Rapid fabrication of modular 3D paper-basedmicrofluidic chips using projection-based 3D printing

Paper-based microchips have different advantages,such as better biocompatibility,simple production,and easy handling,making them promising candidates for clinical diagnosis and other fields.This study describes amethod developed to fabricate modular three-dimensional(3D)paper-based microfluidic chips based on projection-based 3D printing(PBP)technology.A series of two-dimensional(2D)paper-based microfluidic modules was designed and fabricated.After evaluating the effect of exposure time on the accuracy of the flow channel,the resolution of this channel was experimentally analyzed.Furthermore,several 3D paper-based microfluidic chips were assembled based on the 2D ones using different methods,with good channel connectivity.Scaffold-based 2D and hydrogel-based 3D cell culture systems based on 3D paper-based microfluidic chips were verified to be feasible.Furthermore,by combining extrusion 3D bioprinting technology and the proposed 3D paper-based microfluidic chips,multiorgan microfluidic chips were established by directly printing 3D hydrogel structures on 3D paperbased microfluidic chips,confirming that the prepared modular 3D paper-based microfluidic chip is potentially applicable in various biomedical applications.

Advances in microfluidic chips based on islet hormone-sensing techniques

Diabetes mellitus is a global health problem resulting from islet dysfunction or insulin resistance.The mechanisms of islet dysfunction are still under investigation.Islet hormone secretion is the main function of islets,and serves an important role in the homeostasis of blood glucose.Elucidating the detailed mechanism of islet hormone secretome distortion can provide clues for the treatment of diabetes.Therefore,it is crucial to develop accurate,real-time,laborsaving,high-throughput,automated,and cost-effective techniques for the sensing of islet secretome.Microfluidic chips,an elegant platform that combines biology,engineering,computer science,and biomaterials,have attracted tremendous interest from scientists in the field of diabetes worldwide.These tiny devices are miniatures of traditional experimental systems with more advantages of timesaving,reagent-minimization,automation,high-throughput,and online detection.These features of microfluidic chips meet the demands of islet secretome analysis and a variety of chips have been designed in the past 20 years.In this review,we present a brief introduction of microfluidic chips,and three microfluidic chipsbased islet hormone sensing techniques.We focus mainly on the theory of these techniques,and provide detailed examples based on these theories with the hope of providing some insights into the design of future chips or whole detection systems.

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.

Experiments on imbibition mechanisms of fractured reservoirs by microfluidic chips

To solve the problems of long experiment period and difficult measurement in core imbibition experiments,fracture-matrix microfluidic chips of different sizes,boundary conditions and wettability regulated by surface property modification were designed to research the imbibition mechanisms of oil-water,oil-surfactant solution and oil-WinsorⅢtype surfactant solution.In the oil-water,and oil-wettability modification system imbibition process,oil was replaced from the matrix through Haines jump,the capillary back pressure was the main resistance blocking the flow of oil,the reduction of interfacial tension caused the weakening of Haines jump,reduction of oil discharge rate,and increase of oil recovery.The imbibition of oil-water or oil-surfactant solution with low interfacial tension was a counter-current imbibition process dominated by capillary force,in which all boundaries had similar contribution to imbibition,and the recovery data obtained from this experiment fit well with the classic imbibition scaling equation.The imbibition of oil and Winsor III type surfactant solution was a co-current imbibition process dominated by gravity under super-low interfacial tension,and is essentially the formation and re-balance of neutral microemulsion.The imbibition dynamics obtained from this experiment fit well with the modified imbibition scaling equation.

PRESSURE FORCE CONTROL FOR FABRICATION OF PLASTIC MICROFLUIDIC CHIPS WITH HOT EMBOSSING METHOD

A pressure force control system for hot embossing of microfluidic chips is designed with a moment motor and a ball bearing lead screw. Based on the numeric PID technique, the algorithm of pulsant integral accelerated PID control is presented and the negative effects of nonlinearity from friction, clearance and saturation are eliminated. In order to improve the quick-resixmse characteristic, independent thread technique is adopted. The method of pressure force control based on pulsant integral accelerated PID control and independent thread technique is applied with satisfactory control performance.

Highly efficient synthesis and application of aryl diazonium salts via femtosecond laser-tailored 3D flow microfluidic chips

The first example of the microfluidic chips(MFCs) consisting of centimeter-level 3D channels with highdensity and large-volume fabricated by femtosecond laser micromachining were utilized to develop a time-saving, economical and hazardless flow synthesis process, and its advantages have been proved by in situ formation of aryldiazonium salts and subsequent borylation with bis(pinacolato)diboron. There are several important advantages in our 3D MFC-based flow synthesis technology, including the following:(1) the reaction temperature was altered from ice bath to room temperature;(2) the residence time was reduced by 10 times;(3) the yield was greatly improved, that is, several arylboronates were successfully obtained with higher yield compared to traditional batch process. Therefore, it can be envisioned that a novel, simplified flow synthetic protocol will be developed toward green organic synthesis via MFCs.

Femtosecond laser-engineered 3D microfluidic chips: Synthesis system sprouting highly efficient multiphase organic reactions

Recent developments in the utilization of microfluidic chips(MFCs) have shown their potential utility in multiphase organic synthesis by enabling efficient organic reactions in flow chemistry. However, MFCs technology has been wandering in the laboratory of small dose synthetic routes, which is limited to the level of "tiny" fluid flux. To address this issue, we herein report the first case of the chips with highthroughput 3D channels produced by femtosecond laser being used to create a time-saving, cost-effective and risk-free approach suitable for large-scale flow synthesis. Several multiphase reactions have been successfully prepared on demand in our designed flow synthesis system containing 3D MFCs: 1) benzyl alcohol was converted to benzaldehyde in 3 min with a yield of 97.50% by liquid-liquid two-phase transfer catalytic oxidation;2) organozinc reagents and α-cyano carbonyl carbon compounds were synthesized by solid-liquid two-phase metal insertion reaction in 7 min, and the yield was up to 100%;3) benzoic acid was synthesized by gas-liquid two-phase carboxylation reaction in 2.8 s with a yield of 96%. Significant gains in production rate result from the effective scaling of flow reactors from microliters per hour in MFCs to intermediate milliliters per minute without affecting mass transport performance. Meanwhile,our 3D MFCs show excellent mass and heat transfer efficiency in large-scale industrial units, breaking through the bottleneck in this field. As a result, it is possible to imagine the creation of a new, streamlined flow synthetic technique via MFCs for green multiphase organic synthesis.

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.

Droplet microfluidic chip for precise monitoring of dynamic solution changes

In this work,an automated microfluidic chip that uses negative pressure to sample and analyze solutions with high temporal resolution was developed.The chip has a T-shaped channel for mixing the sample with a fluorescent indicator,a flow-focusing channel for generating droplets in oil,and a long storage channel for incubating and detecting the droplets.By monitoring the fluorescence intensity of the droplets,the device could detect changes in solution accurately over time.The chip can generate droplets at frequencies of up to 42 Hz with a mixing ratio of 1:1 and a temporal resolution of 3–6 s.It had excellent linearity in detecting fluorescein solution in the concentration range 1–5μM.This droplet microfluidic chip provides several advantages over traditional methods,including high temporal resolution,stable droplet generation,and faster flow rates.This approach could be applied to monitoring calcium ions with a dynamic range from 102 to 107 nM and a detection limit of 10 nM.

In vitro study of emodin-induced nephrotoxicity in human renal glomerular endothelial cells on a microfluidic chip

Emodin is an effective component of rhubarb with positive pharmacological effects on human health.However,it is also toxic to different cells or tissues to varying degrees.The effects of emodin on glomerular endothelial cells(GECs)remain to be tested,and the documented works were always performed in vitro and hardly reflect the real physiological situation.To study the effects of emodin on GECs in a biomimetic environment,we utilized a microfluidic chip to assess the physiological reaction of human renal glomerular endothelial cells to various concentrations of emodin in this work.The results showed that emodin caused cytotoxicity,impaired glomerular filtration barrier integrity to macromolecules,and increased barrier permeability in a dose-dependent manner.With the increase in emodin concentration,the concentration of the pro-inflammatory cytokine tumor necrosis factor-α,interleukin(IL)-6,transforming growth factor-β1,and monocyte chemoattractant protein(MCP-1)increased while the production of inflammatory cytokine IL-6 first increased and then decreased with the increase in emodin concentration.Our findings shed new light on emodin-induced nephrotoxicity and provide insights for the application of microfluidic chip devices to reveal drug-cell interactions.

Junction matters in hydraulic circuit bio-design of microfluidics

Microfluidic channels are at micrometer scales;thus,their fluid flows are laminar,resulting in the linear dependence of pressure drop on flow rate in the length of the channel.The ratio of the pressure drop to flow rate,referred to as resistance,depends on channel size and dynamic viscosity.Usually,a microfluidic chip is analogous to an electric circuit in design,but the design is adjusted to optimize channel size.However,whereas voltage loss is negligible at the nodes of an electric circuit,hydraulic pressure drops at the nodes of microfluidic chips by a magnitude are comparable to the pressure drops in the straight channels.Here,we prove by experiment that one must fully consider the pressure drops at nodes so as to accurately design a precise microfluidic chip.In the process,we numerically calculated the pressure drops at hydraulic nodes and list their resistances in the range of flows as concerned.We resorted to machine learning to fit the calculated results for complex junctions.Finally,we obtained a library of node resistances for common junctions and used them to design three established chips that work for single-cell analysis and for precision allocation of solutes(in gradient and averaging concentration microfluidic networks).Endothelial cells were stimulated by generating concentrations of adriamycin hydrochloride from the last two microfluidic networks,and we analyzed the response of endothelial cells.The results indicate that consideration of junction resistances in design calculation brings experimental results closer to the design values than usual.This approach may therefore contribute to providing a platform for the precise design of organ chips.

GPU-accelerated OCT imaging: Real-time data processing and artifact suppression for enhanced monitoring of 3D bioprinted tissues and vascular-like networks

Optical coherence tomography(OCT)imaging technology has significant advantages in in situ and noninvasive monitoring of biological tissues.However,it still faces the following challenges:including data processing speed,image quality,and improvements in three-dimensional(3D)visualization effects.OCT technology,especially functional imaging techniques like optical coherence tomography angiography(OCTA),requires a long acquisition time and a large data size.Despite the substantial increase in the acquisition speed of swept source optical coherence tomography(SS-OCT),it still poses significant challenges for data processing.Additionally,during in situ acquisition,image artifacts resulting from interface reflections or strong reflections from biological tissues and culturing containers present obstacles to data visualization and further analysis.Firstly,a customized frequency domainfilter with anti-banding suppression parameters was designed to suppress artifact noises.Then,this study proposed a graphics processing unit(GPU)-based real-time data processing pipeline for SS-OCT,achieving a measured line-process rate of 800 kHz for 3D fast and high-quality data visualization.Furthermore,a GPU-based realtime data processing for CC-OCTA was integrated to acquire dynamic information.Moreover,a vascular-like network chip was prepared using extrusion-based 3D printing and sacrificial materials,with sacrificial material being printed at the desired vascular network locations and then removed to form the vascular-like network.OCTA imaging technology was used to monitor the progression of sacrificial material removal and vascular-like network formation.Therefore,GPU-based OCT enables real-time processing and visualization with artifact suppression,making it particularly suitable for in situ noninvasive longitudinal monitoring of 3D bioprinting tissue and vascular-like networks in microfluidic chips.

Estimation of cancer cell migration in biomimetic random/oriented collagen fiber microenvironments

Increasing data indicate that cancer cell migration is regulated by extracellular matrixes and their surrounding biochemical microenvironment,playing a crucial role in pathological processes such as tumor invasion and metastasis.However,conventional two-dimensional cell culture and animal models have limitations in studying the influence of tumor microenvironment on cancer cell migration.Fortunately,the further development of microfluidic technology has provided solutions for the study of such questions.We utilize microfluidic chip to build a random collagen fiber microenvironment(RFM)model and an oriented collagen fiber microenvironment(OFM)model that resemble early stage and late stage breast cancer microenvironments,respectively.By combining cell culture,biochemical concentration gradient construction,and microscopic imaging techniques,we investigate the impact of different collagen fiber biochemical microenvironments on the migration of breast cancer MDA-MB-231-RFP cells.The results show that MDA-MB-231-RFP cells migrate further in the OFM model compared to the RFM model,with significant differences observed.Furthermore,we establish concentration gradients of the anticancer drug paclitaxel in both the RFM and OFM models and find that paclitaxel significantly inhibits the migration of MDA-MB-231-RFP cells in the RFM model,with stronger inhibition on the high concentration side compared to the low concentration side.However,the inhibitory effect of paclitaxel on the migration of MDA-MB-231-RFP cells in the OFM model is weak.These findings suggest that the oriented collagen fiber microenvironment resembling the late-stage tumor microenvironment is more favorable for cancer cell migration and that the effectiveness of anticancer drugs is diminished.The RFM and OFM models constructed in this study not only provide a platform for studying the mechanism of cancer development,but also serve as a tool for the initial measurement of drug screening.

Wave-shaped microfluidic chip assisted point-of-care testing for accurate and rapid diagnosis of infections

Background:Early diagnosis and classification of infections increase the cure rate while decreasing complications,which is significant for severe infections,especially for war surgery.However,traditional methods rely on laborious operations and bulky devices.On the other hand,point-of-care(POC)methods suffer from limited robustness and accuracy.Therefore,it is of urgent demand to develop POC devices for rapid and accurate diagnosis of infections to fulfill on-site militarized requirements.Methods:We developed a wave-shaped microfluidic chip(WMC)assisted multiplexed detection platform(WMC-MDP).WMC-MDP reduces detection time and improves repeatability through premixing of the samples and reaction of the reagents.We further combined the detection platform with the streptavidin–biotin(SA-B)amplified system to enhance the sensitivity while using chemiluminescence(CL)intensity as signal readout.We realized simultaneous detection of C-reactive protein(CRP),procalcitonin(PCT),and interleukin-6(IL-6)on the detection platform and evaluated the sensitivity,linear range,selectivity,and repeatability.Finally,we finished detecting 15 samples from volunteers and compared the results with commercial ELISA kits.Results:Detection of CRP,PCT,and IL-6 exhibited good linear relationships between CL intensities and concentrations in the range of 1.25–40μg/ml,0.4–12.8 ng/ml,and 50–1600 pg/ml,respectively.The limit of detection of CRP,PCT,and IL-6 were 0.54μg/ml,0.11 ng/ml,and 16.25 pg/ml,respectively.WMC-MDP is capable of good adequate selectivity and repeatability.The whole detection procedure takes only 22 min that meets the requirements of a POC device.Results of 15 samples from volunteers were consistent with the results detected by commercial ELISA kits.Conclusions:WMC-MDP allows simultaneous,rapid,and sensitive detection of CRP,PCT,and IL-6 with satisfactory selectivity and repeatability,requiring minimal manipulation.However,WMC-MDP takes advantage of being a microfluidic device showing the coefficients of variation less than 10%enabling WMC-MDP to be a type of point-of-care testing(POCT).Therefore,WMC-MDP provides a promising alternative to POCT of multiple biomarkers.We believe the practical application of WMC-MDP in militarized fields will revolutionize infection diagnosis for soldiers.

EXPERIMENTAL STUDY ON THE HOT EMBOSSING POLYMER MICROFLUIDIC CHIP

Experiments are used to study the fabrication of polymer microfluidic chip with hot embossing method. The pattern fidelity with respect to the process parameters is analyzed. Experiment results show that the relationship between the imprint temperature and the microchannel width is approximately exponential. However, the depth of micro channel isn＇t sensitive to the imprint temperature. When the imprint pressure is larger than 1 MPa and the imprint time is longer than 2 min, the increasing of imprint pressure and holding time has little impact on the microchannel width. So over long holding time is not needed in hot embossing. Based on the experiment analysis, a series of optimization process parameters is obtained and a fine microfluidic chip is fabricated. The electrophoresis separation experiment are used to verify the microfluidic chip performance after bonding. The results show that 100bp-ladder DNA sample can be separated in less than 5 min successfully.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells,which may be covered by the noises from majority of unmutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multimutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cellswere easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drugrelated mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations,but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.

Simultaneous laser-induced fluorescence and contactless-conductivity detection for microfluidic chip

A combined detection system involving simultaneous LIF and contacfless-conductometric measurements at the same place of the microfluidic chip was described. The LIF measurement was designed according to the confocal principle and a moveable contactless-conduetivity detector was used in C^4D. Both measurements were mutually independent and advantageous in analyses of mixtures. Various experimental parameters affecting the response were examined and optimized. The performances were demonstrated by simultaneous detection of Rhodamine B. And the results showed that the combined detection system could be used sensitively and reliably.

MODELING OF LASER MACHINING ON POLYMETHYL METHACRYLATE TO FABRICATE MICROFLUIDIC CHIP

The use of a CO2 laser system for fabrication of microfluidic chip on polymethyl methacrylate （PMMA） is presented to reduce fabrication cost and time of chip. The grooving process of the laser system and a model for the depth of microchannels are investigated. The relations between the depth of laser-cut channels and the laser beam power, velocity or the number of passes of the beam along the same channel are evaluated. In the experiments, the laser beam power varies from 0 to 50 W, the laser beam scanning velocity varies from 0 to 1 000 mm/s and the passes vary in the range of 1 to 10 times. Based on the principle of conservation of energy, the influence of the laser beam velocity, the laser power and the number of groove passes are examine. Considering the grooving interval energy loss, a modified mathematical model has been obtained and experimental data show good agreement with the theoretical model. This approach provides a simple way of predicting groove depths. The system provides a cost alternative of the other methods and it is especially useful on research work of rnicrofluidic prototyping due to the short cycle time of production.

Sensitive detection of microRNAs using polyadenine-mediated fluorescent spherical nucleic acids and a microfluidic electrokinetic signal amplification chip

The identification of tumor-related microRNAs(miRNAs)exhibits excellent promise for the early diagnosis of cancer and other bioanalytical applications.Therefore,we developed a sensitive and efficient biosensor using polyadenine(polyA)-mediated fluorescent spherical nucleic acid(FSNA)for miRNA analysis based on strand displacement reactions on gold nanoparticle(AuNP)surfaces and electrokinetic signal amplification(ESA)on a microfluidic chip.In this FSNA,polyA-DNA biosensor was anchored on AuNP surfaces via intrinsic affinity between adenine and Au.The upright conformational polyA-DNA recognition block hybridized with 6-carboxyfluorescein-labeled reporter-DNA,resulting in fluorescence quenching of FSNA probes induced by AuNP-based resonance energy transfer.Reporter DNA was replaced in the presence of target miRNA,leading to the recovery of reporter-DNA fluorescence.Subsequently,reporter-DNAs were accumulated and detected in the front of with Nafion membrane in the microchannel by ESA.Our method showed high selectivity and sensitivity with a limit of detection of 1.3 pM.This method could also be used to detect miRNA-21 in human serum and urine samples,with recoveries of 104.0%-113.3% and 104.9%-108.0%,respectively.Furthermore,we constructed a chip with three parallel channels for the simultaneous detection of multiple tumor-related miRNAs(miRNA-21,miRNA-141,and miRNA-375),which increased the detection efficiency.Our universal method can be applied to other DNA/RNA analyses by altering recognition sequences.

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.