Multifunctional Microfluidic Device Research Articles

Droplet-Based Preparation of ZnO-nanostructure Array for Microfluidic Fluorescence Biodetection.

Nanostructure-enhanced biodetection is widely used for early diagnosis and treatment, which plays an essential role in improving the cure rates of cancer patients. ZnO nanostructure-based fluorescence immunoassay has been demonstrated to enable effective and sensitive detection of cancer biomarkers for their excellent biocompatibility, high electrical point, and unique fluorescence enhancement properties. Further optimization of such fluorescence detection technology is still in demand to meet the requirements of highly sensitive, multiplex detection, and user-friendly devices. Droplet microfluidics is a promising platform for high-throughput analysis of biological assays, and they have been intensively used in analytical chemistry and synthesis of nanoparticles. Here, we propose a simple droplet chip, where a static droplet array was successfully obtained for in situ growth of ZnO nanostructures with varied diameters by changing the entire growth time and replenishment interval. This device provides a novel and alternative approach for patterned growth of ZnO nanostructures and understanding the growth condition of ZnO nanostructures in static droplet, which offers some guidance toward the design of multiple fluorescence amplification platforms potentially for biosensing. As a demonstration, we used the patterned grown ZnO nanostructures for multiple detection of cancer biomarkers, achieving a low limit of detection as low as 138 fg/mL in the human α-fetoprotein assay and 218 fg/mL in the carcinoembryonic antigen assay with a large dynamic range of 8 orders. These results suggest that such multifunctional microfluidic devices may be useful tools for efficient fluorescence diagnostic assays.

Artificial neural network-based predictions of surface electrocoalescence of water droplets in hydrocarbon media

Electrowetting (EW) is a widely-studied microfluidic technique to promote coalescence of droplets and also to generate droplets. However, analytical or computational modeling of the intricate phenomena associated with electrocoalescence of water droplets in hydrocarbon media is very challenging. Presently, EW-assisted surface electrocoalescence experiments are used to train machine learning (ML) algorithms. These comprise an artificial neural network (ANN), eXtreme gradient boosting (XGBoost) and polynomial regression. These models are then used to predict the influence of parameters such as applied voltage, frequency, electrode spacing, concentration and initial droplet density (normalized with uncovered area ratio (δi/αi)), to predict nine targets: uncovered area ratio (αf), final droplet density normalized with uncovered area (δf/αf), and seven droplet density distribution (radius) bins. ANN was the most accurate predictive tool among the three ML models with R2 of 0.89. ANN accurately predicted the droplet distribution bins for three distinct cases of good electrocoalescence, poor electrocoalescence and satellite droplet ejection (droplet generation). SHAP (Shapley Additive exPlanations) dependence plots were used to quantify the parametric influence of various parameters on each output. For δf/αf, frequency and electrode spacing were the most and least influential, respectively. Interestingly, the feature influence on droplet density distributions was observed to reverse (magnitude and direction) with droplet radius. The key novelty of this study is the coupling of data from a multifunctional microfluidic device to an ANN model to accurately predict complex stochastic droplet-related phenomena (coalescence, generation). As such, the framework developed here can be utilized for other data-rich droplet-based microfluidic systems. δf/αf

Ion concentration polarization-based multifunctional microfluidic device for analysis of intracellular components

We report a multifunctional microfluidic device that lyses cells and separates intracellular components (including proteins) as a novel cell analysis platform. The device exploits an amplified electric field in an ion-depletion region formed by a nanofluidic phenomenon termed ion concentration polarization (ICP). To enable the multifunctionality of the device, multiple electric streams were employed to generate multiple ICP zones where cell lysis and separation of nano-sized intracellular components were processed sequentially in a semi-continuous manner in a single device. The amplified electric field in the ion-depletion region provides a short processing time (within only a few seconds for each step), and the absence of the internal electrodes offers the advantages of simple, low-cost device fabrication and bubble-free operation compared with conventional electric-force-based microfluidic devices. Due to these unique features, we expect that the developed multifunctional microfluidic device can be a very useful tool as an integrated system for analyzing intracellular components such as DNAs and proteins which is still a challenging and important issue to the biochemist.

Analysis of Single Blood Entities Using an Ultramicroelectrode through Single-Entity Electrochemistry

The research was conducted with the aim of analyzing individual human blood cells in the presence of redox species based on single-entity electrochemistry. Various blood cells (e.g., red blood cell, white blood cell, and platelets) perform a role directly related to survival, such as oxygen transport, hemostasis, and immune response, as they move through the whole body’s blood vessels. However, with the current lab-on-a-chip typed biosensor technology, the factors that can be measured using a small amount of blood are limited to uric acid, cholesterol, glucose, or hemoglobin in the blood. In clinical laboratories, common methods for measuring individual blood counts are automated hematology analyzers based on the automated Coulter cell counting method or flow cytometry, which produce a huge amount of blood count results, but are limited. For this reason, it is proposed a method for detecting individual blood cells that can be applied to a biosensor simply and quickly.References Ran, An. et al. (2021) ‘Emerging point-of-care technologies for anemia detection’, Lab on a Chip, 21(10), pp. 1843-1865.J-H, Lin. et al. (2020) ‘A Multifunctional Microfluidic Device for Blood Typing and Primary Screening of Blood Diseases’ ACS Sensors, 5(10), pp. 3082-3090.J, Lee. et al. (2020) ‘Determination of Serotonin Concentration in Single Human Platelets through Single-Entity Electrochemistry’, ACS Sensors, 5(7), pp. 1943-1948.T.L.T, Ho. et al. (2018) ‘Determining mean corpuscular volume and red blood cell count using electrochemical collision events’, Biosensors and Bioelectronics. 110, pp. 155-159.A-C, Villani. et al. (2017) ‘Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors’, Science. 356, No.6335.

A multi-functional microfluidic device compatible with widefield and light sheet microscopy.

We present a microfluidic device compatible with high resolution light sheet and super-resolution microscopy. The device is a 150 μm thick chamber with a transparent fluorinated ethylene propylene (FEP) cover that has a similar refractive index (1.34) to water (1.33), making it compatible with top-down imaging used in light sheet microscopy. We provide a detailed fabrication protocol and characterize the optical performance of the device. We demonstrate that the device supports long-term imaging of cell growth and differentiation as well as the rapid addition and removal of reagents while simultaneously maintaining sterile culture conditions by physically isolating the sample from the dipping lenses used for imaging. Finally, we demonstrate that the device can be used for super-resolution imaging using lattice light sheet structured illumination microscopy (LLS-SIM) and DNA PAINT. We anticipate that FEP-based microfluidics, as shown here, will be broadly useful to researchers using light sheet microscopy due to the ability to switch reagents, image weakly adherent cells, maintain sterility, and physically isolate the specimen from the optics of the instruments.

EGFR mutation detection of lung circulating tumor cells using a multifunctional microfluidic chip

Microfluidics has become a reliable platform for circulating tumor cells (CTCs) detection because of its high integration, small size, low consumption of reagents and rapid response. Here, we developed a multifunctional microfluidic device consists of three parts, including CTCs capture area, single-layer membrane valves area, and microcavity nucleic acid detection and analysis region based on digital polymerase chain reaction (dPCR), allowing CTCs capture, lysis, and genetic characterization to be performed on a single chip. The CTCs capture chip is coupled to the nucleic acid detection chip via a control valve. CTCs were firstly trapped in the CTC capture area, and then lysed using proteinase K to release nucleic acids. Subsequently CTCs lysate was transferred into nucleic acid detection area consisting of 12800 micro-cavity chambers for nucleic acids detection. To evaluate the performance of this chip, this study detected EGFR-L858R mutation in lung cancer cell lines H1975 and A549 cells, as well as leukocytes from normal donors. The results showed that positive signals were only observed in H1975 cells, and the detected value had a high linear relationship with the expected value (R2 = 0.9897). In conclusion, this multi-functional microfluidic chip that integrates CTCs capture, lysis and nucleic acid detection can successfully detect gene mutations in CTCs, providing reference for tumor-targeted drugs and precise diagnosis and treatment.

Eliminating air bubble in microfluidic systems utilizing integrated in-line sloped microstructures

In most microfluidic systems, formation and accumulation of air and other gas bubbles can be detrimental to their operation. Air bubbles in a microfluidic channel induce a pressure profile fluctuation and therefore disturb the stability of the system. Once an air bubble is generated, itis also extremely difficult to remove such bubbles from the microfluidic systems. In tissue and cell culture microfluidic systems, a single air bubble can completely shear off cells that are being cultured. Air bubbles can be especially problematic in microfluidic systems that have to operate for long periods of time, since completely eliminating the generation of air bubbles for prolonged periods of time, where a single air bubble can ruin an entire multi-day/multi-week experiment, is extremely challenging. Several in-line and off-chip bubble traps have been developed so far, but cannot completely eliminate air bubbles from the system or are relatively difficult to integrate into microfluidic systems. Recent advancements in two-photon polymerization (2PP)-based microfabrication method eliminates the restriction in Z-axis control in conventional two-dimensional microfabricationmethods, and thus enables complex 3D structures to be fabricated at sub-micrometer resolution. In this work, by utilizing this 2PP technique, we developed a sloped microfluidic structure that is capable of both trapping and real-time removal of air bubbles from the system in a consistent and reliable manner. The novel structures and designs developed in this work present a unique opportunity to overcome many limitations of currentmethods, bringstate-of-the-art solutions in air bubble removal, and enable a multifunctional microfluidic device to operate seamlessly free from air bubble disruption. The microfabricated system was tested in both droplet microfluidics and continuous-flow microfluidics applications, and demonstrated to be effective in preventing air bubble aggregation over time. This simple sloped microstructure can be easily integrated into broad ranges ofmicrofluidic devices to minimize bubble introduction, which will contribute to creating a stable and bubble-free microfluidic platform amenable for long-term operation.

A Multifunctional Microfluidic Device for Blood Typing and Primary Screening of Blood Diseases.

In this work, we demonstrate a multifunctional, portable, and disposable microfluidic device for blood typing and primary screening of blood diseases. Preloaded antibodies (anti-A, anti-B, and anti-D) interact with injected whole blood cells to cause an agglutination reaction that blocks a microslit in the microfluidic channel to accumulate red blood cells and form a visible red line that can be easily read to determine the blood type. Moreover, the different blood density and agglutination properties of normal and subtype blood groups, as well as different blood diseases, including anemia and polycythemia vera, generate different lengths of blood agglutination within the channels, which allows us to successfully screen these various conditions in as little as 2 min. The required blood volume for each test is just 1 μL, which can be obtained by minimally invasive finger pricking. This novel method of observing agglutinated red blood cells to distinguish blood types and diseases is both feasible and affordable, suggesting its promise for use in areas with limited resources.

Downscaling screening cultures in a multifunctional bioreactor array-on-a-chip for speeding up optimization of yeast-based lactic acid bioproduction.

A key challenge for bioprocess engineering is the identification of the optimum process conditions for the production of biochemical and biopharmaceutical compounds using prokaryotic as well as eukaryotic cell factories. Shake flasks and bench‐scale bioreactor systems are still the golden standard in the early stage of bioprocess development, though they are known to be expensive, time‐consuming, and labor‐intensive as well as lacking the throughput for efficient production optimizations. To bridge the technological gap between bioprocess optimization and upscaling, we have developed a microfluidic bioreactor array to reduce time and costs, and to increase throughput compared with traditional lab‐scale culture strategies. We present a multifunctional microfluidic device containing 12 individual bioreactors (V t = 15 µl) in a 26 mm × 76 mm area with in‐line biosensing of dissolved oxygen and biomass concentration. Following initial device characterization, the bioreactor lab‐on‐a‐chip was used in a proof‐of‐principle study to identify the most productive cell line for lactic acid production out of two engineered yeast strains, evaluating whether it could reduce the time needed for collecting meaningful data compared with shake flasks cultures. Results of the study showed significant difference in the strains' productivity within 3 hr of operation exhibiting a 4‐ to 6‐fold higher lactic acid production, thus pointing at the potential of microfluidic technology as effective screening tool for fast and parallelizable industrial bioprocess development.

Multifunctional Droplet Microfluidic Platform for Rapid Immobilization of Oligonucleotides on Semiconductor Quantum Dots.

Quantum dot-DNA oligonucleotide (QD-DNA) conjugates have been used in many fields such as nucleic acid bioassays, intracellular probes, and drug delivery systems. A typical solid-phase method that achieves rapid loading of oligonucleotides on surfaces of QDs involves a two-step reaction and is performed in a batch-based approach. In contrast, droplet microfluidics offers many advantages that are unavailable when using batch processing, providing rapid and dense immobilized DNA oligonucleotides on QDs. The presented droplet microfluidic approach allows high-quality QD-DNA conjugates to be produced using one single device, which is designed to have two droplet generators, one droplet merger, and one mixer. One of the droplet generators coencapsulates QDs and magnetic beads (MBs) into nanoliter-sized droplets for the production of QD-MB conjugates and the other encapsulates oligonucleotides in nanoliter-sized droplets. These two streams of droplets then merge at a one-to-one ratio in a chamber. The merged droplets travel along the mixer, which is a serpentine microchannel with 30 turns, resulting in QD-DNA conjugation structures of high quality. This multifunctional microfluidic device provides advantages such as higher degree of control over the reaction conditions, minimized cross-contamination and impurities, and reduction of reagent consumption while eliminating any need for external vortexing and pipetting. To evaluate the quality of the QD-DNA conjugates, they were used as Forster resonance energy transfer (FRET) probes to quantify oligonucleic targets.

Morphology-controllable gold hierarchically micro/nanostructured arrays prepared by electrodeposition on colloidal monolayer and their structurally related wettability

In this paper, we demonstrated a clean fabrication of the morphology-controllable gold hierarchically micro/nanostructured arrays (HMNAs) by electrochemical deposition on polystyrene monolayer colloidal crystal template. Through varying cathodic current density and deposition time to adjust electrocrystallization process, the HMNAs can be controlled from the one with microspheres and nanoparticles to the one with microbowls and nanorods. Further adjusting the nucleating sites via ion-sputtering deposition of gold film over the polystyrene spheres, we designed and prepared a new kind of HMNA with microspheres and nanowimbles. Interestingly, these Au HMNAs exhibited structurally related wettability. Correlated with their different hierarchical micro/nano-structures, they all showed superhydrophobicity after surface modification of low surface free energy material while with different surface adhesions. A water droplet can pine on the surface of the concave Au HMNA with microbowls and nanorods even turned upside down, showing ultrahigh liquid-solid adhesion. However, the convex Au HMNA with microspheres and nanowimbles shows very low surface adhesion with a sliding angle close to zero. These surface adhesion-controlled superhydrophobic surfaces can be found many potential applications, such as self-cleaning surfaces, liquid transportation, and multifunctional microfluidic devices.

Simultaneous microfluidic pumping and mixing using an array of asymmetric 3D ring electrode pairs in a cylindrical microchannel by the AC electroosmosis effect

Abstract The present studies of AC electroosmosis (ACEO) micropumps for simultaneous pumping and mixing are mainly involved in the designs of the planar electrode pairs in a rectangular microchannel, such as the diagonal/herringbone shape of electrodes on top and bottom of the substrates. The two mixing samples usually flow into the left and right half of the inlet, respectively. However, the flow rate is limited because of the less electrodes on the walls, and the mixing is only appropriate for the channel of small diameters owing to the vortices appearing near the surface of electrodes. To solve the problem, we proposed an ACEO micropump with an array of asymmetric ring electrode pairs in a cylindrical microfluidic channel, and established a three-dimensional (3D) theoretical model by the standard Poisson–Boltzmann (PB) theory and convection–diffusion equation. In this paper, the two mixing samples flow into the inner and outer circular pipe of the inlet, respectively. Thus, the samples can be exchanged from the outer pipe area to the inner pipe area, so that it can strengthen the mixing by the vortices around the ring electrode surfaces in the microchannel. The microfluidic velocity field, vorticity field and concentration field are investigated in detail to explain the mechanism of pumping and mixing. To further improve the mixing performance, we designed and researched the combination sequences PnMm of ring electrode pairs based on the pumping mode of “forward driving of electrode pair” and the mixing mode of “reversal driving of electrode pair”. The simulation results show that the ring ACEO micropump with the inner and outer pipe inlet can rapidly pump the microflows and efficiently mix the samples simultaneously compared with the planar multifunctional ACEO micropumps. The numerical simulation of the ring ACEO micropump in this paper is of significant importance for the development of multifunctional microfluidic devices in biochemical field, and feasible fabrication techniques should be experimentally investigated in future studies.

One-Way Particle Transport Using Oscillatory Flow in Asymmetric Traps.

One challenge of integrating of passive, microparticles manipulation techniques into multifunctional microfluidic devices is coupling the continuous-flow format of most systems with the often batch-type operation of particle separation systems. Here, a passive fluidic technique-one-way particle transport-that can conduct microparticle operations in a closed fluidic circuit is presented. Exploiting pass/capture interactions between microparticles and asymmetric traps, this technique accomplishes a net displacement of particles in an oscillatory flow field. One-way particle transport is achieved through four kinds of trap-particle interactions: mechanical capture of the particle, asymmetric interactions between the trap and the particle, physical collision of the particle with an obstacle, and lateral shift of the particle into a particle-trapping stream. The critical dimensions for those four conditions are found by numerically solving analytical mass balance equations formulated using the characteristics of the flow field in periodic obstacle arrays. Visual observation of experimental trap-particle dynamics in low Reynolds number flow (<0.01) confirms the validity of the theoretical predictions. This technique can transport hundreds of microparticles across trap rows in only a few fluid oscillations (<500 ms per oscillation) and separate particles by their size differences.

Cultivation and quantitative single-cell analysis of Saccharomyces cerevisiae on a multifunctional microfluidic device.

Here, we present a multifunctional microfluidic device whose integrative design enables to combine cell culture studies and quantitative single cell biomolecule analysis. The platform consists of 32 analysis units providing two key features; first, a micrometer-sized trap for hydrodynamic capture of a single Saccharomyces cerevisiae (S. cerevisiae) yeast cell; second, a convenient double-valve configuration surrounding the trap. Actuating of the outer valve with integrated opening results in a partial isolation in a volume of 11.8 nL, i.e. the cell surrounding fluid can be exchanged diffusion-based without causing shear stress or cell loss. Actuation of the inner ring-shaped valve isolates the trapped cell completely in a small analysis volume of 230 pL. The device was used to determine the growth rate of yeast cells (S. cerevisiae) under under optimum and oxidative stress conditions. In addition, we successfully quantified the cofactor beta-nicotinamide adenine dinucleotide phosphate (NAD(P)H) in single and few cells exposed to the different microenvironments. In conclusion, the microdevice enables to analyze the influence of an external stress factor on the cellular fitness in a fast and more comprehensive way as cell growth and intracellular biomolecule levels can be investigated.

A Versatile Bonding Method for PDMS and SU-8 and Its Application towards a Multifunctional Microfluidic Device.

This paper reports a versatile and irreversible bonding method for poly(dimethylsiloxane) (PDMS) and SU-8. The method is based on epoxide opening and dehydration reactions between surface-modified PDMS and SU-8. A PDMS replica is first activated via the low-cost lab equipment, i.e., the oxygen plasma cleaner or the corona treater. Then both SU-8 and plasma-treated PDMS samples are functionalized using hydrolyzed (3-aminopropyl)triethoxysilane (APTES). Ultimately, the samples are simply brought into contact and heated to enable covalent bonding. The molecular coupling and chemical reactions behind the bonding occurring at the surfaces were characterized by water contact angle measurement and X-ray photoelectron spectroscopy (XPS) analysis. The reliability of bonded PDMS-SU-8 samples was examined by using tensile strength and leakage tests, which revealed a bonding strength of over 1.4 MPa. The presented bonding method was also applied to create a metal-SU-8-PDMS hybrid device, which integrated SU-8 microfluidic structures and microelectrodes. This hybrid system was used for the effective trapping of microparticles on-chip, and the selective releasing and identification of predefined trapped microparticles. The hybrid fabrication approach presented here, based on the PDMS-SU-8 bonding, enables multifunctional integration in complex microfluidic devices.

Tunable hydrodynamic characteristics in microchannels with biomimetic superhydrophobic (lotus leaf replica) walls

The present work comprehensively addresses the hydrodynamic characteristics through microchannels with lotus leaf replica (exhibiting low adhesion and superhydrophobic properties) walls. The lotus leaf replica is fabricated following an efficient, two-step, soft-molding process and is then integrated with rectangular microchannels. The inherent biomimetic, superhydrophobic surface-liquid interfacial hydrodynamics, and the consequential bulk flow characteristics, are critically analyzed by the micro-particle image velocimetry technique. It is observed that the lotus leaf replica mediated microscale hydrodynamics comprise of two distinct flow regimes even within the low Reynolds number paradigm, unlike the commonly perceived solely apparent slip-stick dominated flows over superhydrophobic surfaces. While the first flow regime is characterized by an apparent slip-stick flow culminating in an enhanced bulk throughput rate, the second flow regime exhibits a complete breakdown of the aforementioned laminar and uni-axial flow model, leading to a predominantly no-slip flow. Interestingly, the critical flow condition dictating the transition between the two hydrodynamic regimes is intrinsically dependent on the micro-confinement effect. In this regard, an energetically consistent theoretical model is also proposed to predict the alterations in the critical flow condition with varying microchannel configurations, by addressing the underlying biomimetic surface-liquid interfacial conditions. Hence, the present research endeavour provides a new design-guiding paradigm for developing multi-functional microfluidic devices involving biomimetic, superhydrophobic surfaces, by judicious exploitation of the tunable hydrodynamic characteristics in the two regimes.