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.

Effects of gas compressibility and surface roughness on the flow in microfluidic devices

With the development of the micro-electro-mechanical system (MEMS),the flow characteristics in micro-channels have drawn increasing attention.In this paper,numerical simulations are conducted to investigate the flow characteristics of compressible flow through micro-channels and micronozzles.An improved surface roughness viscosity model is used to simulate the effect of surface roughness on micro-channels flow characteristics.Using this model,better agreements between the computational results and the experimental data are found.The result indicates that the surface roughness is one of the important factors affecting the flow characteristics of gas through micro-channels.The numerical investigation on the expansion channel shows that by using the laminar model that considers surface roughness,the computational results and experimental data are consistent when Re<450,whereas deviation increases when Re>450.Based on the synthetic model with considerations of turbulence viscosity and surface roughness,the numerical results and the experimental data are identical.

Surface-imprinted polymers in microfluidic devices

Molecularly imprinted polymers are generated by curing a cross-linked polymer in the presence of a template. During the curing process, noncovalent bonds form between the polymer and the template. The interaction sites for the noncovalent bonds become "frozen" in the cross-linking polymer and maintain their shape even after the template is removed. The resulting cavities reproduce the size and shape of the template and can selectively reincorporate the template when a mixture containing it flows over the imprinted surface. In the last few decades the field of molecular imprinting has evolved from being able to selectively capture only small molecules to dealing with all kinds of samples. Molecularly imprinted polymers (MIPs) have been generated for analytes as diverse as metal ions, drug molecules, environmental pollutants, proteins and viruses to entire cells. We review here the relatively new field of surface imprinting, which creates imprints of large, biologically relevant templates. The traditional bulk imprinting, where a template is simply added to a prepolymer before curing, cannot be applied if the analyte is too large to diffuse from the cured polymer. Special methods must be used to generate binding sites only on a surface. Those techniques have solved crucial problems in separation science as well as chemical and biochemical sensing. The implementation of imprinted polymers into microfluidic chips has greatly improved the applicability of microfluidics. We present the latest advances and different approaches of surface imprinting and their applications for microfluidic devices.

Droplet Pattern Formation and Translation in New Microfluidic Flow- Focusing Devices

We conducted experiments on specially designed microfluidic chips that generate droplets through a microfluidic ow-focusing approach. The fluid flow in the microfluidic channel produced a shear flow field at low Reynolds numbers. The droplets in the microfluidic system exhibited special droplet pattern formations similar to periodic crystal-like lattices because of the competition between shear forces and surface tension. By adjusting the flow rate ratio of the water （droplet phase） to oil （continuous phase） phases and changing the outlet channel widths, the droplets formed monolayer dispersion to double-layer formation to monolayer squeezing when the outlet channel widths were 250 or 300 μm. We also obtained droplets with monolayer dispersion, three-layer arrangements, double-layer squeezing, and monolayer squeezing when the outlet channel width was 350 μm. The outlet channel width was increased to 400 μm, and four-layer arrangements were observed. We also studied the translation of droplet formation, which resulted in a detailed strategy to control drop size and droplet pattern formation for emulsi cation in microfluidic devices. We expect that our strategy can provide theoretical guidance to synthesize dispersion or polydisperse colloid particles.

Microfluidic devices as model platforms of CNS injury-ischemia to study axonal regeneration by regulating mitochondrial transport and bioenergetic metabolism

Central nervous system(CNS)neurons typically fail to regenerate their axons after injury leading to neurological impairment.Axonal regeneration is a highly energy-demanding cellular program that requires local mitochondria to supply most energy within injured axons.Recent emerging lines of evidence have started to reveal that injury-triggered acute mitochondrial damage and local energy crisis contribute to the intrinsic energetic restriction that accounts for axon regeneration failure in the CNS.Characterizing and reprogramming bioenergetic signaling and mitochondrial maintenance after axon injury-ischemia is fundamental for developing therapeutic strategies that can restore local energy metabolism and thus facilitate axon regeneration.Therefore,establishing reliable and reproduc-ible neuronal model platforms is critical for assessing axonal energetic metabolism and regeneration capacity after injury-ischemia.In this focused methodology article,we discuss recent advances in applying cutting-edge microflu-idic chamber devices in combination with state-of-the-art live-neuron imaging tools to monitor axonal regeneration,mitochondrial transport,bioenergetic metabolism,and local protein synthesis in response to injury-ischemic stress in mature CNS neurons.

In vitro engineered models of neurodegenerative diseases

Neurodegeneration is a catastrophic process that develops progressive damage leading to functional andstructural loss of the cells of the nervous system and is among the biggest unavoidable problems of our age.Animalmodels do not reflect the pathophysiology observed in humans due to distinct differences between the neuralpathways,gene expression patterns,neuronal plasticity,and other disease-related mechanisms in animals andhumans.Classical in vitro cell culture models are also not sufficient for pre-clinical drug testing in reflecting thecomplex pathophysiology of neurodegenerative diseases.Today,modern,engineered techniques are applied to developmulticellular,intricate in vitro models and to create the closest microenvironment simulating biological,biochemical,and mechanical characteristics of the in vivo degenerating tissue.In THIS review,the capabilities and shortcomings ofscaffold-based and scaffold-free techniques,organoids,and microfluidic models that best reflect neurodegeneration invitro in the biomimetic framework are discussed.

Microfluidics-based assay on the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors

Abstract A new microfluidic system with four different microchambers （a circle and three equilateral concave polygons） was designed and fabricated using poly（dimethylsiloxane） （PDMS） and the soft lithography method. Using this microfluidic device at six flow rates （5, 10, 20, 30, 40, and 50 μL/h）, the effects of microenvironmental geometry and aqueous flow on bacterial adhesion behaviors were investigated. Escherichia coli HB101 pGLO, which could produce a green fluorescent protein induced by L-arabinose, was utilized as the model bacteria. The results demonstrated that bacterial adhesion was significantly related to culture time, microenvironment geometry, and aqueous flow rates. Adhered bacterial density increased with the culture time. Initially, the adhesion occurred at the microchamber sides, and then the entire chamber was gradually covered with increased culture time. Adhesion densities in the side zones were larger than those in the center zones because of the lower shearing force in the side zone. Also, the adhesion densities in the complex chambers were larger than those in the simple chambers. At low flow rates, the orientation of adhered bacteria was random and disorderly. At high flow rates, bacterial orientation became close to the streamline and oriented toward the flow direction; All these results implied that bacterial adhesion tended to occur in complicated aqueous flow areas.The present study provided an on-chip flow system for physiological behavior of biological cells, as well as provided a strategic cue for the prevention of bacterial infection and biofilm formation.

Current overview of induced pluripotent stem cell-based blood-brain barrier-on-a-chip

BACKGROUND Induced pluripotent stem cells(iPSCs)show great ability to differentiate into any tissue,making them attractive candidates for pathophysiological investigations.The rise of organ-on-a-chip technology in the past century has introduced a novel way to make in vitro cell cultures that more closely resemble their in vivo environments,both structural and functionally.The literature still lacks consensus on the best conditions to mimic the blood-brain barrier(BBB)for drug screening and other personalized therapies.The development of models based on BBB-on-achip using iPSCs is promising and is a potential alternative to the use of animals in research.AIM To analyze the literature for BBB models on-a-chip involving iPSCs,describe the microdevices,the BBB in vitro construction,and applications.METHODS We searched for original articles indexed in PubMed and Scopus that used iPSCs to mimic the BBB and its microenvironment in microfluidic devices.Thirty articles were identified,wherein only 14 articles were finally selected according to the inclusion and exclusion criteria.Data compiled from the selected articles were organized into four topics:(1)Microfluidic devices design and fabrication;(2)characteristics of the iPSCs used in the BBB model and their differentiation conditions;(3)BBB-on-a-chip reconstruction process;and(4)applications of BBB microfluidic three-dimensional models using iPSCs.RESULTS This study showed that BBB models with iPSCs in microdevices are quite novel in scientific research.Important technological advances in this area regarding the use of commercial BBB-on-a-chip were identified in the most recent articles by different research groups.Conventional polydimethylsiloxane was the most used material to fabricate in-house chips(57%),whereas few studies(14.3%)adopted polymethylmethacrylate.Half the models were constructed using a porous membrane made of diverse materials to separate the channels.iPSC sources were divergent among the studies,but the main line used was IMR90-C4 from human fetal lung fibroblast(41.2%).The cells were differentiated through diverse and complex processes either to endothelial or neural cells,wherein only one study promoted differentiation inside the chip.The construction process of the BBB-on-a-chip involved previous coating mostly with fibronectin/collagen Ⅳ(39.3%),followed by cell seeding in single cultures(36%)or co-cultures(64%)under controlled conditions,aimed at developing an in vitro BBB that mimics the human BBB for future applications.CONCLUSION This review evidenced technological advances in the construction of BBB models using iPSCs.Nonetheless,a definitive BBB-on-a-chip has not yet been achieved,hindering the applicability of the models.

3D tumor model biofabrication

Animal models have been extensively used in cancer pathology studies and drug discovery.These models,however,fail to reflect the complex human tumor microenvironment and do not allow for high-throughput drug screening in more human-like physiological conditions.Three-dimensional(3D)cancer models present an alternative to automated high-throughput cancer drug discovery and oncology.In this review,we highlight recent technology innovations in building 3D tumor models that simulate the complex human tumor microenvironment and responses of patients to treatment.We discussed various biofabrication technologies,including 3D bioprinting techniques developed for characterizing tumor progression,metastasis,and response to treatment.

Axonal regeneration and sprouting as a potential therapeutic target for nervous system disorders

Nervous system disorders are prevalent health issues that will only continue to increase in frequency as the population ages.Dying-back axonopathy is a hallmark of many neurologic diseases and leads to axonal disconnection from their targets,which in turn leads to functional impairment.During the course of many of neurologic diseases,axons can regenerate or sprout in an attempt to reconnect with the target and restore synapse function.In amyotrophic lateral sclerosis(ALS),distal motor axons retract from neuromuscular junctions early in the disease-course before significant motor neuron death.There is evidence of compensatory motor axon sprouting and reinnervation of neuromuscular junctions in ALS that is usually quickly overtaken by the disease course.Potential drugs that enhance compensatory sprouting and encourage reinnervation may slow symptom progression and retain muscle function for a longer period of time in ALS and in other diseases that exhibit dying-back axonopathy.There remain many outstanding questions as to the impact of distinct disease-causing mutations on axonal outgrowth and regeneration,especially in regards to motor neurons derived from patient induced pluripotent stem cells.Compartmentalized microfluidic chambers are powerful tools for studying the distal axons of human induced pluripotent stem cells-derived motor neurons,and have recently been used to demonstrate striking regeneration defects in human motor neurons harboring ALS disease-causing mutations.Modeling the human neuromuscular circuit with human induced pluripotent stem cells-derived motor neurons will be critical for developing drugs that enhance axonal regeneration,sprouting,and reinnervation of neuromuscular junctions.In this review we will discuss compensatory axonal sprouting as a potential therapeutic target for ALS,and the use of compartmentalized microfluidic devices to find drugs that enhance regeneration and axonal sprouting of motor axons.

SERS sensing for cancer biomarker:Approaches and directions

These days,cancer is thought to be more than just one illness,with several complex subtypes that require different screening approaches.These subtypes can be distinguished by the distinct markings left by metabolites,proteins,miRNA,and DNA.Personalized illness management may be possible if cancer is categorized according to its biomarkers.In order to stop cancer from spreading and posing a significant risk to patient survival,early detection and prompt treatment are essential.Traditional cancer screening techniques are tedious,time-consuming,and require expert personnel for analysis.This has led scientists to reevaluate screening methodologies and make use of emerging technologies to achieve better results.Using time and money saving techniques,these methodologies integrate the procedures from sample preparation to detection in small devices with high accuracy and sensitivity.With its proven potential for biomedical use,surface-enhanced Raman scattering(SERS)has been widely used in biosensing applications,particularly in biomarker identification.Consideration was given especially to the potential of SERS as a portable clinical diagnostic tool.The approaches to SERS-based sensing technologies for both invasive and non-invasive samples are reviewed in this article,along with sample preparation techniques and obstacles.Aside from these significant constraints in the detection approach and techniques,the review also takes into account the complexity of biological fluids,the availability of biomarkers,and their sensitivity and selectivity,which are generally lowered.Massive ways to maintain sensing capabilities in clinical samples are being developed recently to get over this restriction.SERS is known to be a reliable diagnostic method for treatment judgments.Nonetheless,there is still room for advancement in terms of portability,creation of diagnostic apps,and interdisciplinary AI-based applications.Therefore,we will outline the current state of technological maturity for SERS-based cancer biomarker detection in this article.The review will meet the demand for reviewing various sample types(invasive and non-invasive)of cancer biomarkers and their detection using SERS.It will also shed light on the growing body of research on portable methods for clinical application and quick cancer detection.

A titanium dioxide nanorod array as a high-affinity nano-bio interface of a microfluidic device for efficient capture of circulating tumor cells

Nanomaterials show promising opportunities to address clinical problems （such as insufficient capture of circulating tumor cells; CTCs） via the high surface area-to-volume ratio and high affinity for biological cells. However, how to apply these nanomaterials as a nano-bio interface in a microfluidic device for efficient CTC capture with high specificity remains a challenge. In the present work, we first found that a titanium dioxide （TiO2） nanorod array that can be conveniently prepared on multiple kinds of substrates has high affinity for tumor cells. Then, the TiO2 nanorod array was vertically grown on the surface of a microchannel with hexagonally patterned Si micropillars via a hydrothermal reaction, forming a new kind of a micro-nano 3D hierarchically structured microfluidic device. The vertically grown TiO2 nanorod array was used as a sensitive nano-bio interface of this 3D hierarchically structured microfluidic device, which showed high efficiency of CTC capture （76.7% ± 7.1%） in an artificial whole-blood sample.

Highly precise Ti/Pt/Cr/Au thin-film temperature sensor embedded in a microfluidic device

A multilayer(Ti/Pt/Cr/Au)resistive temperature sensor was proposed and investigated to precisely measure the temperature characteristic in microfluidic devices.The Ti/Pt/Cr/Au sensor was fabricated by direct current(DC)sputtering,vacuum evaporation and liftoff process.The thermal annealing test was conducted in the temperature range of 200-800℃for obtaining an appropriate property of the multilayer.Based on the experimental results,400℃was selected as the experimental annealing temperature for the Ti/Pt/Cr/Au layer.The redistribution of structural imperfections and recrystallization promote the density and adhesion of multilayer during the annealing process.With the annealing temperature rising,the annealing process leads to through-thickness migration of chromium and partial depletion of the adhesive layer.The Ti also diffuses into the Pt,which makes the interface disappear.Nevertheless,the layer remains continuous.The temperature coefficient of resistance(TCR)of the sensors was investigated through the microfluidic testing system.The excellent stability and sensitivity of the Ti/Pt/Cr/Au thin-film temperature sensor are verified.Furthermore,the capability of the Ti/Pt/Cr/Au thin-film temperature sensor detecting the sudden temperature change caused by bubble effect is very meaningful to the microfluidic devices.

A microfluidic device for accurate detection of hs-cTnI

This device is aimed at ensuring that the sample is uniformly and equivalently reacted with the antibody on the NC membrane in each test when the microfluidic liquid system is introduced to the chip.In this study,the developed microfluidic chip can avoid the presence of the sample and conjugate pads in the chip,while the precision of the chro matography system can be greatly improved using the same particles,NC membrane and antibody alongside the traditional strip.The results,taking the detection of cTnI as an example,revealed that the coefficient of variation(CV)is controlled within 4%,while the maximum record of the contrast chromatographic reagent strip can reach 15%.Additionally,the detection sensitivity can maintain the same order of magnitudes with that of the traditional chromatographic strip.With the results,the determination correlation of the developed microfluidic chip has been greatly improved.In addition,the CV of the chip in this study is greatly improved in comparison with that of the traditional strip.The biggest improvement lies in the mixing between the sample and the microspheres,indicating that this is a new approach to improve the CV of the traditional strip.

Microfluidic Device： A Miniaturized Platform for Chemical Reactions

Microfluidic devices, as a new miniaturized platform stemming from the field of micro-electromechanical sys-tems, have been used in many disciplines. In the field of chemical reactions, microfluidic device-based microreac-tors have shown great promise in building new chemical technologies and processes with increased speed and reli- ability and reduced sample consumption and cost. This technology has also become a new and effective tool for precise, high-throughput, and automatic analysis of chemical synthesis processes. Compared with conventional chemical laboratory batch methodologies, microfluidic reactors have a number of features, such as high mixing ef- ficiency, short reaction time, high heat-transfer coefficient, small reactant volume, controllable residence time, and high surface-to-volume ratio, among others. Combined with recent advances in microfluidic devices for chemical reactions, this review aims to give an overview of the features and applications of microfluidic devices in the field of chemical synthesis. It also aims to stimulate the development of microfluidic device applications in the field of chemical reactions.

Hierarchical Porous Polymer Beads Prepared by Polymerization-induced Phase Separation and Emulsion-template in a Microfluidic Device

Porous polymer beads（PPBs） containing hierarchical bimodal pore structure with gigapores and meso-macropores were prepared by polymerization-induced phase separation（PIPS） and emulsion-template technique in a glass capillary microfluidic device（GCMD）. Fabrication procedure involved the preparation of water-in-oil emulsion by emulsifying aqueous solution into the monomer solution that contains porogen. The emulsion was added into the GCMD to fabricate the（water-in-oil）-in-water double emulsion droplets. The flow rate of the carrier continuous phase strongly influenced the formation mechanism and size of droplets. Formation mechanism transformed from dripping to jetting and size of droplets decreased from 550 μm to 250 μm with the increase in flow rate of the carrier continuous phase. The prepared droplets were initiated for polymerization by on-line UV-irradiation to form PPBs. The meso-macropores in these beads were generated by PIPS because of the presence of porogen and gigapores obtained from the emulsion-template. The pore morphology and pore size distribution of the PPBs were investigated extensively by scanning electron microscopy and mercury intrusion porosimetry（MIP）. New pore morphology was formed at the edge of the beads different from traditional theory because of different osmolarities between the water phase of the emulsion and the carrier continuous phase. The morphology and proportion of bimodal pore structure can be tuned by changing the kind and amount of porogen.

Distance-Based Detection of Ag^(+) with Gold Nanoparticles-Coated Microfluidic Paper

In this study,we developed a microfluidic paper analysis device(μPAD)for distance-based detection of Ag^(+)in water.TheμPAD was manufactured by wax printing method on filter paper.Then,a layer of gold nanoparticles(AuNPs)was deposited and ascorbic acid was printed on the channel.In the detection,Ag^(+)was reduced by ascorbic acid and coated on the surface of the AuNPs on the channel,forming Au@Ag core/shell nanoparticles.Based on the capillary flow principle,diff erent concentrations of Ag^(+)formed diff erent distances of color ribbons.Thus,quantitative detection of Ag^(+)can be achieved by measuring the distance of the color ribbon.The detection limit of this method was as low as 1 mg·L^(-1)within 15 min and the interference of common metal ions in water can be eliminated.In conclusion,this method had successfully realized the leap from colorimetry to direct reading,realizing fast read and easy manipulation with low-cost.

Utilizing a microfluidic device to enrich and fluorescently detect circulating tumor cells

Circulating tumor cells(CTCs)are cancer cells that have propagated from primary tumor sites,spreading into the bloodstream as the cellular origin of fatal metastasis,and to secondary tumor sites.Capturing and analyzing CTCs is a kind of‘‘liquid biopsy'of the tumor that provides information about cancer changes over time and tailoring treatment[1].CTC enrichment and detection remains technologically challenging due to their extremely low concentra-

Ricinus communis agglutinin I functionalisation of poly(methyl methacrylate) (PMMA) as a substrate for microfluidic device

We report a functionalisation strategy which is able to generate Ricinus communis agglutinin 1 （RCA 120） modified PMMA microfluidic device for binding and culturing living cells. The functionalisation is achieved by standard aminealdehyde （Schiff base） reaction through the crosslinker, glutaraldehyde. To prove the ability of the RCA 120 modified PMMA surface, the PC 12 cell line （rat pheochromocytoma ceils） has been captured and cultured by the microfluidic device. In the presence of tunicamycin, the dose/timedependence on decreasing of binding affinity of RCA 120 modified device with PC 12 cell is also observed. The experimental results demonstrate that the lectin-functionalized microfluidic device can be employed as efficient cell culturing platform, and has a great promise of being used as a powerful tool for monitoring the interaction of drug with living cell.

Theoretical, Simulation and Experimental Analysis of Microfluidic Droplet Generation and Recovering Process with Applications in Frying Oil Assessments

The research on microfluidic droplet size prediction has been extensive and fruitful, while the droplet deforming process has been seldom studied. In this paper, a frying-oil-assessing microfluidic device was designed to study the droplet deforming and recovering processes, which were dominated by channel geometry, flow rates,sheath flow viscosity and interfacial tension of the two phases. Theoretical expressions of the deforming process and its extreme value were obtained for the first time, supported by simulation and experiments. Theoretical,simulation and experimental results indicated that the steady-state droplet length could be a useful parameter for frying oil assessment.