Facile Fabrication of Silk Fibroin/Off-Stoichiometry Thiol-Ene (OSTE) Microneedle Array Patches.

Microneedles have been used in various applications in biomedical engineering, including drug delivery, biosensing, and vaccine delivery. In this study, we develop a novel protocol to fabricate silk fibroin/off-stoichiometry thiol-ene (OSTE) hybrid microneedle array patches. Silk fibroin, as a natural biomaterial, has been proven to be suitable as a drug carrier. Firstly, drug (we use insulin in this experiment) dissolved in silk fibroin solution is deposited on a microneedle mold and dried thoroughly. After that, silk fibroin needle tips are formed on the OSTE base by replica molding. We investigated the influence of the silk fibroin concentration on the length of silk needle tips and found that the silk concentration had a small influence on the tip length. We also tested the mechanical strength of the microneedles by inserting them into gelatin gel for dummy drug delivery tests. Such composite structures have the potential to increase the delivery efficiency by delivering the whole silk tip into the dermis.

Droplet Microfluidics Enables Tracing of Target Cells at the Single-Cell Transcriptome Resolution.

The rapid promotion of single-cell omics in various fields has begun to help solve many problems encountered in research, including precision medicine, prenatal diagnosis, and embryo development. Meanwhile, single-cell techniques are also constantly updated with increasing demand. For some specific target cells, the workflow from droplet screening to single-cell sequencing is a preferred option and should reduce the impact of operation steps, such as demulsification and cell recovery. We developed an all-in-droplet method integrating cell encapsulation, target sorting, droplet picoinjection, and single-cell transcriptome profiling on chips to achieve labor-saving monitoring of TCR-T cells. As a proof of concept, in this research, TCR-T cells were encapsulated, sorted, and performed single-cell transcriptome sequencing (scRNA-seq) by injecting reagents into droplets. It avoided the tedious operation of droplet breakage and re-encapsulation between droplet sorting and scRNA-seq. Moreover, convenient device operation will accelerate the progress of chip marketization. The strategy achieved an excellent recovery performance of single-cell transcriptome with a median gene number over 4000 and a cross-contamination rate of 8.2 ± 2%. Furthermore, this strategy allows us to develop a device with high integrability to monitor infused TCR-T cells, which will promote the development of adoptive T cell immunotherapy and their clinical application.

Fabrication of Concave Microwells and Their Applications in Micro-Tissue Engineering: A Review.

At present, there is an increasing need to mimic the in vivo micro-environment in the culture of cells and tissues in micro-tissue engineering. Concave microwells are becoming increasingly popular since they can provide a micro-environment that is closer to the in vivo environment compared to traditional microwells, which can facilitate the culture of cells and tissues. Here, we will summarize the fabrication methods of concave microwells, as well as their applications in micro-tissue engineering. The fabrication methods of concave microwells include traditional methods, such as lithography and etching, thermal reflow of photoresist, laser ablation, precision-computerized numerical control (CNC) milling, and emerging technologies, such as surface tension methods, the deformation of soft membranes, 3D printing, the molding of microbeads, air bubbles, and frozen droplets. The fabrication of concave microwells is transferring from professional microfabrication labs to common biochemical labs to facilitate their applications and provide convenience for users. Concave microwells have mostly been used in organ-on-a-chip models, including the formation and culture of 3D cell aggregates (spheroids, organoids, and embryoids). Researchers have also used microwells to study the influence of substrate topology on cellular behaviors. We will briefly review their applications in different aspects of micro-tissue engineering and discuss the further applications of concave microwells. We believe that building multiorgan-on-a-chip by 3D cell aggregates of different cell lines will be a popular application of concave microwells, while integrating physiologically relevant molecular analyses with the 3D culture platform will be another popular application in the near future. Furthermore, 3D cell aggregates from these biosystems will find more applications in drug screening and xenogeneic implantation.

Generation and Screening of Antigen-Specific Nanobodies from Mammalian Cells Expressing the BCR Repertoire Library Using Droplet-Based Microfluidics.

Nanobodies, also known as VHHs, originate from the serum of Camelidae. Nanobodies have considerable advantages over conventional antibodies, including smaller size, more modifiable, and deeper tissue penetration, making them promising tools for immunotherapy and antibody-drug development. A high-throughput nanobody screening platform is critical to the rapid development of nanobodies. To date, droplet-based microfluidic systems have exhibited improved performance compared to the traditional phage display technology in terms of time and throughput. In realistic situations, however, it is difficult to directly apply the technology to the screening of nanobodies. Requirements of plasma cell enrichment and high cell viability, as well as a lack of related commercial reagents, are leading causes for impeding the development of novel methods. We overcame these obstacles by constructing a eukaryotic display system that secretes nanobodies utilizing homologous recombination and eukaryotic transformation technologies, and the significant advantages are that it is independent of primary cell viability and it does not require plasma cell enrichment in advance. Next, a signal capture system of "SA-beads + Biotin-antigen + nanobody-6 × His + fluorescence-labeled anti-6 × His (secondary antibody)" was designed for precise localization of the eukaryotic-expressed nanobodies in a droplet. Based on this innovation, we screened 293T cells expressing anti-PD-L1 nanobodies with a high positive rate of targeted cells (up to 99.8%). Then, single-cell transcriptomic profiling uncovered the intercellular heterogeneity and BCR sequence of target cells at a single-cell level. The complete complementarity determining region (CDR3) structure was obtained, which was totally consistent with the BCR reference. This study expanded the linkage between microfluidic technology and nanobody applications and also showed potential to accelerate the rapid transformation of nanobodies in the large-scale market.

High-Performance Passive Plasma Separation on OSTE Pillar Forest.

Plasma separation is of high interest for lateral flow tests using whole blood as sample liquids. Here, we built a passive microfluidic device for plasma separation with high performance. This device was made by blood filtration membrane and off-stoichiometry thiol-ene (OSTE) pillar forest. OSTE pillar forest was fabricated by double replica moldings of a laser-cut polymethylmethacrylate (PMMA) mold, which has a uniform microstructure. This device utilized a filtration membrane to separate plasma from whole blood samples and used hydrophilic OSTE pillar forest as the capillary pump to propel the plasma. The device can be used to separate blood plasma with high purity for later use in lateral flow tests. The device can process 45 µL of whole blood in 72 s and achieves a plasma separation yield as high as 60.0%. The protein recovery rate of separated plasma is 85.5%, which is on par with state-of-the-art technologies. This device can be further developed into lateral flow tests for biomarker detection in whole blood.

Controlling Capillary Flow Rate on Lateral Flow Test Substrates by Tape.

Controlling capillary flow rate of sample liquid is of high interest for lateral flow tests, since the flow rate can affect the dissolution and mixing of the immunoreagents and the efficiency of immunoreactions. Here we develop a facile method to adjust the capillary flow rate on lateral flow test substrates by using tape to cover the surface of substrates. We test this method on the traditional lateral flow test substrate-nitrocellulose and a novel lateral flow test substrate-synthetic paper, which is a porous media made by interlocked off-stoichiometry thiol-ene (OSTE) micropillars. We found that after the surface was covered by tape, the average flow rate decreased to 61% of the original flow rate on nitrocellulose, while the average flow rate increased to at least 320% of the original flow rate on synthetic paper. More interesting, besides the increase of flow rate, the volume capacity of synthetic paper also increases after covered by tape. Furthermore, we investigated the influence of length and position of tape on the capillary flow rate for nitrocellulose. A longer tape will lead to a smaller flow rate. The influence of tape of same length on the flow rate is bigger when the tape is placed closer to the loading pad. These results can help in the flow rate control on lateral flow test substrates, and potentially improve the performance of lateral flow tests.

Immunoassays on thiol-ene synthetic paper generate a superior fluorescence signal.

The fluorescence-based detection of biological complexes on solid substrates is widely used in microarrays and lateral flow tests. Here, we investigate thiol-ene micropillar scaffold sheets ("synthetic paper") as the solid substrate in such assays. Compared to state-of-the-art glass and nitrocellulose substrates, assays on synthetic paper provide a stronger fluorescence signal, similar or better reproducibility, lower limit of detection (LOD), and the possibility of working with lower immunoreagent concentrations. Using synthetic paper, we detected the antibiotic enrofloxacin in whole milk with a LOD of 1.64 nM, which is on par or better than the values obtained with other common tests, and much lower than the maximum level allowed by European Union regulations. The significance of these results lays in that they indicate that synthetically-derived microstructured substrate materials have the potential to improve the performance of diagnostic assays.

Synthetic Paper Separates Plasma from Whole Blood with Low Protein Loss.

The separation of plasma from whole blood is the first step in many diagnostic tests. Point-of-care tests often rely on integrated plasma filters, but protein retention in such filters limits their performance. Here, we investigate plasma separation on interlocked micropillar scaffolds ("synthetic paper") by the local agglutination of blood cells coupled with the capillary separation of the plasma. We separated clinically relevant volumes of plasma with high efficiency in a separation time on par with that of state of the art techniques. We investigated different covalent and noncovalent surface treatments (PEGMA, HEMA, BSA, O2 plasma) on our blood filter and their effect on protein recovery and identified O2 plasma treatment and 7.9 µg/cm2 agglutination antibody as most suitable treatments. Using these treatments, we recovered at least 82% of the blood plasma proteins, more than with state-of-the-art filters. The simplicity of our device and the performance of our approach could enable better point-of-care tests.

Facile Nanoimprinting of Robust High-Aspect-Ratio Nanostructures for Human Cell Biomechanics.

High-aspect-ratio and hierarchically nanostructured surfaces are common in nature. Synthetic variants are of interest for their specific chemical, mechanic, electric, photonic, or biologic properties but are cumbersome in fabrication or suffer from structural collapse. Here, we replicated and directly biofunctionalized robust, large-area, and high-aspect-ratio nanostructures by nanoimprint lithography of an off-stoichiometric thiol-ene-epoxy polymer. We structured-in a single-step process-dense arrays of pillars with a diameter as low as 100 nm and an aspect ratio of 7.2; holes with a diameter of 70 nm and an aspect ratio of >20; and complex hierarchically layered structures, all with minimal collapse and defectivity. We show that the nanopillar arrays alter mechanosensing of human hepatic cells and provide precise spatial control of cell attachment. We speculate that our results can enable the widespread use of high-aspect-ratio nanotopograhy applications in mechanics, optics, and biomedicine.

E-Beam Nanostructuring and Direct Click Biofunctionalization of Thiol-Ene Resist.

Electron beam lithography (EBL) is of major importance for ultraminiaturized biohybrid system fabrication, as it allows combining biomolecular patterning and mechanical structure definition on the nanoscale. Existing methods are limited by multistep biomolecule immobilization procedures, harsh processing conditions that are harmful to sensitive biomolecules, or the structural properties of the resulting protein monolayers or hydrogel-based resists. This work introduces a thiol-ene EBL resist with chemically reactive thiol groups on its native surface that allow the direct and selective "click" immobilization of biomolecules under benign processing conditions. We constructed EBL structured features of size down to 20 nm, and direct functionalized the nanostructures with a sandwich of biotin and streptavidin. The facile combination of polymer nanostructuring with biomolecule immobilization enables mechanically robust biohybrid components of interest for nanoscale biomedical, electronic, photonic, and robotic applications.

Capillary pumping independent of the liquid surface energy and viscosity.

Capillary pumping is an attractive means of liquid actuation because it is a passive mechanism, i.e., it does not rely on an external energy supply during operation. The capillary flow rate generally depends on the liquid sample viscosity and surface energy. This poses a problem for capillary-driven systems that rely on a predictable flow rate and for which the sample viscosity or surface energy are not precisely known. Here, we introduce the capillary pumping of sample liquids with a flow rate that is constant in time and independent of the sample viscosity and sample surface energy. These features are enabled by a design in which a well-characterized pump liquid is capillarily imbibed into the downstream section of the pump and thereby pulls the unknown sample liquid into the upstream pump section. The downstream pump geometry is designed to exert a Laplace pressure and fluidic resistance that are substantially larger than those exerted by the upstream pump geometry on the sample liquid. Hence, the influence of the unknown sample liquid on the flow rate is negligible. We experimentally tested pumps of the new design with a variety of sample liquids, including water, different samples of whole blood, different samples of urine, isopropanol, mineral oil, and glycerol. The capillary filling speeds of these liquids vary by more than a factor 1000 when imbibed to a standard constant cross-section glass capillary. In our new pump design, 20 filling tests involving these liquid samples with vastly different properties resulted in a constant volumetric flow rate in the range of 20.96-24.76 µL/min. We expect this novel capillary design to have immediate applications in lab-on-a-chip systems and diagnostic devices.

A microfluidic device for isolation and characterization of transendothelial migrating cancer cells.

Transendothelial migration of cancer cells is a critical stage in cancer, including breast cancer, as the migrating cells are generally believed to be highly metastatic. However, it is still challenging for many existing platforms to achieve a fully covering endothelium and to ensure transendothelial migration capability of the extracted cancer cells for analyses with high specificity. Here, we report a microfluidic device containing multiple independent cell collection microchambers underneath an embedded endothelium such that the transendothelial-migrated cells can be selectively collected from only the microchambers with full coverage of an endothelial layer. In this work, we first optimize the pore size of a microfabricated supporting membrane for the endothelium formation. We quantify transendothelial migration rates of a malignant human breast cell type (MDA-MB-231) under different shear stress levels. We investigate characteristics of the migrating cells including morphology, cytoskeletal structures, and migration (speed and persistence). Further implementation of this endothelium-embedded microfluidic device can provide important insights into migration and intracellular characteristics related to cancer metastasis and strategies for effective cancer therapy.

Clinicopathological Features and Immunohistochemical Alterations of Keratinocyte Proliferation, Melanocyte Density, Smooth Muscle Hyperplasia and Nerve Fiber Distribution in Becker's Nevus.

BACKGROUND: Although Becker's nevus (BN) is a relatively common disease, the systematic studies of clinicopathological and immunohistochemical results are poorly reported. OBJECTIVE: To investigate the clinicopathological features and immunohistochemical alterations of keratinocyte proliferation, melanocyte density, smooth muscle hyperplasia and nerve fiber distribution in BN. METHODS: Clinical and pathological data were collected in 60 newly-diagnosed BN cases. Immunohistochemical stain of Ki-67, Melan-A, keratin 15, smooth muscle actin and protein gene product 9.5 was performed in 21 cases. RESULTS: The median diagnostic and onset age was 17 and 12 years, respectively. Skin lesions usually appeared on the upper trunk and upper limbs. The pathological features included the rete ridge elongation and fusion and basal hyperpigmentation. Epidermal Ki-67, Melan-A and keratin 15 expression and dermal nerve fiber length were significantly higher in lesional and perilesional skin than in normal skin (p<0.05~0.01), while smooth muscle actin expression was upregulated only in skin lesion (p<0.05). CONCLUSION: Although the clinical diagnosis of BN is often straightforward, histopathology is helpful to differentiate from other pigmentary disorders. The hyperproliferation of keratinocytes, melanocytes, arrector pili muscle and dermal nerve fibers could be involved in the pathogenesis of BN.

Capillary Pumping Independent of Liquid Sample Viscosity.

Capillary flow is a dominating liquid transport phenomenon on the micro- and nanoscale. As described at the beginning of the 20th century, the flow rate during imbibition of a horizontal capillary tube follows the Washburn equation, i.e., decreases over time and depends on the viscosity of the sample. This poses a problem for capillary driven systems that rely on a predictable flow rate and where the liquid viscosity is not precisely known. Here we introduce and successfully experimentally verify the first compact capillary pump design with a flow rate constant in time and independent of the liquid viscosity that can operate over an extended period of time. We also present a detailed theoretical model for gravitation-independent capillary filling, which predicts the novel pump performance to within measurement error margins, and in which we, for the first time, explicitly identify gas inertia dominated flow as a fourth distinct flow regime in capillary pumping. These results are of potential interest for a multitude of applications and we expect our results to find most immediate applications within lab-on-a-chip systems and diagnostic devices.

High-throughput dental biofilm growth analysis for multiparametric microenvironmental biochemical conditions using microfluidics.

Dental biofilm formation is not only a precursor to tooth decay, but also induces more serious systematic health problems such as cardiovascular disease and diabetes. Understanding the conditions promoting colonization and subsequent biofilm development involving complex bacteria coaggregation is particularly important. In this paper, we report a high-throughput microfluidic 'artificial teeth' device offering controls of multiple microenvironmental factors (e.g. nutrients, growth factors, dissolved gases, and seeded cell populations) for quantitative characteristics of long-term dental bacteria growth and biofilm development. This 'artificial teeth' device contains multiple (up to 128) incubation chambers to perform parallel cultivation and analyses (e.g. biofilm thickness, viable-dead cell ratio, and spatial distribution of multiple bacterial species) of bacteria samples under a matrix of different combinations of microenvironmental factors, further revealing possible developmental mechanisms of dental biofilms. Specifically, we applied the 'artificial teeth' to investigate the growth of two key dental bacteria, Streptococci species and Fusobacterium nucleatum, in the biofilm under different dissolved gas conditions and sucrose concentrations. Together, this high-throughput microfluidic platform can provide extended applications for general biofilm research, including screening of the biofilm properties developing under combinations of specified growth parameters such as seeding bacteria populations, growth medium compositions, medium flow rates and dissolved gas levels.