analysis have made significant progress in this area, but their dilemma is the destruction of cellular activity, the introduction of exogenetic probes and the resulting cellular metabolism perturbation, or the steric hindrance of the cells to the substrate. Here, we develop a non-destructive, probe-free, and hindrance-free photoelectrochemical (PEC) single-cell microsensor. First, a patterned PEC microelectrode with low-toxic and near-infrared AgInS2 microdots is simply fabricated by using a polydimethylsiloxane (PDMS) microstencil, exhibiting high photoelectric efficiency and low S2- detection limit (1.0 nM). Then, a PDMS microwell array is prepared for single cells trapping, matching the size of the microelectrode and with high single-cell occupancy (>93%). Finally, a sealed sandwiching platform is formed by aligning and clamping the two arrays, each microdot contacts with a single cell in one microwell and reacts with the secreted H2S, preventing the neighboring microwell’s cross-contamination, cellular analyte’s dilution, and cell’s steric effect. The different light-addressable recorded photocurrent signals of all single cells directly reflect the 55.6% cell-to-cell differences. This report provides a useful and powerful tool to study single-cell heterogeneity.

One-stop assembly of adherent 3D retinal organoids from hiPSCs based on 3D-printed derived PDMS microwell platform

The three-dimensional (3D) retinal organoids (ROs) derived from human induced pluripotent stem cells (hiPSCs), mimicking the growth and development of the human retina, is a promising model for investigating inherited retinal diseases in vitro. However, the efficient generation of homogenous ROs remains a challenge. Here we introduce a novel polydimethylsiloxane (PDMS) microwell platform containing 62 V-bottom micro-cavities for the ROs differentiation from hiPSCs. The uniform adherent 3D ROs could spontaneously form using neural retina (NR) induction. Our results showed that the complex of NR (expressing VSX2), ciliary margin (CM) (expressing RDH10), and retinal pigment epithelium (RPE) (expressing ZO-1, MITF, and RPE65) developed in the PDMS microwell after the differentiation. It is important to note that ROs in PDMS microwell platforms not only enable one-stop assembly but also maintain homogeneity and mature differentiation over a period of more than 25 weeks without the use of BMP4 and Matrigel. Retinal ganglion cells (expressing BRN3a), amacrine cells (expressing AP2a), horizontal cells (expressing PROX1 and AP2α), photoreceptor cells for cone (expressing S-opsin and L/M-opsin) and rod (expressing Rod opsin), bipolar cells (expressing VSX2 and PKCα), and Müller glial cells (expressing GS and Sox9) gradually emerged. Furthermore, we replaced fetal bovine serum with human platelet lysate and established a xeno-free culture workflow that facilitates clinical application. Thus, our PDMS microwell platform for one-stop assembly and long-term culture of ROs using a xeno-free workflow is favorable for retinal disease modeling, drug screening, and manufacturing ROs for clinical translation.

Enhanced Differentiation Capacity and Transplantation Efficacy of Insulin-Producing Cell Clusters from Human iPSCs Using Permeable Nanofibrous Microwell-Arrayed Membrane for Diabetes Treatment.

Although pancreatic islet transplantation is a potentially curative treatment for insulin-dependent diabetes, a shortage of donor sources, low differentiation capacity, and transplantation efficacy are major hurdles to overcome before becoming a standard therapy. Stem cell-derived insulin-producing cells (IPCs) are a potential approach to overcoming these limitations. To improve the differentiation capacity of the IPCs, cell cluster formation is crucial to mimic the 3D structure of the islet. This study developed a biodegradable polycaprolactone (PCL) electrospun nanofibrous (NF) microwell-arrayed membrane permeable to soluble factors. Based on the numerical analysis and experimental diffusion test, the NF microwell could provide sufficient nutrients, unlike an impermeable PDMS (polydimethylsiloxane) microwell. The IPC clusters in the NF microwells showed higher gene expression of insulin and PDX1 and insulin secretion than the PDMS microwells. The IPC clusters in the NF microwell-arrayed membrane could be directly transplanted. Transplanted IPC clusters in the microwells survived well and expressed PDX1 and insulin. Additionally, human c-peptide was identified in the blood plasma at two months after transplantation of the membranes. The NF microwell-arrayed membrane can be a new platform promoting IPC differentiation capacity and realizing an in situ transplantation technique for diabetic patients.

Calcium Peroxide-Containing Polydimethylsiloxane-Based Microwells for Inhibiting Cell Death in Multicellular Spheroids Through Improved Oxygen Supply

Multicellular spheroids are expected to be used for in vivo-like tissue models and cell transplantation. Microwell devices are useful for the fabrication of multicellular spheroids to improve productivity and regulate their size. However, the high cell density in microwell devices and densely packed cells in the spheroids limit oxygen supply to the core region of the spheroids, which leads to accelerated cell death. In this study, we developed O2-generating microwells by incorporating calcium peroxide (CaO2) into polydimethylsiloxane (PDMS)-based microwells. The CaO2-containing PDMS was shown to react with water and generate O2 for 3 days. Then, CaO2-containing PDMS was used to fabricate O2-generating microwells using a micro-molding technique. HepG2 spheroids were then prepared using either conventional PDMS microwells or O2-generating microwells. Using the conventional PDMS microwells, the O2 concentration in the culture medium reduced to ~67% of the cell-free level. In contrast, the O2-generating microwells maintained O2 at constant levels. The HepG2 spheroids prepared using the O2-generating microwells were greater in size and had a larger number of live cells than those prepared using the conventional microwells. In addition, the O2-generating microwells rescued hypoxia in the HepG2 spheroids and increased cell viability in the core region of the spheroids. Lastly, the O2-generating microwells were also useful for the preparation of multicellular spheroids of other cell types (i.e., MIN6, B16-BL6, and adipose-derived stem cells) with high cell viability. These results showed that the O2-generating microwells are useful for preparing multicellular spheroids with high functional activity.

High-throughput generation of a concentration gradient on open arrays by serial and parallel dilution for drug testing and screening

In this study, we successively and parallelly slide a solvent strip on a polydimethylsiloxane microwell array to dilute the solution within the microwells. A total of 60 concentration gradients were generated one by one along the sliding direction within minutes while simultaneously forming 60 microwells with the same concentration perpendicular to the sliding direction. Using theoretical calculations, simulated predictions and experimental verification, we could regulate the concentration gradient on the microwell array by tuning the parameters of the sliding head and sliding speed. Afterwards, we cultured human non-small cell lung cancer cells and produced an anticancer drug concentration gradient on the microwell array by this sliding method. The cell viability was examined to demonstrate the potential of this method for high-throughput cell-based drug testing and screening, and this open microwell array method may enable further analysis during a drug-screening process.

A 25 micron-thin microscope for imaging upconverting nanoparticles with NIR-I and NIR-II illumination.

Rationale: Intraoperative visualization in small surgical cavities and hard-to-access areas are essential requirements for modern, minimally invasive surgeries and demand significant miniaturization. However, current optical imagers require multiple hard-to-miniaturize components including lenses, filters and optical fibers. These components restrict both the form-factor and maneuverability of these imagers, and imagers largely remain stand-alone devices with centimeter-scale dimensions.Methods: We have engineered INSITE (Immunotargeted Nanoparticle Single-Chip Imaging Technology), which integrates the unique optical properties of lanthanide-based alloyed upconverting nanoparticles (aUCNPs) with the time-resolved imaging of a 25-micron thin CMOS-based (complementary metal oxide semiconductor) imager. We have synthesized core/shell aUCNPs of different compositions and imaged their visible emission with INSITE under either NIR-I and NIR-II photoexcitation. We characterized aUCNP imaging with INSITE across both varying aUCNP composition and 980 nm and 1550 nm excitation wavelengths. To demonstrate clinical experimental validity, we also conducted an intratumoral injection into LNCaP prostate tumors in a male nude mouse that was subsequently excised and imaged with INSITE.Results: Under the low illumination fluences compatible with live animal imaging, we measure aUCNP radiative lifetimes of 600 μs - 1.3 ms, which provides strong signal for time-resolved INSITE imaging. Core/shell NaEr0.6Yb0.4F4 aUCNPs show the highest INSITE signal when illuminated at either 980 nm or 1550 nm, with signal from NIR-I excitation about an order of magnitude brighter than from NIR-II excitation. The 55 μm spatial resolution achievable with this approach is demonstrated through imaging of aUCNPs in PDMS (polydimethylsiloxane) micro-wells, showing resolution of micrometer-scale targets with single-pixel precision. INSITE imaging of intratumoral NaEr0.8Yb0.2F4 aUCNPs shows a signal-to-background ratio of 9, limited only by photodiode dark current and electronic noise.Conclusion: This work demonstrates INSITE imaging of aUCNPs in tumors, achieving an imaging platform that is thinned to just a 25 μm-thin, planar form-factor, with both NIR-I and NIR-II excitation. Based on a highly paralleled array structure INSITE is scalable, enabling direct coupling with a wide array of surgical and robotic tools for seamless integration with tissue actuation, resection or ablation.

Highly Multiplexed Single-Cell Protein Profiling with Large-Scale Convertible DNA-Antibody Barcoded Arrays.

Highly multiplexed detection of proteins secreted by single cells is always challenging. Herein, a multiplexed in situ tagging technique based on single‐stranded DNA encoded microbead arrays and multicolor successive imaging for assaying single‐cell secreted proteins with high throughput and high sensitivity is presented. This technology is demonstrated to be capable of increasing the multiplexity exponentially. Upon integration with polydimethylsiloxane microwells, this platform is applied to detect ten immune effector proteins from differentiated single macrophages stimulated with lipopolysaccharide. Significant heterogeneity is observed when the derived human primary macrophages are analyzed. This versatile technology is expected to open new opportunities in systems biology, immune regulation studies, signaling analysis, and molecular diagnostics.

Microwell-assisted filtration with anodic aluminum oxide membrane for Raman analysis of algal cells

In this study, rapid and direct Raman analysis of microalgae was demonstrated through measurement of algal cells (or algal paste) prepared by microwell-assisted filtration using an anodic aluminum oxide (AAO) membrane. To obtain the algal paste, an array of polydimethylsiloxane (PDMS) microwells with through holes was placed on top of the AAO membrane, after which a vacuum was initiated and a droplet of algal solution dispensed inside the PDMS microwells for a 5-min filtration process. The results showed that the AAO membrane has the lowest background noise compared to other types of filter papers or membranes. Compared to the droplet evaporation method, the “coffee-ring” effect was effectively suppressed when applying our proposed filtration method. The algal cells became stacked inside the microwells, which led to relatively uniform and strong Raman signals across individual algal paste and consistency among different algal pastes.

Gold-coated polydimethylsiloxane microwells for high-throughput electrochemiluminescence analysis of intracellular glucose at single cells.

In this communication, agold-coated polydimethylsiloxane (PDMS) chip with cell-sized microwells was prepared through a stamping and spraying process that was applied directly for high-throughput electrochemiluminescence (ECL) analysis of intracellular glucose at single cells. As compared with the previous multiple-step fabrication of photoresist-based microwells on the electrode, the preparation process is simple and offers fresh electrode surface for higher luminescence intensity. More luminescence intensity was recorded from cell-retained microwells than that at theplanar region among the microwells that was correlated with the content of intracellular glucose. The successful monitoring of intracellular glucose at single cells using this PDMS chip will provide an alternative strategy for high-throughput single-cell analysis. Graphical abstract ᅟ.

Single-step fabrication of polydimethylsiloxane microwell arrays with long-lasting hydrophilic inner surfaces

We propose a single-step fabrication method for polydimethylsiloxane (PDMS) cell-adhesive microwell arrays with long-lasting (>10 months in aqueous medium) hydrophilic inner surfaces without the need for any chemical treatment such as development. Irradiation of a PDMS film with a low-energy electron beam (55 kV) in air generated a ∼40-μm-thick hydrophilic silica-like layer on the PDMS surface, which was the key to the prolonged hydrophilicity. Moreover, the concomitant compaction of the irradiated area produced dozens-of-micrometers-deep concave wells. The hydrophilic microwells generated on the hydrophobic non-irradiated PDMS surface easily trapped nano-/picoliter droplets and cells/single-cells. In addition, the surfaces of the microwells offered stable and favorable cell-adherent environments. The method presented here can realize stable and reliable lab-on-chips and cater to the expanding demand in biological and medical applications.

Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.

While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25–400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38− cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38− cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38− cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.

A Simple Method for Fabrication of Microstructures Using a PDMS Stamp.

We report a simple method to fabricate PDMS (polydimethylsiloxane) microwell arrays on glass by using a PDMS stamp to study cell-to-cell adhesion. In the cell-to-cell study, a glass substrate is required since glass has better cell attachment. The microwell arrays are replicated from an SU-8 master mold, and then are transferred to a glass substrate by lifting the PDMS stamp, followed by oxygen plasma bonding of the PDMS stamp on the glass substrate. For the cell-to-cell adhesion, four different types of PDMS arrays (e.g., rectangle, bowtie, wide-rhombus, and rhombus) were designed to vary the cell-to-cell contact length. The transfer success rates of the microwell arrays were measured as a function of both the contact area of the PDMS and the glass substrate and the different ratios between the base polymers and the curing agent. This method of generating the microwell arrays will enable a simple and robust construction of PDMS-based devices for various biological applications.

Advanced micromachining of concave microwells for long term on-chip culture of multicellular tumor spheroids.

A novel approach based on advanced micromachining is demonstrated to fabricate concave microwell arrays for the formation of high quality multicellular tumor spheroids. Microfabricated molds were prepared using a state-of-the-art CNC machining center, containing arrays of 3D convex micropillars with size ranging from 150 μm to 600 μm. Microscopic imaging of the micropillars machined on the mold showed smooth, curved microfeatures of a dramatic 3D shape. Agarose microwells could be easily replicated from the metallic molds. EMT-6 tumor cells seeded in the primary macrowell sedimented efficiently to the bottom of the concave microwells and formed multicellular spheroids within 48 h. Dense and homogeneous multicellular spheroids were obtained after 10 days of culture, confirming the suitability of the proposed approach. To facilitate long term spheroid culture and reliable on-chip drug assay, polydimethylsiloxane microwells were also replicated from the metallic molds. A solvent swelling method was adapted and optimized to Pluronic F127 towards physically entrapping the block copolymer molecules within the polydimethylsiloxane network and in turn to improve long term cell-binding resistance. Homogeneous multicellular spheroids were efficiently formed in the concave microwells and on-chip drug assays could be reliably carried out using curcumin as a model anti-cancer drug. Advanced micromachining provides an excellent technological solution to the fabrication of high quality concave microwells.

Reusable conductimetric array of interdigitated microelectrodes for the readout of low-density microarrays

Low-density protein microarrays are emerging tools in diagnostics whose deployment could be primarily limited by the cost of fluorescence detection schemes. This paper describes an electrical readout system of microarrays comprising an array of gold interdigitated microelectrodes and an array of polydimethylsiloxane microwells, which enabled multiplexed detection of up to thirty six biological events on the same substrate. Similarly to fluorescent readout counterparts, the microarray can be developed on disposable glass slide substrates. However, unlike them, the presented approach is compact and requires a simple and inexpensive instrumentation. The system makes use of urease labeled affinity reagents for developing the microarrays and is based on detection of conductivity changes taking place when ionic species are generated in solution due to the catalytic hydrolysis of urea. The use of a polydimethylsiloxane microwell array facilitates the positioning of the measurement solution on every spot of the microarray. Also, it ensures the liquid tightness and isolation from the surrounding ones during the microarray readout process, thereby avoiding evaporation and chemical cross-talk effects that were shown to affect the sensitivity and reliability of the system. The performance of the system is demonstrated by carrying out the readout of a microarray for boldenone anabolic androgenic steroid hormone. Analytical results are comparable to those obtained by fluorescent scanner detection approaches. The estimated detection limit is 4.0ngmL−1, this being below the threshold value set by the World Anti-Doping Agency and the European Community.

Vacuum-Induced Wrinkle Arrays of InGaAs Semiconductor Nanomembranes on Polydimethylsiloxane Microwell Arrays

Tunable surface morphology in III-V semiconductor nanomembranes provides opportunities to modulate electronic structures and light interactions of semiconductors. Here, we introduce a vacuum-induced wrinkling method for the formation of ordered wrinkles in InGaAs nanomembranes (thickness, 42 nm) on PDMS microwell arrays as a strategy for deterministic and multidirectional wrinkle engineering of semiconductor nanomembranes. In this approach, a vacuum-induced pressure difference between the outer and inner sides of the microwell patterns covered with nanomembranes leads to bulging of the nanomembranes at the predefined microwells, which, in turn, results in stretch-induced wrinkle formation of the nanomembranes between the microwells. The direction and geometry of the nanomembrane wrinkles are well controlled by varying the PDMS modulus, depth, and shape of microwells, and the temperature during the transfer printing of nanomembrane onto heterogeneous substrates. The wrinkling method shown here can be applied to other semiconductor nanomembranes and may create an important platform to realize unconventional electronic devices with tunable electronic properties.

In vitro generation of colonic epithelium from primary cells guided by microstructures

The proliferative compartment of the colonic epithelium in vivo is located in the basal crypt where colonic stem cells and transit-amplifying cells reside and fuel the rapid renewal of non-proliferative epithelial cells as they migrate toward the gut lumen. To mimic this tissue polarity, microstructures composed of polydimethylsiloxane (PDMS) microwells and Matrigel micropockets were used to guide a combined 2-dimensional (2D) and 3-dimensional (3D) hybrid culture of primary crypts isolated from the murine colon. The 2D and 3D culture of crypts on a planar PDMS surface was first investigated in terms of cell proliferation and stem cell activity. 3D culture of crypts with overlaid Matrigel generated enclosed, but highly proliferative spheroids (termed colonoids). 2D culture of crypts produced a spreading monolayer of cells, which were non-proliferative. A combined 2D/3D hybrid culture was generated in a PDMS microwell platform on which crypts were loaded by centrifugation into microwells (diameter = 150 μm, depth = 150 μm) followed by addition of Matrigel that formed micropockets locking the crypts within the microwells. Embedded crypts first underwent 3D expansion inside the wells. After the cells filled the microwells, they migrated onto the surrounding surface forming a 2D monolayer in the array regions without Matrigel. This unique 2D/3D hybrid culture generated a continuous, millimeter-scale colonic epithelial tissue in vitro, which resembled the polarized architecture (i.e. distinct proliferative and non-proliferative zones) and geometry of the colonic epithelium in vivo. This work initiates the construction of a "colon-on-a-chip" using primary cells/tissues with the ultimate goal of producing the physiologic structure and organ-level function of the colon.

Application of Concave Microwells to Pancreatic Tumor Spheroids Enabling Anticancer Drug Evaluation in a Clinically Relevant Drug Resistance Model

Intrinsic drug resistance of pancreatic ductal adenocarcinoma (PDAC) warrants studies using models that are more clinically relevant for identifying novel resistance mechanisms as well as for drug development. Tumor spheroids (TS) mimic in vivo tumor conditions associated with multicellular resistance and represent a promising model for efficient drug screening, however, pancreatic cancer cells often fail to form spheroids using conventional methods such as liquid overlay. This study describes the induction of TS of human pancreatic cancer cells (Panc-1, Aspc-1, Capan-2) in concave polydimethylsiloxane (PDMS) microwell plates and evaluation of their usefulness as an anticancer efficacy test model. All three cell lines showed TS formation with varying degree of necrosis inside TS. Among these, Panc-1 spheroid with spherical morphology, a rather rough surface, and unique adhesion structures were successfully produced with no notable necrosis in concave microwell plates. Panc-1 TS contained growth factors or enzymes such as TGF-β1, CTGF, and MT1-MMP, and extracellular matrix proteins such as collagen type I, fibronectin, and laminin. Panc-1 cells grown as TS showed changes in stem cell populations and in expression levels of miRNAs that may play roles in chemoresistance. Visualization of drug penetration and detection of viability indicators, such as Ki-67 and MitoSOX, were optimized for TS for quantitative analysis. Water-soluble tetrazolium (MTS) and acid phosphatase (APH) assays were also successfully optimized. Overall, we demonstrated that concave PDMS microwell plates are a novel platform for preparation of TS of weakly aggregating cells and that Panc-1 spheroids may represent a novel three-dimensional model for anti-pancreatic cancer drug screening.

Abstract 2722: Characteristics of pancreatic cancer spheroids as a model for anticancer efficacy test.

Abstract Compared to the conventional monolayers or suspension cultures, 3D culture models have drawn attention as a vivo-mimic model which can produce clinically relevant data. Among the several types of 3D culture models, multicellular spheroid (MCS) is a appropriate model for study of penetration and efficacy of anticancer agents. Purpose of this study was to establish MCS of human pancreatic cancer cell (Panc-1) and evaluate its usefulness as anticancer efficacy test. Panc-1 MCS was produced successfully by using poly-dimethylsiloxane(PDMS) microwell instead of agarose-coated 96-well system. MCS with average diameter of 375 μm was prepared in 700 μm microwells after 4 days of culture. Spherical morphology with rather rough surface and junctional structures in outer cell layers were observed with no notable necrotic area. Extracellular matrix proteins such as collagen I, fibronectin, and laminin were detected in the interstitial space of MCS. Stem cell population with CD44+/CD24+/ESA+ expression increased from 0.1% to 2.1% when cultured as MCS. miRNA expression profiling showed a difference between monolayers and MCS, i.e., 36 and 85 genes were up- and down-regulated in MCS, respectively. Imaging of drug penetration and viability indicators including MitoSOX, Edu incorporation, and Ki-67 expression were optimized for microwell format. Colorimetric assays of MTS and APH were performed after transferring MCS to 96-well plates. Overall, we demonstrated that PDMS microwell is an appropriate platform for preparation of MCS of Panc-1 cells and MCS showed different biological features from monolayers, suggesting that Panc-1 MCS prepared using PDMS microwell may be a useful 3D model for anticancer efficacy test. Citation Format: Sang-Eun Yeon, Sang-Hoon Lee, Hyo-Jeong Kuh. Characteristics of pancreatic cancer spheroids as a model for anticancer efficacy test. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2722. doi:10.1158/1538-7445.AM2013-2722 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Trapping cells on a stretchable microwell array for single-cell analysis.

There is a need for a technology that can be incorporated into routine laboratory procedures to obtain a continuous, quantitative, fluorescence-based measurement of the dynamic behaviors of numerous individual living cells in parallel, while allowing other manipulations, such as staining, rinsing, and even retrieval of targeted cells. Here, we report a simple, low-cost microarray platform that can trap cells for dynamic single-cell analysis of mammalian cells. The elasticity of polydimethylsiloxane (PDMS) was utilized to trap tens of thousands of cells on an array. The PDMS microwell array was stretched by a tube through which cells were loaded on the array. Cells were trapped on the array by removal of the tube and relaxation of the PDMS. Once that was accomplished, the cells remained trapped on the array without continuous application of an external force and permitted subsequent manipulations, such as staining, rinsing, imaging, and even isolation of targeted cells. We demonstrate the utility of this platform by multicolor analysis of trapped cells and monitoring in individual cells real-time calcium flux after exposure to the calcium ionophore ionomycin. Additionally, a proof of concept for target cell isolation was demonstrated by using a microneedle to locally deform the PDMS membrane in order to retrieve a particular cell from the array.

Modular plastic chip for one-shot human papillomavirus diagnostic analysis

In this article, we report the design and development of a plastic modular chip suitable for one-shot human papillomavirus (HPV) diagnostics, namely detection of the viral presence and relative genotyping, by two sequential steps performed directly on the same device. The device is composed of two modular and disposable plastic units that can be assembled or used separately. The first module is represented by a polydimethylsiloxane (PDMS) microreactor that is exploited for real-time polymerase chain reaction (PCR) and, thus, is suitable for detecting the presence of virus. The second unit is a PDMS microwell array that allows virus genotyping by a colorimetric assay, based on DNA hybridization technology developed on plastic, requiring simple inspection by the naked eye. The two modules can be easily coupled to reusable hardware, enabling the heating/cooling processes and the real-time detection of HPV. By coupling real-time assay and colorimetric genotyping on the same chip, the assembled device may provide a low-cost tool for HPV diagnostics, thereby favoring the prediction of cancer risk in patients.