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 deep-reinforcement learning approach for optimizing homogeneous droplet routing in digital microfluidic biochips

Over the past two decades,digital microfluidic biochips have been in much demand for safety-critical and biomedical applications and increasingly important in point-of-care analysis,drug discovery,and immunoassays,among other areas.However,for complex bioassays,finding routes for the transportation of droplets in an electrowetting-on-dielectric digital biochip while maintaining their discreteness is a challenging task.In this study,we propose a deep reinforcement learning-based droplet routing technique for digital microfluidic biochips.The technique is implemented on a distributed architecture to optimize the possible paths for predefined source–target pairs of droplets.The actors of the technique calculate the possible routes of the source–target pairs and store the experience in a replay buffer,and the learner fetches the experiences and updates the routing paths.The proposed algorithm was applied to benchmark suitesⅠand Ⅲ as two different test benches,and it achieved significant improvements over state-of-the-art techniques.

DMF-scMT-seq linking methylome and transcriptome within single cells with digital microfluidics

Single-cell joint analysis of methylome and transcriptome reveals how the methylation regulates the transcriptional activity.However,traditional bench-top protocols for single-cell DNA methylation and RNA transcription co-detection are laborintensive,cost-ineffective and contaminant-prone.Herein,we establish the DMF-sc MT-seq,a highly-efficient and cost-effective method to simultaneously analyze single-cell DNA methylation and transcriptional activity based on digital microfluidics.DMFsc MT-seq automates the workflow of single-cell isolation,cellular hypotonic lysis,nucleic acid separation and methylome/transcriptome library construction in a contactless and addressable way.The system ensures high accuracy(R>0.85),high gene detection ability(14,697 genes per cell at 4 million sequencing depth),and high CpG coverage(677,198 CpG sites per cell at 1million sequencing depth).By using DMF-sc MT-seq,the relationship of DNA methylation and RNA transcription under different genomic contexts is resolved.We further apply DMF-sc MT-seq to study the dynamics of transcription regulation with methylation-inhibiting anti-tumor Decitabine,and identify the methylated promoter/gene body driven genes in response to Decitabine treatment.DMF-sc MT-seq facilitates the construction of the correlation of DNA methylation and transcriptional activity at the single-cell level in a flexible,sensitive and accurate way,which is anticipated to be a powerful tool in studying single-cell biological systems.

Numerical Study of the Evaporation Effects on the Microdroplet Dynamics in Digital Microfluidic Biochips

In this paper, an electrohydrodynamic approach is used to model and study dynamics of evaporating microdroplets in digital microfluidic systems. A numerical eleetrohydrodynamic approach is used to calculate the driving force and shear force （due to the walls）. Effects of contact line pinning is considered by adding a three-phase contact line force, and also considering dynamic contact angle which modifies the mierodroplet boundary conditions. Since air is used as the filler fluid, the drag force is neglected. Although energy equation is not solved （constant temperature assumption）, effects of the evaporation is considered from two aspects： It is shown that an additional force is needed to balance the dynamic equation of the mierodroplet motion. Also, at each time step the microdroplet interface has to be deformed due to the change in the microdroplet radius. Important findings of the proposed model includes the transient velocity and displacement of the microdroplet as well as the driving and opposing forces acting on the microdroplet as functions of time. It is shown that mass loss due to evaporation tends to accelerate the droplet; whereas the competitive effect of the reduced driving force decelerates the droplet at the end of motion. The modeling results indicate that evaporation plays a crucial role in microdroplet motion by changing the force balance and the microdroplet boundary condition.

Automatic measurement of three-phase contact angles in pore throats based on digital images

With the help of digital image processing technology, an automatic measurement method for the three-phase contact angles in the pore throats of the microfluidic model was established using the microfluidic water flooding experiment videos as the data source. The results of the new method were verified through comparing with the manual measurement data.On this basis, the dynamic changes of the three-phase contact angles under flow conditions were clarified by the contact angles probability density curve and mean value change curve. The results show that, for water-wetting rocks, the mean value of the contact angles is acute angle during the early stage of the water flooding process, and it increases with the displacement time and becomes obtuse angle in the middle-late stage of displacement as the dominant force of oil phase gradually changes from viscous force to capillary force. The droplet flow in the remaining oil occurs in the central part of the pore throats, without three-phase contact angle. The contact angles for the porous flow and the columnar flow change slightly during the displacement and present as obtuse angles in view of mean values, which makes the remaining oil poorly movable and thus hard to be recovered. The mean value of the contact angle for the cluster flow tends to increase in the flooding process, which makes the remaining oil more difficult to be recovered. The contact angles for the membrane flow are mainly obtuse angles and reach the highest mean value in the late stage of displacement, which makes the remaining oil most difficult to be recovered. After displacement, the remaining oils under different flow regimes are just subjected to capillary force, with obtuse contact angles, and the wettability of the pore throat walls in the microfluidic model tends to be oil-wet under the action of crude oil.

基于DMF直扩系统捕获性能的研究

对以数字匹配滤波器(DMF)为核心的捕获电路的性能进行了深入研究,推导出单次、两次驻留判决方式的平均捕获时间的表达式,并进行了数值分析。得出的主要结论是:基于DMF的捕获电路具有很快的捕获速度,适合于猝发通信;低信噪比下,捕获速度对门限更为敏感,且两次驻留判决在低信噪比下略好于单次驻留判决。

基于DMF捕获系统频率选择性信道下捕获性能的分析

对基于数字匹配滤波器(DMF)捕获系统在频率选择性信道下的捕获性能进行了深入讨论,利用状态转移图推导出单次驻留判决方式时平均捕获时间的表达式,对平均捕获时间与多径分量的关系进行了数字分析。得出的主要结论是对于多径分量为非衰落信号时,多径分量之间的能量差别越大,系统捕获性能越好,多径分量的能量相等时,系统捕获性能最差,且随着可以分离的多径路数的增加下降;对于多径分量为慢衰落信号时,多径信道的捕获性能好于单径信道(非频率选择性衰落信道),且多径分量之间的能量差别越大,系统捕获性能越差,当多径分量的能量相等时,捕获性能最好,这与非衰落信道的情形相反。因此在频率选择性衰落信道中,采用分集接收可以改善系统的捕获性能。

基于CNN-LSTM的数字微流控系统液滴运动时间预测模型

由于数字微流控芯片在制造和使用过程中可能存在潜在故障,并且芯片的应用领域对安全性要求极高,因此需要对芯片进行全面的测试以确保其可靠性和安全性。提出融合卷积神经网络(CNN)算法和长短期记忆(LSTM)神经网络的预测模型。利用机器视觉技术获取数字微流控驱动控制系统数据,基于CNN-LSTM模型对数字微流控系统液滴运动时间变化进行预测及相关性分析,并与传统的LSTM神经网络模型进行对比。实验结果表明,在驱动电压90 V下,CNN-LSTM模型在预测精度上明显优于LSTM神经网络模型,决定系数(R^(2))达到0.9543,均方误差(MSE)仅为6.5301,平均绝对误差(MAE)为1.9179,平均绝对百分比误差(MAPE)为5.0520,预测值与实验值之间的拟合程度高。结果表明,CNN-LSTM的预测准确率高于传统的LSTM神经网络模型,能有效识别实验器件健康状态、较好预测潜在故障时间,具有有效性和优越性。

Microfluidic bubble-generator enables digital light processing 3D printing of porous structures

Three-dimensional(3D)printing is an emerging technique that has shown promising success in engineering human tissues in recent years.Further development of vatphotopolymerization printing modalities has significantly enhanced the complexity level for 3D printing of various functional structures and components.Similarly,the development of microfluidic chip systems is an emerging research sector with promising medical applications.This work demonstrates the coupling of a digital light processing(DLP)printing procedure with a microfluidic chip system to produce size-tunable,3D-printable porosities with narrow pore size distributions within a gelatin methacryloyl(GelMA)hydrogel matrix.It is found that the generation of size-tunable gas bubbles trapped within an aqueous GelMA hydrogel-precursor can be controlled with high precision.Furthermore,the porosities are printed in two-dimensional(2D)as well as in 3D using the DLP printer.In addition,the cytocompatibility of the printed porous scaffolds is investigated using fibroblasts,where high cell viabilities as well as cell proliferation,spreading,and migration are confirmed.It is anticipated that the strategy is widely applicable in a range of application areas such as tissue engineering and regenerative medicine,among others.

基于微流控平台的SARS-CoV-2核酸检测的研究进展

介绍了微流控技术的检测原理,综述了基于微流控平台的实时荧光定量逆转录聚合酶链式反应(reverse transcription quantitative real-time polymerase chain reaction,RT-qPCR)、数字PCR、等温扩增、成簇规律间隔短回文重复序列及其相关蛋白等技术应用于的SARS-CoV-2核酸检测的研究现状,分析了基于微流控平台的SARS-CoV-2核酸检测的局限性,指出了基于微流控的SARS-CoV-2核酸检测系统在特异度、敏感度、检测限、重复性、信息化、质量控制及试剂成本等方面仍需要进一步的优化改进和临床验证。

A 3D-printed microfluidic gradient concentration chip for rapid antibiotic-susceptibility testing

The rise of antibiotic resistance as one of the most serious global public health threats has necessitated the timely clinical diagnosis and precise treatment of deadly bacterial infections.To identify which types and doses of antibiotics remain effective for fighting against multi-drug-resistant pathogens,the development of rapid and accurate antibiotic-susceptibility testing(AST)is of primary importance.Conventional methods for AST in well-plate formats with disk diffusion or broth dilution are both labor-intensive and operationally tedious.The microfluidic chip provides a versatile tool for evaluating bacterial AST and resistant behaviors.In this paper,we develop an operationally simple,3D-printed microfluidic chip for AST which automatically deploys antibiotic concentration gradients and fluorescence intensity-based reporting to ideally reduce the report time for AST to within 5 h.By harnessing a commercially available,digital light processing(DLP)3D printing method that offers a rapid,high-precision microfluidic chip-manufacturing capability,we design and realize the accurate generation of on-chip antibiotic concentration gradients based on flow resistance and diffusion mechanisms.We further demonstrate the employment of the microfluidic chip for the AST of E.coli to representative clinical antibiotics of three classes:ampicillin,chloramphenicol,and kanamycin.The determined minimum inhibitory concentration values are comparable to those reported by conventional well-plate methods.Our proposed method demonstrates a promising approach for realizing robust,convenient,and automatable AST of clinical bacterial pathogens.

Microfluidics-based strategies for molecular diagnostics of infectious diseases

Traditional diagnostic strategies for infectious disease detection require benchtop instruments that are inappropriate for point-of-care testing(POCT). Emerging microfluidics, a highly miniaturized, automatic, and integrated technology,are a potential substitute for traditional methods in performing rapid, low-cost, accurate, and on-site diagnoses.Molecular diagnostics are widely used in microfluidic devices as the most effective approaches for pathogen detection.This review summarizes the latest advances in microfluidics-based molecular diagnostics for infectious diseases from academic perspectives and industrial outlooks. First, we introduce the typical on-chip nucleic acid processes,including sample preprocessing, amplification, and signal read-out. Then, four categories of microfluidic platforms are compared with respect to features, merits, and demerits. We further discuss application of the digital assay in absolute nucleic acid quantification. Both the classic and recent microfluidics-based commercial molecular diagnostic devices are summarized as proof of the current market status. Finally, we propose future directions for microfluidics-based infectious disease diagnosis.

基于微流控技术的数字PCR的发展及其应用

作为一种新兴的扩增检测技术,基于微流控的数字聚合酶链式反应(PCR)有着高通量、高灵敏度及高耐受性等优势,因而得到了研究者的广泛关注,其相关技术也在不断的发展中。综述了基于微流控的数字PCR的研究进展,重点讨论了基于微流控的数字PCR的液滴打印技术、多层微流控芯片技术、微珠技术、聚二甲基硅氧烷(PDMS)技术等的不同实现方式。介绍了基于微流控的数字PCR在定量检测、精准分子诊断、肿瘤个性化诊断以及食品安全检测中的应用。最后,对基于微流控的数字PCR技术目前存在的不足和问题进行了阐述,并对其未来的研究方向和发展趋势进行了展望。

大视场dPCR芯片荧光检测系统的设计与优化

数字PCR(dPCR)技术作为传统PCR技术的更新迭代,有着绝对定量的特点,在新冠病毒核酸序列检测、癌细胞探测等生物学领域有着巨大的潜力。现有的dPCR荧光检测系统在检测通量、检测速度以及成本之间难以做出合理的权衡。由于成像视野小、需多次图像拼接极大影响了dPCR荧光检测系统效率和时间。文章构建了一套采用大视场荧光显微设计的多通道dPCR检测系统实验平台,依托该实验平台,运用Zemax软件优化结构,改进显微成像物镜设计,能够实现直径18mm的全视场范围FAM、HEX、ROX三种荧光通道分辨率在6μm的多通道成像。采用随机霍夫变换算法(RHT)图像处理分析方法,对常规均匀“圆孔”和新型“雪花”、“树枝”等非均匀结构的微流控荧光芯片,均能实现高效检测,得到清晰、稳定的荧光图像。

基于数字图像处理的孔喉内三相接触角自动测量方法

借助数字图像处理技术,以微流控水驱油实验视频为数据源,建立了微流控模型孔喉内三相接触角的自动批量测量计算方法;采用人工测量数据验证了新方法测量结果的可靠性,在此基础上,利用接触角概率密度曲线、均值变化曲线揭示了三相接触角在流动条件下的动态变化规律。研究表明:对于水湿岩石,水驱油过程中,驱替前期接触角均值以锐角为主,随驱替时间的延长,油相受力逐渐由黏滞力为主导向毛细管力为主导过渡,接触角均值不断增大,至驱替中后期以钝角为主。剩余油中的滴状流赋存于孔喉通道中央,不存在三相接触角;多孔流和柱状流在整个驱替过程中接触角变化幅度较小且均值皆为钝角,该类剩余油形成后流动性差,动用难度较大;簇状流在驱替过程中三相接触角均值整体呈上升趋势,剩余油动用难度逐渐升高;膜状流整体呈钝角,水驱后期接触角均值最大,动用难度最大。驱替结束后,不同流态剩余油仅受毛细管力作用的影响,接触角均为钝角,在原油作用下模型孔喉壁润湿性呈油湿特征。

An automated nucleic acid detection platform using digital microfluidics with an optimized Cas12a system

Outbreaks of both influenza virus and the novel coronavirus SARS-CoV-2 are serious threats to human health and life. It is very important to establish a rapid, accurate test with large-scale detection potential to prevent the further spread of the epidemic. An optimized RPA-Cas12a-based platform combined with digital microfluidics(DMF), the RCD platform, was established to achieve the automated, rapid detection of influenza viruses and SARS-CoV-2. The probe in the RPA-Cas12a system was optimized to produce maximal fluorescence to increase the amplification signal. The reaction droplets in the platform were all at the microliter level and the detection could be accomplished within 30 min due to the effective mixing of droplets by digital microfluidic technology. The whole process from amplification to recognition is completed in the chip, which reduces the risk of aerosol contamination. One chip can contain multiple detection reaction areas, offering the potential for customized detection.The RCD platform demonstrated a high level of sensitivity, specificity(no false positives or negatives), speed(≤30 min),automation and multiplexing. We also used the RCD platform to detect nucleic acids from influenza patients and COVID-19 patients. The results were consistent with the findings of q PCR. The RCD platform is a one-step, rapid, highly sensitive and specific method with the advantages of digital microfluidic technology, which circumvents the shortcomings of manual operation. The development of the RCD platform provides potential for the isothermal automatic detection of nucleic acids during epidemics.

基于LabVIEW for Arduino的数字微流控液滴系统研究

针对数字微流控系统成本高和程序可移植性差的问题,文中设计一种基于LabVIEW for Arduino的新型数字微流控系统,该系统包括上位机LabVIEW软件、Arduino单片机、DC-DC升压模块、开关三极管、电极板,实现软件硬件相结合,其中可以通过调节DC-DC升压模块的输出电压,实现0~600 V直流电压连续调节。经过实验测得,当电压为400 V时,液滴移动速度为1.7 mm/s;电压为500 V时,液滴移动速度为3.5 mm/s。当频率为50 Hz时,液滴移动速度为1.5 mm/s;频率为100 Hz时,液滴移动速度为0.9 mm/s,液滴操控效果明显。与传统数字微流控系统相比,该系统具有成本低、可移植性好等优点,对于提升数字微流控系统性能具有重要意义。

基于数字微流控芯片平台进行动物源性成分快速初筛的应用研究

基于数字微流控芯片平台并结合聚合酶链反应法(PCR),对鸡、鸭、鹅、猪、牛、羊6项动物源性成分进行快速初筛检测,利用GB/T 38164—2019和SN/T 2051—2008分析方法对初筛有问题的样品进行确证检测。实验对随机购买的市售牛肉、羊肉、鸭血(共计100批次)样品进行快速检测,发现14批问题样品,确证检测后发现有3组样品检测结果存在偏差,引起偏差的原因可能是确证检测法较快速初筛检测法的检出限更低。实验结果表明,数字微流控芯片平台初筛检测法具有快速、即时、高效、成本低的优势,便于现场进行大批量筛查检测。

数字化微喷射用直列微喷嘴制作工艺

为解决玻璃微管道拉制工艺制作的单孔微喷嘴存在容易脆断,微管道长度不易控制,微喷射效率低等问题,提出了一种新的微喷嘴制作工艺。该工艺延续了微喷嘴拉制工艺中非IC工艺和低成本的制作特点,仍以低流阻的圆截面为喷口形状,通过材料流变运动和冷却时收缩率不同,将多根微管道紧密嵌入到塑料管中;通过毛细腐蚀作用,制作出变内径的锥形管,并实现微喷管长度的控制。制作了内径50μm的4孔微喷嘴和10孔微喷嘴,锥管长为750μm,微喷管长2.5mm。使用制作的微喷嘴在微流体数字化驱动平台上进行了水性液体在油相和气相中的微喷射实验,制备了80μm的微滴和微滴阵列。实验表明,该工艺制作的直列微喷嘴可以在微流体数字化驱动下实现较好的数字化微喷射效果。

微流体数字化技术制备基因芯片微阵列

采用以脉冲为微流动基本形态、脉冲当地惯性力为主动力的微流体数字化技术进行了基因芯片微阵列制备实验。在搭建的基于微流体数字化技术的基因芯片微阵列制备系统上,实验验证了脉冲点样系统参量(收敛角2θ、微喷嘴内径d、电压幅值U和驱动频率f)对样点直径和脉冲点样稳定性的影响规律。以实验规律为依据,提出了制备样点直径约为100μm的中等密度微阵列的实验路线,制备出了样点平均直径为102.2μm、微阵列密度约为4 000spot/cm2的基因芯片微阵列(点样溶液为3×SSC柠檬酸盐缓冲液)。得到的研究结果可为建立高密度基因芯片脉冲点样技术提供实验研究基础。