Microfluidic systems for axonal growth and regeneration research

Damage to the adult mammalian central nervous system （CNS） often results in persistent neurological deficits with limited recovery of functions. The past decade has seen in- creasing research efforts in neural regeneration research with the ultimate goal of achieving functional recovery. Many studies have focused on prevention of further neural damage and restoration of functional connections that are com- promised after iniurY or pathological damage.

Engineering organoid microfluidic system for biomedical and health engineering:A review

In recent years,organoid technology,i.e.,in vitro three-dimensional(3D)tissue culture,has attracted increasing attention in biomedical engineering.Organoids are cell complexes induced by differentiation of stem cells or organ-progenitor cells in vitro using 3D culture technology.They can replicate the key structural and functional characteristics of the target organs in vivo.With the opening up of this new field of health engineering,there is a need for engineering-system approaches to the production,control,and quantitative analysis of organoids and their microenvironment.Traditional organoid technology has limitations,including lack of physical and chemical microenvironment control,high heterogeneity,complex manual operation,imperfect nutritional supply system,and lack of feasible online analytical technology for the organoids.The introduction of microfluidic chip technology into organoids has overcome many of these limitations and greatly expanded the scope of applications.Engineering organoid microfluidic system has become an interdisciplinary field in biomedical and health engineering.In this review,we summarize the development and culture system of organoids,discuss how microfluidic technology has been used to solve the main technical challenges in organoid research and development,and point out new opportunities and prospects for applications of organoid microfluidic system in drug development and screening,food safety,precision medicine,and other biomedical and health engineering fields.

Microfluidic System for Synthesis of Trigonal Selenium Nanowires

A microfluidic system was developed for the synthesis of trigonal selenium（t-Se） nanowires, which was composed of a glass microchip coupled with a poly（methyl methacrylate）（PMMA） microchip. In the glass microchip, amorphous selenium（a-Se） colloid was prepared by reducing selenious acid with an excess amount of hydrazine at a temperature of 100 ℃. In the coupled PMMA microchip, a-Se was transformed into more stable t-Se seeds via sonication at room temperature. The residence time of the reactants in both microchips was optimized by varying the dimension and length of the microchannel each. The t-Se nanowires were formed by anisotropic growth of selenium crystallite during sonication and aging under the assistance of β-cyclodextrin（β-CD）. Various stages of the nanowires＇ growth were investigated. The as-synthesized products were characterized by powder X-Ray diffraction（XRD）, Raman spectroscopy, scanning electron microscopy（SEM）, transmission electron microscopy（TEM） and selected-area electron diffraction（SAED）.

A Microfluidic System with Active Mixing for Improved Real-Time Isothermal Amplification

To improve the performance of real-time recombinase polymerase amplification(RPA),a microfluidic system with active mixing is developed to optimize the reaction dynamics.Instead of adopting a single typical reaction chamber,a specific reactor including a relatively large chamber in center with two adjacent zig-zag channels at two sides is integrated into the microfluidic chip.Active mixing is achieved by driving the viscous reagent between the chamber and the channel back and forth periodically with an outside compact peristaltic pump.To avoid reagent evapora-tion,one end of the reactor is sealed with paraffin oil.A hand-held companion device is developed to facilitate real-time RPA amplification within 20 min.The whole area of the reactor is heated with a resistance heater to provide uniform reaction temperature.To achieve real-time monitoring,a compact fluorescence detection module is integrated into the hand-held device.A smartphone with custom application software is adopted to control the hand-held device and display the real-time fluorescence curves.The performances of two cases with and without active on-chip mixing are compared between each other by detecting African swine fever viruses.It has been demonstrated that,with active on-chip mixing,the amplification efficiency and detection sensitivity can be signifi-cantly improved.

3D Microfluidic System for Evaluating Inhibitory Effect of Chinese Herbal Medicine Oldenlandia diffusa on Human Malignant Glioma Invasion Combined with Network Pharmacology Analysis

Objective:To investigate the anti-invasion efficacy of the ethanol extract of Oldenlandia diffusa Will.(EEOD) on a three-dimensional(3D) human malignant glioma(MG) cell invasion and perfusion model based on microfluidic chip culture and the possible mechanism of action of Oldenlandia diffusa Will.(OD).Methods:The comprehensive pharmacodynamic analysis method in this study was based on microfluidic chip 3D cell perfusion culture technology,and the action mechanism of Chinese medicine(CM) on human MG cells was investigated through network pharmacology analysis.First,the components of EEOD were analyzed by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF/MS).Then,cell viability and apoptosis were assessed to determine the optimum concentration of EEOD for invasion experiments,and two-dimensional(2D) migration and invasion abilities of U87 and U251 MG cells were evaluated using scratch wound and Transwell assays.The possible mechanism underlying the effects of EEOD on glioma was analyzed through a network pharmacology approach.Results:Thirty-five compounds of EEOD were detected by UPLC-Q-TOF/MS.EEOD suppressed the viability of MG cells,promoted their apoptosis,and inhibited their migratory and invasive potentials(all P<0.05).Network pharmacology analysis showed that OD inhibited the invasion of MG cells by directly regulating MAPK and Wnt pathways through MAPK,EGFR,MYC,GSK3B,and other targets.The anti-invasion effect of OD was also found to be related to the indirect regulation of microtubule cytoskeleton organization.Conclusion:EEOD could inhibit the invasion of human MG cells,and the anti-invasion mechanism of OD might be regulating MAPK and Wnt signaling pathways and microtubule cytoskeleton organization.

A digital microfluidic single-cell manipulation systemoptimized by extending-depth-of-field device

Microfuidic systems have been widely utilized in high-throughput biology analysis,but thedificulties in iquid manipulation and cell cultivation limit its application.This work has developed a new digital microfluidic(DMF)system for on-demand droplet control.By adopting anextending-depth-of-field(EDoF)phase modulator to the optical system,the entire depth of themicrofluidic channel can be covered in one image without any refocusing process,ensuring that 95%of the particles in the droplet are captured within three shots together with shaking pro-cesses.With this system,suspension droplets are generated and droplets containing only oneyeast cll can be recognized,then each single cell is cultured in the array of the chip.Byobservingtheir growth in cell numbers and the green fluorescence protein(GFP)production via fluorescence imaging,the single cell with the highest production can be identified.The results haveproved the heterogeneity of yeast cells,and showed that the combined system can be applied forrapid single-cell sorting,cultivation,and analysis.

A novel integrated microfluidic chip for on-demand electrostatic droplet charging and sorting

On-demand droplet sorting is extensively applied for the efficient manipulation and genome-wide analysis of individual cells.However,state-of-the-art microfluidic chips for droplet sorting still suffer from low sorting speeds,sample loss,and labor-intensive preparation procedures.Here,we demonstrate the development of a novel microfluidic chip that integrates droplet generation,on-demand electrostatic droplet charging,and high-throughput sorting.The charging electrode is a copper wire buried above the nozzle of the microchannel,and the deflecting electrode is the phosphate buffered saline in the microchannel,which greatly simplifies the structure and fabrication process of the chip.Moreover,this chip is capable of high-frequency droplet generation and sorting,with a frequency of 11.757 kHz in the drop state.The chip completes the selective charging process via electrostatic induction during droplet generation.On-demand charged microdroplets can arbitrarilymove to specific exit channels in a three-dimensional(3D)-deflected electric field,which can be controlled according to user requirements,and the flux of droplet deflection is thereby significantly enhanced.Furthermore,a lossless modification strategy is presented to improve the accuracy of droplet deflection or harvest rate from 97.49% to 99.38% by monitoring the frequency of droplet generation in real time and feeding it back to the charging signal.This chip has great potential for quantitative processing and analysis of single cells for elucidating cell-to-cell variations.

Microfluidic-oriented synthesis of enriched iridium nanodots/carbon architecture for robust electrocatalytic nitrogen fixation

Electrocatalytic nitrogen reduction reaction(NRR)is considered as a promising candidate to achieve ammonia synthesis because of clean electric energy,moderate reaction condition,safe operating process and harmless by-products.However,the chemical inertness of nitrogen and poor activated capacity on catalyst surface usually produce low ammonia yield and faradic efficiency.Herein,the microfluidic technology is proposed to efficiently fabricate enriched iridium nanodots/carbon architecture.Owing to in-situ co-precipitation reaction and microfluidic manipulation,the iridium nanodots/carbon nanomaterials possess small average size,uniform dispersion,high conductivity and abundant active sites,producing good proton activation and rapid electrons transmission and moderate adsorption/desorption capacity.As a result,the as-prepared iridium nanodots/carbon nanomaterials realize large ammonia yield of 28.73 μg h^(-1) cm^(-2) and faradic efficiency of 9.14%in KOH solution.Moreover,the high ammonia yield of 11.21 μg h^(-1) cm^(-2) and faradic efficiency of 24.30%are also achieved in H_(2)SO_(4) solution.The microfluidic method provides a reference for large-scale fabrication of nano-sized catalyst materials,which may accelerate the progress of electrocatalytic NRR in industrialization field.

A novel method for fast and continuous preparation of superfine titanium dioxide nanoparticles in microfluidic system

Previously we had developed a microfluidic system that can be easily fabricated by bending a stainless-steel tube into large circular loops.In this study,a fast and continuous preparation method for superfine TiO_(2) nanoparticles(TiO_(2)-NPs)was developed for the aforementioned microfluidic system.The proposed method can yield anatase TiO_(2) in 3.5 min,in contrast to the traditional hydrothermal reaction method,which requires hours or even days.Different reaction conditions,such as reaction temperature(120-200℃),urea concentration(20-100 g/L),and tube length(5-20 m)were investigated.X-ray diffraction and Brunauer-Emmett-Teller analysis indicate that the as-prepared TiO_(2)-NPs have crystalline sizes of 4.1-5.8 nm and specific surface areas of 250.7-330.7 m^(2)/g.Transmission electron microscopy images show that these TiO_(2)-NPs have an even diameter of approximately 5 nm.Moreover,because of their small crystalline sizes and large specific surface areas,most of these as-prepared TiO_(2)-NPs exhibit considerably better absorption and photocatalytic performance with methylene blue than commercial P5 TiO_(2) does.

A fully automated centrifugal microfluidic system for sample-to-answer viral nucleic acid testing

The outbreak of virus-induced infectious diseases poses a global public-health challenge.Nucleic acid amplification testing(NAAT)enables early detection of pandemic viruses and plays a vital role in preventing onward transmission.However,the requirement of skilled operators,expensive instrumentation,and biosafety laboratories has hindered the use of NAAT for screening and diagnosis of suspected patients.Here we report development of a fully automated centrifugal microfluidic system with sample-in-answer-out capability for sensitive,specific,and rapid viral nucleic acid testing.The release of nucleic acids and the subsequent reverse transcription loop-mediated isothermal amplification(RT-LAMP)were integrated into the reaction units of a microfluidic disc.The whole processing steps such as injection of reagents,fluid actuation by rotation,heating and temperature control,and detection of fluorescence signals were carried out automatically by a customized instrument.We validate the centrifugal microfluidic system using oropharyngeal swab samples spiked with severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)armored RNA particles.The estimated limit of detection for armored RNA particles is 2 copies per reaction,the throughput is 21 reactions per disc,and the assay sample-to-answer time is approximately 70 min.This enclosed and automated microfluidic system efficiently avoids viral contamination of aerosol,and can be readily adapted for virus detection outside the diagnostic laboratory.

Microfluidic chips for the endothelial biomechanics and mechanobiology of the vascular system

Endothelial cells arranged on the vessel lumen are constantly stimulated by blood flow,blood pressure and pressureinduced cyclic stretch.These stimuli are sensed through mechanical sensory structures and converted into a series of functional responses through mechanotransduction pathways.The process will eventually affect vascular health.Therefore,there has been an urgent need to establish in vitro endothelial biomechanics and mechanobiology of models,which reproduce three-dimensional structure vascular system.In recent years,the rapid development in microfluidic technology makes it possible to replicate the key structural and functionally biomechanical characteristics of vessels.Here,we summarized the progress of microfluidic chips used for the investigation of endothelial biomechanics and mechanobiology of the vascular system.Firstly,we elucidated the contribution of shear stress and circumferential stress,to vascular physiology.Then,we reviewed some applications using microfluidic technology in angiogenesis and vasculogenesis,endothelial permeability and mechanotransduction,as well as the blood-brain barrier under these physical forces.Finally,we discussed the future obstacles in terms of the development and application of microfluidic vascular chips.

Multiple exosome RNA analysis methods for lung cancer diagnosis through integrated on-chip microfluidic system

Exosomes are now raising focus as a prospective biomarker for cancer diagnostics and prognosis owing to its unique bio-origin and composition.Exosomes take part in cellular communication and receptor mediation and transfer their cargos(e.g.,proteins,m RNA and DNA).Quantitative analysis of tumor-related nucleic acid mutations can be a potential method to cancer diagnosis and prognosis in early stages.Here we present an integrated microfluidic system for exosome on-chip isolation and lung cancer RNA analysis through droplet digital PCR(dd PCR).Gradient dilution experiments show great linearity over a large concentration range with R^(2)=0.9998.Utilizing the system,four cell lines and two mutation targets were parallelly detected for mutation analysis.The experiments demonstrated mutation heterogeneity and the results were agree with cell researches.These results proved our integrated microfluidic system as a promising means for early cancer diagnosis and prognosis in the era of liquid biopsy.

Neurotoxicity mechanism of aconitine in HT22 cells studied by microfluidic chip-mass spectrometry

Aconitine,a common and main toxic component of Aconitum,is toxic to the central nervous system.However,the mechanism of aconitine neurotoxicity is not yet clear.In this work,we had the hypothesis that excitatory amino acids can trigger excitotoxicity as a pointcut to explore the mechanism of neurotoxicity induced by aconitine.HT22 cells were simulated by aconitine and the changes of target cell metabolites were real-time online investigated based on a microfluidic chip-mass spectrometry system.Meanwhile,to confirm the metabolic mechanism of aconitine toxicity on HT22 cells,the levels of lactate dehydrogenase,intracellular Ca^(2+),reactive oxygen species,glutathione and superoxide dismutase,and ratio of Bax/Bcl-2 protein were detected by molecular biotechnology.Integration of the detected results revealed that neurotoxicity induced by aconitine was associated with the process of excitotoxicity caused by glutamic acid and aspartic acid,which was followed by the accumulation of lactic acid and reduction of glucose.The surge of extracellular glutamic acid could further lead to a series of cascade reactions including intracellular Ca^(2+)overload and oxidative stress,and eventually result in cell apoptosis.In general,we illustrated a new mechanism of aconitine neurotoxicity and presented a novel analysis strategy that real-time online monitoring of cell metabolites can provide a new approach to mechanism analysis.

Microfluidic assisted 90%loading CL-20 spherical particles:Enhancing self-sustaining combustion performance

The performance of the chemical fuel determines the altitude,range and longevity of spacecraft in air and space exploration.Promising alternatives(e.g.,hypergolic ionic liquids or high-energy composites)with high-energy density,heat of formation and fast initial rate are considered as potential chemical fuels.As the high-energy density material,hexanitrohexaazaisowurtzitane(CL-20)often serves as secondary explosive with poor self-propagating combustion behaviors.Herein,90%loading CL-20 microspheres with uniform particle sizes are precisely prepared by microfluid method,which exhibit unique hierarchical structure.The morphology,thermal behaviors,as well as combustion performance were further investigated.The results demonstrated that as-prepared spherical particles exhibit prominent thermal compatibility,and the enhanced self-sustaining combustion performance.This work provides an efficient method achieving the uniform high-energy density particles with excellent self-sustaining combustion performance.

A Camouflaged Film Imitating the Chameleon Skin with Color-Changing Microfluidic Systems Based on the Color Information Identification of Background

To adapt to a complex and variable environment,self-adaptive camouflage technology is becoming more and more important in all kinds of military applications by overcoming the weakness of the static camouflage.In nature,the chameleon can achieve self-adaptive camouflage by changing its skin color in real time with the change of the background color.To imitate the chameleon skin,a camouflaged film controlled by a color-changing microfluidic system is proposed in this paper.The film with microfluidic channels fabricated by soft materials can achieve dynamic cloaking and camouflage by circulating color liquids through channels inside the film.By sensing and collecting environmental color change information,the control signal of the microfluidic system can be adjusted in real time to imitate chameleon skin.The microstructure of the film and the working principle of the microfluidic color-changing system are introduced.The mechanism to generate the control signal by information processing of background colors is illustrated.“Canny”double-threshold edge detection algorithm and color similarity are used to analyze and evaluate the camouflage.The tested results show that camouflaged images have a relatively high compatibility with environmental backgrounds and the dynamic cloaking eff ect can be achieved.

Thermal and ignition properties of hexanitrostilbene(HNS) microspheres prepared by droplet microfluidics

HNS-IV(Hexanitrostilbene-IV) is the main charge of the exploding foil initiators(EFI), and the microstructure of the HNS will directly affect its density, flowability, sensitivity, and stability. HNS microspheres were prepared using droplet microfluidics, and the particle size, morphology, specific surface area, thermal performance, and ignition threshold of the HNS microspheres were characterized and tested. The results shown that the prepared HNS microspheres have high sphericity, with an average particle size of 20.52 μm(coefficient of variation less than 0.2), and a specific surface area of 21.62 m^(2)/g(6.87 m^(2)/g higher than the raw material). Without changing the crystal structure and thermal stability of HNS-IV, this method significantly enhances the sensitivity of HNS-IV to short pulses and reduces the ignition threshold of the slapper detonator to below 1000 V. This will contribute to the miniaturization and low cost of EFI.

Green microfluidics in microchemical engineering for carbon neutrality

The concept of“carbon neutrality”poses a huge challenge for chemical engineering and brings great opportunities for boosting the development of novel technologies to realize carbon offsetting and reduce carbon emissions.Developing high-efficient,low-cost,energy-efficient and eco-friendly microfluidicbased microchemical engineering is of great significance.Such kind of“green microfluidics”can reduce carbon emissions from the source of raw materials and facilitate controllable and intensified microchemical engineering processes,which represents the new power for the transformation and upgrading of chemical engineering industry.Here,a brief review of green microfluidics for achieving carbon neutral microchemical engineering is presented,with specific discussions about the characteristics and feasibility of applying green microfluidics in realizing carbon neutrality.Development of green microfluidic systems are categorized and reviewed,including the construction of microfluidic devices by bio-based substrate materials and by low carbon fabrication methods,and the use of more biocompatible and nondestructive fluidic systems such as aqueous two-phase systems(ATPSs).Moreover,low carbon applications benefit from green microfluidics are summarized,ranging from separation and purification of biomolecules,high-throughput screening of chemicals and drugs,rapid and cost-effective detections,to synthesis of fine chemicals and novel materials.Finally,challenges and perspectives for further advancing green microfluidics in microchemical engineering for carbon neutrality are proposed and discussed.

Fabrication of Microfluidic Fiber Chip Detection System

The diameter of the excitation beam was decreased greatly by integrating the fiber on the microfluidic chip as light propagation medium.The coupling efficiency of the fiber was improved with optical fiber collimation device coupling beam. The chip was placed in the darkroom to avoid the interference of the external light.The cost of the instrument was decreased with a high brightness blue LED as excitation source;the performance of the system was valuated by the determination of FITC fluorescein with a minimum detectable concentration of 2.2×10^(-8) mol/L,the Signal-to-Noise Ratio (SNR) S/N=5.The correlation coefficient of the detection system within the range of 1.8×10^(-7) mol/L～4×10^(-5)mol/L was 0.9972.

Continuous medium exchange and optically induced electroporation of cells in an integrated microfluidic system

Optically induced electroporation(OIE)is a promising microfluidic-based approach for the electroporation of cell membranes.However,previously proposed microfluidic cell-electroporation devices required tedious sample pre-treatment steps,specifically,periodic media exchange.To enable the use of this OIE process in a practical protocol,we developed a new design for a microfluidic device that can perform continuous OIE;i.e.,it is capable of automatically replacing the culture medium with electroporation buffers.Integrating medium exchanges on-chip with OIE minimises critical issues such as cell loss and damage,both of which are common in traditional,centrifuge-based approaches.Most importantly,our new system is suitable for handling small or rare cell populations.Two medium exchange modules,including a micropost array railing structure and a deterministic lateral displacement structure,were first adopted and optimised for medium exchange and then integrated with the OIE module.The efficacy of these integrated microfluidic systems was demonstrated by transfecting an enhanced green fluorescent protein(EGFP)plasmid into human embryonic kidney 293T cells,with an efficiency of 8.3%.This result is the highest efficiency reported for any existing OIE-based microfluidic system.In addition,successful co-transfections of three distinct plasmids(EGFP,DsRed and ECFP)into cells were successfully achieved.Hence,we demonstrated that this system is capable of automatically performing multiple gene transfections into mammalian cells.

Enhancing gene editing efficiency for cells by CRISPR/Cas9 system-loaded multilayered nanoparticles assembled via microfluidics

Most non-viral carriers for in vitro delivery of nucleic acids suffer from low efficiency of introducing m RNA and other nucleic acids,especially large m RNA.Cas9 protein is the nuclease part of the powerful gene-editing tool,CRISPR/Cas9 system,Cas9 m RNA is particularly large,thus presents a big challenge for delivery.We assembled a multilayered biodegradable nanocarrier to load Cas9 m RNA inside to protect Cas9 m RNA from degradation.We used a microfluidic chip to synthesize a small,positively charged,and degradable core to attract negatively charged Cas9 m RNA.The microfluidic assembly allows the core to be small enough to incorporate into a cationic liposome.The multilayered nanocarriers elevated the delivery efficiency of Cas9 m RNA by over 2 folds and increased the expression by over 5 folds compared to commercially used non-viral carriers.In addition,the multilayered nanocarriers do not require reduced serum medium for transfection.When using the standard complete medium for transfection,the multilayered nanocarriers could increase the expression of Cas9 m RNA by over 15 folds compared to commercially used non-viral carriers.The co-delivery of Cas9 m RNA and sg RNA via LRC elevated the gene-editing efficiency by 3 folds compared to that via commercially used non-viral carriers.Based on the higher transfection efficiency of Cas9 m RNA/sg RNA than commercially used non-viral carriers,these multilayered nanocarriers may have a good prospect as efficient commercial delivery carriers for Cas9 m RNA/sg RNA and other nucleic acids.