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 thermotaxic selection of highlymotile sperm and in vitro fertilization

The selection of the most motile and functionally competent sperm is an essential basis for in vitro fertilization(IVF)and normal embryonic development.Widely adopted clinical approaches for sperm sample processing intensely rely on centrifugation and wash steps that may induce mechanical damage and oxidative stress to sperm.Although a few microfluidic sperm sorting devices may avoid these adverse effects by exploiting intrinsic guidance mechanisms of sperm swimming,none of these approaches have been fully validated by clinical-grade assessment criteria.In this study,a microfluidic sperm sorting device that enables the selection of highly motile and functional sperm via their intrinsic thermotaxis is presented.Bioinspired by the temperature microenvironment in the fallopian tube during natural sperm selection,a microfluidic device with controllable temperature gradients along the sperm separation channel was designed and fabricated.This study investigated the optimal temperature conditions for human sperm selection and fully characterized thermotaxis-selected sperm with 45 human sperm samples.Results indicated that a temperature range of 35–36.5℃along the separation channel significantly improves human sperm motility rate((85.25±6.28)%vs.(60.72±1.37)%;P=0.0484),increases normal sperm morphology rate((16.42±1.43)%vs.(12.55±0.88)%;P<0.0001),and reduces DNA fragmentation((7.44±0.79)%vs.(10.36±0.72)%;P=0.0485)compared to the nonthermotaxis group.Sperm thermotaxis is species-specific,and selected mouse sperm displayed the highest motility in response to a temperature range of 36–37.5℃ along the separation channel.Furthermore,IVF experiments indicated that the selected sperm permitted an increased fertilization rate and improved embryonic development from zygote to blastocyst.This microfluidic thermotaxic selection approach will be translated into clinical practice to improve the IVF success rate for patients with oligozoospermia and asthenozoospermia.

Microfluidic platform for circulating tumor cells isolation and detection

Circulating tumor cells(CTCs)are essential biomarkers for liquid biopsies,which are important in the early screening,prognosis,and real-time monitoring of cancer.However,CTCs are less abundant in the peripheral blood of patients,therefore,their isolation is necessary.Recently,the use of microfluidics for CTC sorting has become a research hotspot owing to its low cost,ease of integration,low sample consumption,and unique advantages in the manipulation of micron-sized particles.Herein,we review the latest research on microfluidics-based CTC sorting.Specifically,we consider active sorting using external fields(electric,magnetic,acoustic,and optical tweezers)and passive sorting using the flow effects of cells in specific channel structures(microfiltration sorting,deterministic lateral displacement sorting,and inertial sorting).The advantages and limitations of each method and their recent applications are summarized here.To conclude,a forward-looking perspective is presented on future research on the microfluidic sorting of CTCs.

From Droplets and Particles to Hierarchical Spatial Organization： Nanotechnology Challenges for Microfluidics

The compartimentation of fluids in the microliter, nanoliter and picoliter range leads recently to many applications of microfluidics in material development, diagnostics and biological screenings. Droplet-based microfluidics allows the improvement of nanoparticle homogeneity and the tuning of particle properties. It supports combinatorial synthesis of inorganic as well as organic substances and can be applied for the cultivation and screening of bacteria, eucaryotic cells and fish embryos. The well-ordered handling and the addressing of microfluid segments improves the information transfer between chemical, biological and electronic systems. Despite this remarkable technical progress, there is a particular importance of microfluidics for future nanotechnological solutions. The hierarchical spatial organization of liquids, particles and gels in microfluidics represents a fundamental biomimetic principle which overcomes the limits of planar technology and opens the gate for realizing complex structured threedimensional nanoarchitectures. Recent applications of microstructured fluids in chemistry and biology and concepts for future developments will be discussed.

Emerging microfluidic technologies for sperm sorting

Sorting high-quality sperm with intact DNA,normal morphology,and active motility is crucial for clinically assisted reproductive technology,which influences the success of treatment and the health of offspring.Currently,microfluidic technology has been developed as a powerful platform for sperm sorting owing to its ability to manipulate fluid at the microscale and handle small samples.Specifically,microfluidic technology provides the necessary stimuli including fluid stimulus,chemical induction,and shape sift,which supports researchers in developing various sperm-sorting devices.According to the sorting principle,these devices can be divided into three categories:active sorting devices based on sperm rheological properties,passive sorting devices based on sperm physical properties,and external stimuli-induced sorting devices.Hence,we review a broad range of researches about sperm sorting with microfluidics and briefly present the properties of sperm and female reproductive tract to assist the design of microfluidic sperm sorting devices.

Phospholipase C-Zeta Reveals the Underlying Pathological Influence after Density Gradient Centrifugation, Microfluidic Sorting, and Cryopreservation of Human Sperm

Objective:Sperm preparation techniques and cryopreservation are widely used in assisted reproductive techniques(ART).How to improve the quality of sperm management is a matter of great concern.Phospholipase C-zeta(PLCζ)is considered a sperm-specific agent that activates oocyte activation and thus playing a crucial role in male fertility.However,the potential mechanisms by which semen processing and cryopreservation on PLCζcontribute to keyhole have not been addressed.Methods:In this study,semen samples were taken from have not been addressed 10 normozoospermic men.Each semen sample was assigned to the following groups:density gradient centrifugation(DGC)as control,microfluidic sorting,and cryopreservation.Sperm parameters of molity,viability,membrane integrity,and intracellular ROS were evaluated during sperm preparation and cryopreservation.The expression of PLCζin human sperm was determined by immunofluorescence and western blotting.Results:The results showed that molity,viability,and membrane integrity decreased in cryopreservation group.Intracellular ROS were also significantly increased compared to the the control group.There was no significant difference between DGC and microfluidic sorting group.Our investigation revealed that total levels of PLCζwere comparable between DGC and microfluidic sorting,but there were significantly reduced levels of PLCζafter cryopreservation as quantified by both immunofluorescenceand immunoblotting.PLCζimmunofluorescence in sperm revealed different PLCζlocalization patterns around the acrosomal(Ac),equatorial(Eq),post-acrosomal(PA)areas of sperm heads,and their combination.The predominant patterns of PLCζlocalization in DGC were similar to that of microfluidic sorting,with strong,with staining.In contrast,PLCζstaining in freeze-thawed sperm was considerably weaker fluorescence intensity.Conclusion:This study clarified the mechanism of sperm preparation and cryopreservation underlying effect on sperm characteristic,accompanied with PLCζexpresion.We demonstrated that microfluidic sorting provides a highly efficient preparation method for clinical selection of PLCζ-expressing sperm comparable to DGC gene expression.It is suggested that the cryopreservation of sperm has a significant detrimental effect on PLCζ.

微流控粒子分选中圆形微凹槽容纳特性研究

微流控技术由于具备操控微通道中微小体积流体的能力,已成为操控粒子和细胞的新平台.基于粒子惯性迁移和微凹槽涡胞捕获的粒子分选方法,是一种重要的微流控粒子操控技术.目前,微凹槽容纳的粒子数量不高,制约了该方法的效率.为了提高微凹槽粒子容量,对圆形微凹槽进行结构设计,并利用高速显微成像技术和数值模拟,研究了不同圆形微凹槽的粒子容纳能力.研究发现,相同入口雷诺数(Re=37~555)下,带底腔的圆形微凹槽相较于普通圆形微凹槽容纳的粒子数量提升了45%,这是因为增加底腔后使得涡流线向下延展,形成更深的“U形”结构,可以容纳更多的粒子;当Re=482时,带底腔的直径500μm的圆凹槽比直径600μm的圆凹槽的粒子容量提高了93.9%,原因是前者凹槽内粒子运动轨道与流线更加吻合,粒子轨道面积与凹槽面积占比达到了97%;随着Re增加,从侧通道收集到的粒子富集浓度整体呈现先缓慢增大后减小的趋势,收集到的20μm粒子的最大富集浓度为初始悬浮液的126.7倍;不同微凹槽内粒子群的轨道运动受到涡流场特性、粒子物性及粒子间相互作用和壁面限制作用等因素的共同影响.研究结果对微凹槽结构设计和提高粒子分选性能有重要指导意义.

流式细胞仪样品预处理系统的设计与实现

根据流式细胞仪对样品“均一化”的需求,研制了一套基于微流控细胞分选技术的流式细胞仪样品预处理系统,装置可以实现对粒径小于100µm细胞或微粒的驱动和分选.系统主要包含微流控分选芯片和样品驱动模块两部分,通过聚焦不同位置,实现了对不同粒径细胞/颗粒的有效分离.系统无需对细胞进行标记处理,经分选的细胞便于后续流式细胞仪检测.经验证,系统能够有效去除牡蛎血淋巴细胞样品中的大粒子杂质,提高细胞样品的稳定性和均一性,增加流式细胞仪检测结果的准确性.

深度学习驱动的液滴微流控单细胞分选系统

单细胞操作和分析对于研究许多基本生物学过程和揭示细胞异质性至关重要,并且在生物医学领域具有巨大的应用潜力。液滴微流控技术在单细胞分析方面具有显著优势。研制了一种深度学习驱动的液滴微流控单细胞分选系统,主要以液滴内所包含的生物样本种类以及数量作为标准分选目标液滴。根据实验需求制作好相应生物样本的数据集,在服务器上训练好对应的网络模型,并将该网络模型转移到NVIDIA Jetson TX2开发板上,利用该网络模型对实验过程中拍摄到的液滴图像进行实时检测判断,最后根据算法对包含特定物质的液滴进行分选,从而得到目标液滴。此方法能够有效地判断并分选出液滴内图像特征有差异的不同生物样本,可以实现对包含单个及2个细胞液滴的分选。该研究为液滴微流控单细胞分选技术在生物学和医学等领域的广泛应用提供了支撑。

基于微流控芯片的循环肿瘤细胞检测系统研究

目的实现人体外周血液中循环肿瘤细胞(circulating tumor cells,CTCs)的高通量、高检出率的分选富集与检测,为癌症患者提供高效、精准的早期筛查。方法设计一款分选富集与检测集成化的微流控芯片,采用荧光检测系统进行单细胞水平的CTCs检测,得到样本中的CTCs数量。结果使用裂解红细胞后的外周血液样本进行6次实验,在进样速度达到0.2 mL/h时,CTCs能够达到78.6%的最佳检测率,组内相关系数达到0.8以上。结论CTCs检测系统能够实现较高的检出率并具有良好的可靠性,可以为相关领域的临床研究提供可靠的参考。

微流控技术在过继细胞免疫治疗中的应用研究进展

过继细胞疗法(ACT)是一种个体化疗法,通过采集患者自身或供体的T细胞,在体外进行基因增强后重新引入患者体内,以对抗特定的癌细胞或感染因子。然而,ACT存在许多的局限性,如多步骤耗时且成本高昂等。微流控技术(MT)是以微观通道流动为基础,借助微加工技术制造的微流体芯片和微阀门系统进行分析和操作的技术。微流控平台具有集成度高、效率高、高通量、样品需求低等优点,能够有效简化ACT制备步骤,降低生产成本,探索药物开发过程中的多种因素,以及模拟肿瘤微环境。现对MT在ACT疗法中的细胞分选、细胞增殖和临床前测定及药物评估方面以及在其他肿瘤免疫治疗中的应用进行综述。

A nano-liter droplet-based microfluidic reactor serves as continuous large-scale production of inorganic perovskite nanocrystals

Lead halide perovskite nanocrystals(NCs)exhibit high photoluminescence quantum yield(PLQY),high defect tolerance,narrow half peak width,and wide luminous gamut,making them the ideal optoelectronic materials in numerous fields.Nonetheless,their production still suffers from the limited productivity at the bench level.In this work,we fabricated CsPbX3(X=Cl,Br,I)NCs within droplet-based micro-reactors,where both the nucleation and growth processes could be precisely controlled inside 130-nL microdroplets.This provides a new paradigm for the large-scale synthesis of perovskite NCs with high PLQY.Compared with other synthetic methods,this method can increase the concentration of reactant precursors by 3±116 times,while lowering the ligand to reactant ratio to 2%±50%of the commonly used hot-injection method.By modulating the reaction temperature and residence time,the structure-function relationship between the morphology of NCs and PL properties was extensively investigated.The microfluidic-based process allows the flexible adjustment in the proportion of PbX2 precursors to achieve the fabrication of perovskite NCs whose luminescence range covers the entire visible spectrum(406±677 nm)within one reaction.Finally,perovskite NCs with different halide ions were encapsulated in polymethyl methacrylate to prepare a colored light-emitting diode strip.

流式细胞分选仪研究进展与展望

流式细胞技术一直是生物细胞研究的重要手段,近年来流式细胞分选仪在细胞富集、筛选后再利用等方面的应用使得对能够满足高通量、高细胞信息提取、高细胞活性要求的流式细胞分选仪的需求增加。本文阐述基于光学探测方法的流式细胞分选仪结构、工作原理及其发展现状出发;提出在光学流式细胞分选仪研究领域出现无标记化和微流控芯片化两种新的发展趋势。在两种发展趋势下,无标记成像方法、微流控片上分选系统和片上压电致动器集成分选等新兴技术越来越多地应用到光学流式细胞分选仪的研发设计中,本文从技术特性、性能优势、应用前景等方面对这些技术的研究进展以及面临的挑战进行了总结和展望,供流式细胞分选技术的使用者及研究者参考。

微液滴高通量筛选方法的研究与应用进展

在单细胞层面对生物功能进行高通量的分析和分选是对关键基因、元件、途径与细胞工厂进行优化的重要技术。基于微液滴的筛选方法因其低成本、超高通量等优势,已被广泛应用于生物、医药、食品和工业等各个领域。本文针对目前主流的荧光激活的液滴分选、吸光度激活的液滴分选,以及无标记液滴分选等微液滴筛选设备的进展进行综述,主要包括基于质谱、拉曼、核磁共振、电化学、图像识别等。并总结了近年来微液滴筛选设备在酶进化、微生物育种等领域应用成功的案例。此外还对不同的微液滴筛选设备的优势与面临的挑战进行了讨论,未来各种新的荧光探针的开发以及质谱等非标记检测方法的进一步发展,将是微液滴筛选设备的主要发展方向,在蛋白质工程、抗体工程、细胞分选及临床研究等方面具有重要的应用潜力。

AI-assisted cell identification and optical sorting[Invited]

Cell identification and sorting have been hot topics recently.However,most conventional approaches can only predict the category of a single target,and lack the ability to perform multitarget tasks to provide coordinate information of the targets.This limits the development of high-throughput cell screening technologies.Fortunately,artificial intelligence(AI)systems based on deep-learning algorithms provide the possibility to extract hidden features of cells from original image information.Here,we demonstrate an AI-assisted multitarget processing system for cell identification and sorting.With this system,each target cell can be swiftly and accurately identified in a mixture by extracting cell morphological features,whereafter accurate cell sorting is achieved through noninvasive manipulation by optical tweezers.The AI-assisted model shows promise in guiding the precise manipulation and intelligent detection of high-flux cells,thereby realizing semiautomatic cell research.

利用激光在微流控芯片中实现细胞分选的研究

光学操纵由于其无机械损伤的特点在生物医学微芯片中的应用较为广泛,可实现细胞培养、捕获、分选和检测等功能。采用COMSOL Multiphysics仿真分析软件,利用光学操纵,在微通道结构中,模拟细胞群随鞘流的流动并分析细胞受激光力后的运动过程。选用聚苯乙烯微球进行鞘流实验,在100mw激光器的作用下,观察粒子在微流控芯片中的运动情况。结果表明,细胞在鞘流中的偏转受其物理尺寸和激光功率的影响。同时聚苯乙烯实验结果与模拟实验结果相符。根据模拟结果,可以确定用于操纵不同直径细胞的激光功率大小。利用COMSOL Multiphysics建立的细胞操纵模型可进行集成光流控细胞分选,并为光流控细胞分选实验提供参考。

Numerical Study on Separation of Circulating Tumor Cell Using Dielectrophoresis in a Four-Electrode Microfluidic Device

This numerical study proposes a cell sorting technique based on dielectrophoresis(DEP)in a microfluidic chip.Under the joint effect of DEP and fluid drag,white blood cells and circulating tumor cells are separated because of different dielectric properties.First,the mathematical models of device geometry,single cell,DEP force,electric field,and flow field are established to simulate the cell motion.Based on the simulation model,important boundary parameters are discussed to optimize the cell sorting ability of the device.A proper matching relationship between voltage and flow rate is then provided.The inlet and outlet conditions are also investigated to control the particle motion in the flow field.The significance of this study is to verify the cell separating ability of the microfluidic chip,and to provide a logistic design for the separation of rare diseased cells.

一种微流控细胞分离芯片及其流场分析

微流控细胞分离芯片中流场的分布对混合细胞的有效分离有非常重要的意义,是细胞分离芯片结构设计的一个重要因素。本文设计了一种细胞分离芯片,用于将细胞融合实验后的融合细胞从大量未融合细胞中自动分离出来。为进一步研究芯片中流场分布与芯片结构的关系,利用CFD分析软件Fluent对微流控细胞分离芯片中的流场进行仿真分析,得到其流速分布。针对微通道中的流动情况实验分析表明,实验与仿真结果相比误差在10%以内,证明了CFD软件仿真在细胞分离芯片结构的设计中的指导作用。通过对仿真结果的对比分析,选择了一个优化的芯片结构模型并制作了Y型通道的微流控分离芯片。

微流控芯片流式细胞术

流式细胞术在单细胞分析领域应用广泛,但常规仪器体积大、价格昂贵。基于微机电(MEMS)技术的微流控芯片流式细胞术具有耗样少、集成度高、体积小等特点,可以更好地实现细胞操控、检测与分选。综述了微流控芯片流式细胞术,包括样品聚焦、信号检测、细胞分选等技术的研究进展。

微流控芯片技术在循环肿瘤细胞分离中的研究进展

循环肿瘤细胞(circulating tumor cells,CTCs)是指从原发肿瘤或转移灶脱落、发生上皮-间质转化进入患者外周血血液循环的恶性肿瘤细胞.CTCs在肿瘤研究和临床诊断上的作用逐渐得到认可,外周血中CTCs存在与否以及数量多少不但可以用于肿瘤的早期诊断,还可以用于评估肿瘤预后、监测肿瘤的转移和复发.微流控芯片作为一个高通量、小型化的细胞实验平台,已被应用于CTCs的分选当中.本文综述了用于CTCs捕获的微流控芯片系统的最新研究进展,着重介绍各类芯片的捕获原理、芯片结构和捕获效率,最后对微流控芯片技术在CTCs分选中的应用前景进行了展望.