Gravity-Oriented Microfluidic Device for Biocompatible End-to-End Fabrication of Cell-Laden Microgels.

Droplet microfluidics are extensively utilized to generate monodisperse cell-laden microgels in biomedical applications. However, maintaining cell viability is still challenging due to overexposure to harsh conditions in subsequent procedures that recover the microgels from the oil phase. Here, a gravity-oriented microfluidic device for end-to-end fabrication of cell-laden microgels is reported, which integrates dispersion, gelation, and extraction into a continuous workflow. This innovative on-chip extraction, driven by native buoyancy and kinetically facilitated by pseudosurfactant, exhibits 100% retrieval efficiency for microgels with a wide range of sizes and stiffnesses. The viability of encapsulated cells is perfectly maintained at ≈98% with minimal variations within and between batches. The end-to-end fabrication remarkably enhances the biocompatibility and practicality of microfluidics-based cell encapsulation and is promising to be compatible with various applications ranging from single-cell analysis to clinical therapy.

Paper-Based Flow Sensor for the Detection of Hyaluronidase via an Enzyme Hydrolysis-Induced Viscosity Change in a Polymer Solution.

Hyaluronidase (HAase) is implicated in inflammation, cancer development, and allergic reaction. The detection of HAase is significantly important in clinical diagnosis and medical treatment. Herein, we propose a new principle for the development of equipment-free and label-free paper-based flow sensors based on the enzymatic hydrolysis-induced viscosity change in a stimuli-responsive polymer solution, which increases the water flow distance on the pH indicator paper. The detection of HAase is demonstrated as an example. This facile and versatile method can overcome the potential drawbacks of traditional hydrogel-based sensors, including complex preparation steps, slow response time, or low sensitivity. Moreover, it can also avoid the use of specialized instruments, labeled molecules, or functionalized nanoparticles in the sensors developed using the polymer solutions. Using this strategy, the detection of HAase is achieved with a limit of detection as low as 0.2 U/mL. Also, it works well in human urine. Additionally, the detection of tannic acid, which is an inhibitor of HAase, is also fulfilled. Overall, a simple, efficient, high-throughput, and low-cost detection method is developed for the rapid and quantitative detection of HAase and its inhibitor without the use of labeled molecules, synthetic particles, and specialized instruments. As only minimal reagents of HAase, HA, and paper are used, it is very promising in the development of commercial kits for point-of-care testing.

Combination Stiffness Gradient with Chemical Stimulation Directs Glioma Cell Migration on a Microfluidic Chip.

Tumor cells respond actively to the extracellular microenvironment via biomechanical and biochemical stimulation. Microchips provide a flexible platform to integrate multiple factors for cell research. In this work, we developed a subtle microfluidic chip that can generate a controllable stiffness gradient and orthogonal chemical stimulation to study the behaviors of glioma cells. Fibronectin-conjugated polyacrylamide (PAA) hydrogel with a longitudinal stiffness gradient ranging from about 1 kPa to 40 kPa is integrated within the cell culture chamber while lateral diffusion-based chemical stimulation is generated through circumambient microchannel arrays. The synergistic effect of epidermal growth factor (EGF) stimulation and hydrogel stiffness gradient on U87-MG cell migration was studied. By tracing cell migration, we discovered that hydrogel stiffness can promote cell chemotaxis, while the EGF gradient can accelerate cell migration. In addition, cell morphology showed typical cell spreading, increased aspect ratios, and decreased circularity in response to a stiffer substrate and plateaued at a certain stiffness level. Meanwhile, the content of intracellular reactive oxygen species (ROS) on the hydrogel soft end is enhanced by approximately 2 fold compared with that on the hydrogel stiff end. The enhancement of substrate stiffness on cell chemotaxis is very significant for in vitro model simulation and tissue engineering.

Monitoring H2 O2 on the Surface of Single Cells with Liquid Crystal Elastomer Microspheres.

Live-imaging of signaling molecules released from living cells is a fundamental challenge in life sciences. Herein, we synthesized liquid crystal elastomer microspheres functionalized with horse-radish peroxidase (LCEM-HRP), which can be immobilized directly on the cell membrane to monitor real-time release of H2 O2 at the single-cell level. LCEM-HRP could report H2 O2 through a concentric-to-radial (C-R) transfiguration, which is due to the deprotonation of LCEM-HRP and the break of inter or intra-chain hydrogen bonding in LCEM-HRP caused by HRP-catalyzed reduction of H2 O2 . The level of transfiguration of LCEM-HRP revealed the different amounts of H2 O2 released from cells. The estimated detection sensitivity was ≈2.2×10-7  µm for 10 min of detection time. The cell lines and cell-cell heterogeneity was explored from different configurations. LCEM-HRP presents a new approach for in situ real-time imaging of H2 O2 release from living cells and can be the basis for seeking more advanced chemical probes for imaging of various signaling molecules in the cellular microenvironment.

Measurement of Cell-Matrix Adhesion at Single-Cell Resolution for Revealing the Functions of Biomaterials for Adherent Cell Culture.

Cell adhesion is essential for a cell to maintain its functions, and biomaterials acting as the extracellular matrix (ECM) play a vital role. However, conventional methods for evaluating the functions of biomaterials become insufficient and sometimes incorrect when we give a deeper insight into single-cell research. In this work, we reported a novel methodology for the measurement of cell-matrix adhesion at single-cell resolution that could precisely evaluate the functions of biomaterials for adherent cell culture. A microfludic device, a live single-cell extractor (LSCE), was used for cell extraction. We applied this method to evaluate various modified biomaterials. The results indicated that poly(l-polylysine) (PLL)-coated glass and fibronection (FN)-coated glass slides showed the best biocompatibility for adherent cell culture following by the (3-aminopropyl)triethoxysilane (APTES)-coated glass, while piranha solution treated glass slide and octadecyltrichlorosilane (OTS)-coated glass showed weak biocompatibilities. Furthermore, APTES, PLL, and FN modifications enhanced the cell heterogeneity, while the OTS modification weakened the cell heterogeneity compare to the initial piranha solution treated glass. The method not only clarified the cell-matrix adhesion strength at single-cell resolution but also revealed the influences of biomaterials on cell-matrix adhesion and heterogeneity of cell-matrix adhesion for adherent cell culture. It might be a general strategy for precise evaluation of biomaterials.

A novel approach for precisely controlled multiple cell patterning in microfluidic chips by inkjet printing and the detection of drug metabolism and diffusion.

In this work we report the use of inkjet printing as a precise and convenient means for microscale cell patterning in microfluidic chips followed by cell co-culture, stimulation and analysis. A self-made inkjet printing device was manufactured with adjustable parameters, which was capable of multiple cell printing within biocompatible materials. Sodium alginate was used as a printing matrix for cell encapsulation, and precisely distributed cell arrays on glass slides were obtained by accurate software controlled printing. By covering a PDMS layer with the corresponding microchannels onto the cell array substrate and subsequently injecting an ion cross-linking reagent, the cells containing alginate arrays gelated immediately and were immobilized on the bottom of the microchip, which could be utilized for cell culture and analysis. HepG2 cells and U251 cells were successfully co-patterned in the microchip and used for drug metabolism and diffusion experiment to imitate the in vivo situation, as a means to ascertain the capability of the system for precise microscale cell patterning in a microchip. The prodrug tegafur was metabolized by HepG2 cells into the active anticancer compound 5-fluorouracil and this produced an adverse gradient effect on U251 cells according to the distance from the HepG2 cells. The developed approach presented a feasible way to integrate inkjet cell printing and microfluidic chips for the first time, which is proved to be capable of spatially controlled printing of multiple kinds of cells into a microchip for cell culture, stimulation and analysis, which could be applied to tissue engineering, drug testing and related areas. We envision that the approach will help significantly increase the cell patterning efficacy in microfluidic chips as well as reduce the extent of laborious experimental work.

Inkjet nanoinjection for high-thoughput chemiluminescence immunoassay on multicapillary glass plate.

We report a novel chemiluminescence diagnosis system for high-throughput human IgA detection by inkjet nanoinjection on a multicapillary glass plate. As proof-of-concept, microhole-based polydimethylsiloxane (PDMS) sheets were aligned on a multicapillary glass plate to form a microwell array as microreactors for enzyme-linked immunosorbent assay (ELISA). The multicapillary glass plate was utilized as a switch that controlled the holding/passing of the solution. Further, anti-IgA-labeled polystyrene (PS) microbeads was assembled into the microwell array, and an inkjet nanoinjection was specially used to distribute the sample and reagent solution for chemiluminescence ELISA, enabling high-throughput detection of human IgA. As a result, the performance of human IgA tests revealed a wider range for the calibration curve and a lower limit of detection (LOD) of 0.1 ng mL(-1) than the ELISA by a standard 96-well plate. The analysis time and reagent consumption were significantly decreased. The IgA concentrations in saliva samples were determined after 10000-fold dilution by the developed ELISA system showing comparable results by conventional immune assay with 96-wells. Thus, we believe that the inkjet nanoinjection for high-throughput chemiluminescence immunoassay on a multicapillary glass plate will be promising in disease diagnosis.

Microfluidic Engineering of Crater-Terrain Hydrogel Microparticles: Toward Novel Cell Carriers.

Fabrication and application of novel anisotropic microparticles are of wide interest. Herein, a new method for producing novel crater-terrain hydrogel microparticles is presented using a concept of droplet-aerosol impact and regional polymerization. The surface pattern of microparticles is similar to the widespread "crater" texture on the lunar surface and can be regulated by the impact morphology of aerosols on the droplet surface. Methodological applicability was demonstrated by producing ionic-cross-linked (alginate) and photo-cross-linked (poly(ethylene glycol) diacrylate, PEGDA) microparticles. Additionally, the crater-terrain microparticles (CTMs) can induce nonspecific protein absorption on their surface to acquire cell affinity, and they were exploited as cell carriers to load living cells. Cells could adhere and proliferate, and a special cellular adhesion fingerprint was observed on the novel cell carrier. Therefore, the scalable manufacturing method and biological potential make the engineered microparticles promising to open a new avenue for exploring cell-biomaterial crosstalk.

Room-temperature synthesis of nitrogen-rich conjugated microporous polymers for solid-phase extraction of trace synthetic musks.

This study synthesized a conjugated microporous polymer (CMP) at room temperature, which has high surface area, large conjugate system, and nitrogen-rich features. The material was explored as a solid-phase extraction (SPE) column, and it showed a higher extraction efficiency for nitro-musks compared to most commercial columns. Under optimal SPE conditions, a sensitive and efficient method for determining five nitro-musks was established based on gas chromatography-mass spectrometry (GC-MS). The method showed excellent linearity (R2 ≥ 0.996), low limits of detection (0.13-0.57 ng·L-1), good repeatability (1.1-4.0 %, n = 6) and was applied to the detection of trace nitro-musks in water and milk samples. The mechanism was further discussed combined with a simulation calculation. The advantages of the proposed method were mainly reflected in the extraction efficiency and sensitivity, which also indicated the potential of CMPs as a sample pretreatment material.

Composite SPE Paper Membrane Based on the Functional Superstructure of Metal-Organic Frameworks and Ionic Liquids for Detection of Tetracycline-like Antibiotics.

Composite adsorbents based on metal-organic frameworks (MOFs) are excellent candidates for solid-phase extraction (SPE) due to their diverse chemical functionality and multilevel porosity. MOF superstructures based on self-assembly at room temperature (RT) could have less energy consumption and easier manipulation due to the larger complex geometry. The π-π stacking of the benzene ring could not only enhance the interaction toward hydrophobic or plane-structured targets but also be expected to promote the formation of the MOF superstructure. In this work, in the established RT self-assembly synthesis system, several factors were investigated to see how to obtain functional MOF superstructures with a regular geometry, among which the number of benzene rings in the ligand was mainly tested for its impact on self-assembly and adsorption capacity. By means of adsorption experiments and computational fluid dynamics (CFD) simulation, the relationship between structure and activity (SARs) was further explored. Interestingly, the MOF unit with the lowest specific surface area performed the best in adsorption. Then, the selected functional MOF superstructure and ionic liquid were used to produce the composite paper membrane facilely applied in the SPE device. After optimization of the preparation conditions and operation parameters, the established SPE-HPLC-UV method could selectively analyze tetracycline-like antibiotics in the range of 16.6-833.3 ng/g (ppb) in a meat sample. This work provided an RT synthesis method to produce a microsize MOF superstructure, with experimental and theoretical insights into the SARs, which could be expanded in the design of other MOF-based SPE composite membranes toward one group of analogues.

Fluorine-functionalized conjugated microporous polymer as adsorbents for solid-phase extraction of nine perfluorinated alkyl substances.

Perfluorinated alkyl substances (PFASs) are persistent, toxic, ubiquitously distributed, and bioaccumulated substances, which have attracted increasing concern. To investigate the environmental effects of PFASs, there is a need to develop a sensitive, rapid, and efficient method for detecting trace level PFASs. In this study, a conjugated microporous polymer (CMP) with loading of fluorine, fabricated by Sonogashira-Hagihara cross-coupling, was exploited as a solid-phase extraction (SPE) adsorbent. The prepared fluorine-functionalized CMP (FCMP), which showed a large surface area of 1089 m2·g-1, high porosity, and good chemical stability, was used to extract PFASs from water samples. The adsorption mechanism was investigated using a sorption isotherm model, and the main interactions were fluorous and hydrophobic affinity. The FCMP-based SPE combined with high-performance liquid chromatography-tandem mass spectrometry achieved low limits of detection (0.19-0.97 ng·L-1), wide linear range (2-1600 ng·L-1), and good reproducibility (3.4%-12.9%) under the optimal conditions. Furthermore, the approach was utilized for the analysis of three water samples (snow, river water, and irrigation water) to evaluate its reliability, and satisfactory recovery (70.5%-127.5%) was obtained. Thus, FCMP was feasible SPE adsorbents for the selective extraction of PFASs.

Cell signaling analysis by mass spectrometry under coculture conditions on an integrated microfluidic device.

A microfluidic device was integrated in a controlled coculture system, in which the secreted proteins were qualitatively and semiquantitatively determined by a directly coupled mass spectrometer. PC12 cells and GH3 cells were cocultured under various conditions as a model of the regulation of the organism by the nervous system. A micro-solid phase extraction (SPE) column was integrated in order to remove salts from the cells secretion prior to mass spectrometry detection. A three layer polydimethylsiloxane (PDMS) microfluidic device was fabricated to integrate valves for avoiding contamination between the cells coculture zone and the pretreatment zone. Electrospray ionization (ESI)-quadrupole (Q)-time of flight (TOF)-mass spectrometry was employed to realize highly sensitive qualitative analysis and to implement semiquantitative analysis. Furthermore, cell migrations under various coculture conditions were observed and discussed. The inhibition on growth hormone secretion from GH3 cells by dopamine released from PC12 cells was investigated and demonstrated. Thus, the developed platform provides a useful tool on cell to cell signaling studies for disease monitoring and drug delivery control.

On-chip sample pretreatment using a porous polymer monolithic column for solid-phase microextraction and chemiluminescence determination of catechins in green tea.

A porous polymer monolithic column for solid-phase microextraction and chemiluminescence detection was integrated into a simple microfluidic chip for the extraction and determination of catechins in green tea. The porous polymer was prepared by poly(glycidyl methacrylate-co-ethylene dimethacrylate) and modified with ethylenediamine. Catechins can be concentrated in the porous polymer monolithic column and react with potassium permanganate to give chemiluminescence. The microfluidic chip is reusable with high sensitivity and very low reagent consumption. The on-line preconcentration and detection can be realized without an elution step. The enrichment factor was calculated to be about 20 for catechins. The relative chemiluminescence intensity increased linearly with concentration of catechin from 5.0 × 10(-9) to 1.0 × 10(-6) M and the limit of detection was 1.0 × 10(-9) M. The proposed method was applied to determine catechin in green tea. The recoveries are from 90% to 110% which benefits the actual application for green tea samples.

Determination of parabens in cosmetic products by solid-phase microextraction of poly(ethylene glycol) diacrylate thin film on fibers and ultra high-speed liquid chromatography with diode array detector.

The fabrication of a solid-phase microextraction (SPME) fiber through UV-induced polymerization of poly(ethylene glycol) diacrylate (PEG-DA) for determination of parabens in cosmetic products is presented in this work. The PEG-DA polymer coating was covalently attached to the fiber by introducing a surface modification with 3-(trichlorosilyl)propyl methacrylate (TPM). The PEG-DA polymer thin film coated on the fiber was homogeneous and wrinkled, which led to an increase of the surface area and high extraction efficiency. The extraction performances of the prepared SPME fibers were assessed by preconcentration of parabens including methylparaben, ethylparaben, propylparaben and benzylparaben from cosmetic products. The analysis was performed on an ultra high-speed liquid chromatography with diode array detector. The prepared SPME fibers exhibited good repeatability (for one fiber) and reproducibility (fiber-to-fiber) with RSDs of 5.4 and 6.9%, respectively. The optimized SPME method supported a wide linear range of 0.50-160 µg/mL and the detection limits for parabens were in the range of 0.12-0.15 µg/mL (S/N=3). The developed method was successfully applied for determination of parabens in cosmetic products with different natures.

Preparation of surface imprinting polymer capped Mn-doped ZnS quantum dots and their application for chemiluminescence detection of 4-nitrophenol in tap water.

In this paper, Mn-doped ZnS quantum dots (QDs) capped by a molecularly imprinted polymer (MIP) were synthesized. The results showed a high selectivity of the MIP-capped Mn-doped ZnS QDs toward the template molecule (4-nitrophenol) by QD fluorescence quenching. The application of MIP-capped Mn-doped ZnS QDs to the chemiluminescence (CL) system was also studied using a KIO(4)-H(2)O(2) system. This application combines the good selectivity of MIP with the high sensitivity of CL. The linear range of this CL system is from 0.1 to 40 microM, and the detection limit (DL) for 4-nitrophenol in the water can reach 76 nM. The method was also used in the real water samples, and the recoveries can fall in the range of 91-96%.

Fabrication of microwell arrays based on two-dimensional ordered polystyrene microspheres for high-throughput single-cell analysis.

This paper describes a method of fabricating rounded bottom microwell arrays (MA) in poly(dimethylsiloxane) (PDMS) by molding a monolayer of ordered polystyrene (PS) microspheres. PS microspheres were self-assembled on a glass slide and partially melted mainly from the bottom at 240 °C to increase adhesive force with the substrate. The partially melted PS arrays were used as master to generate MA. Microwell sizes are tunable in the 10-20 µm range with rounded bottoms; such a 3D structure is not readily obtainable through conventional soft lithography. Both adherent and nonadherent cell types can be retained in the microwells with high efficiency. As a demonstration of the advantage of real-time cell screening with this MA, single cell enzyme kinetic analysis was also carried out on trapped single cells. The PDMS MA may find applications in high-throughput drug screening, guided formation of cell clusters, and multicellular communication.

Stable and Biocompatible Carbon Nanotube Ink Mediated by Silk Protein for Printed Electronics.

Ink-based processes, which enable scalable fabrication of flexible devices based on nanomaterials, are one of the practical approaches for the production of wearable electronics. However, carbon nanotubes (CNTs), which possess great potential for flexible electronics, are facing challenges for use in inks due to their low dispersity in most solvents and suspicious cytotoxicity. Here, a stable and biocompatible CNT ink, which is stabilized by sustainable silk sericin and free from any artificial chemicals, is reported. The ink shows stability up to months, which can be attributed to the formation of sericin-CNT (SSCNT) hybrid through non-covalent interactions. It is demonstrated that the SSCNT ink can be used for fabricating versatile circuits on textile, paper, and plastic films through various techniques. As proofs of concept, electrocardiogram electrodes, breath sensors, and electrochemical sensors for monitoring human health and activity are fabricated, demonstrating the great potential of the SSCNT ink for smart wearables.

Assay of bradykinin metabolites in human body fluids by CE-LIF coupled with transient ITP preconcentration.

A CE with LIF detection was developed for the separation and determination of three bradykinin (BK) metabolites: BK2-9, BK1-7 and BK1-5. BK fragments were derivatized with 5-(4, 6-dichloro-s-triazin-2-ylamino) fluorescein before CE-LIF analysis. Eighty millimolar of Tris-H3PO4 (pH 9.0) was selected as the derivatization media. Three BK fragments were baseline separated within 10 min by using 0.2 M TAPS-Tris buffer (pH 8.5) as the running buffer. Meanwhile we have also developed a simple, quick, and sensitive on-column transient ITP preconcentration for CE-LIF detection of three BK fragments. Ten millimolar of Tris-HCl (pH 9.0) was chosen as the leading electrolyte and 0.2 M TAPS-Tris (pH 8.5) containing 10 mM 1-butyl-3-methylimidazolium tetrafluoroborate as the terminating electrolyte and also served as the running buffer during CZE separation. By using this transient ITP coupled with CE-LIF, concentration detection limits (S/N=3) for BK2-9, BK1-7 and BK1-5 were 0.3, 0.1 and 0.1 pmol/L, respectively. This method has been applied to the assay of human saliva and plasma samples with satisfactory results.

Latent Redox Reporter of 4-Methoxyphenol as Electrochemical Signal Proxy for Real-Time Profiling of Endogenous H2O2 in Living Cells.

Hydrogen peroxide (H2O2) plays a persuasive role in the human cell physiology. Developing an efficient assay platform and a highly sensitive tracking and quantification of H2O2 in a physiological system is crucial to understand the neoplastic changes and/or redox homeostasis of cells. In this study, a novel turn-on latent electrochemical redox probe coupled with electrocatalytic signal amplification strategy is proposed. A custom-made readily available turn-on latent electrochemical probe 4-methoxyphenylboronic acid pinacol ester (4-MPBP) have been designed for the selective detection of endogenous H2O2 in live cells. The electrochemical probe composed of a latent electrochemical reporter (4-methoxy phenol, 4-MP) bearing a recognition unit (boronic acid pinacol ester) for H2O2 sensing. The selective analyte-triggered chemical transformation releases free electrochemical reporter 4-MP. The amount of H2O2 was evaluated electrochemically at glassy carbon electrode (GCE) with a broad detection range of 0.5 µM-1 mM. An amplified signal response of released 4-MP to build a highly sensitive assay tool has been achieved via replacing the GCE transducer electrode with polydopamine@carbonnanotube-molebtinumdisulfie hybrid modified GCE as it delivered an exceptional dynamic detection range of 0.01-100 µM. The innovative blend of electrochemical molecular probe strategy, with electrocatalytic signal amplification technique has delivered outstanding assay performance at trace level sensing of H2O2. Next, we set up a platform for real-time in vivo monitoring of the endogenously produced H2O2 in Caco-2 and MCF-7 cells through spermine-polyamine analogue and phorbol 12-myristate 13-acetate induction in SSAT/PAO gene and protein kinase C, respectively. As expected, the 4-MPBP latent probe coupled with electrocatalytic signal amplification strategy delivered outstanding performance for in situ H2O2 release and tracking over time.

Near-physiological microenvironment simulation on chip to evaluate drug resistance of different loci in tumour mass.

Developing a bio-functional model in vitro to study cancer resistance, which is a big challenge for clinical cancer therapy, is of great interest. Such reliable model requires appropriate drug diffusion kinetics simulation and a microenvironment that allows cell-cell and cell-matrix interactions. In this work, a special hydrogel-based three-dimensional (3D) microfluidic chip was constructed to simulate tumour-vascular microenvironment. The self-healing hydrogel supports long-time cell survival and proliferation, effective cellular metabolism of cancer drugs and cell-cell interaction between different types of cells. In the effective near-physiological tumour-vascular microenvironment, the endothelial and fibroblast cells are spread on different sides of a porous membrane, while sensitive and resistant breast tumour cells are separately cultured in the dynamic hydrogel consisting of glycol chitosan and telechelic difunctional poly (ethylene glycol) in the upper chambers. Nutrients and drugs are introduced through the bottom channel and diffuse into the cancer cells. Doxorubicin molecules pass first through blood vessel endothelial cells and act on the tumour cells surrounded by fibroblasts. Tumour cells respond differently to drug when they are cultured in the microenvironment. Sensitive breast tumour cells have a 47% increase in viability than those cultured without fibroblasts and endothelial cells. Both sensitive and resistant tumour cells can be analysed under the same chemical environment. This work represents a multi-functional in vitro platform that allows near-physiological simulation, effective drug metabolism and cellular response to extracellular stimuli and has great potential to make drug discovery speedy and precise.