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Hypersound-Assisted Size Sorting of Microparticles on Inkjet-Patterned Protein Films
Abstract
Hydrodynamic approaches are important for biomedical diagnostics, chemical analysis, and a broad range of industrial applications. Size-based separation and sorting is an important tool for these applications. We report the integration of hypersound technology with patterned protein films to provide efficient sorting of microparticles based on particle charge and size. We employed a hypersonic resonator for the acoustic streaming of the fluidic system to generate microvortices that exert drag forces on the objects on the surface that are dictated by their radius of curvature. We demonstrate a size-based sorting of anionic silica particles using protein patterns and gradients fabricated using attractive cationic and repulsive anionic proteins.
... Drop-on-demand (DOD) inkjet printing technique is usually applying a controllable external force for inducing a sharp change in the local pressure of the ink in the solid channel. The underlying idea is to control the fluidic ink for overcoming the viscous resistance and surface tension to form droplets at the nozzle to achieve jetting (Kuang et al. 2014;Li et al. 2019a, b;Singh et al. 2010;Wijshoff 2010), with a wide range of applications in biomedical engineering (Gopalakrishnan et al. 2021;Jutila et al. 2020;Kim et al. 2010;Lee et al. 2016;Yoon et al. 2020), electronic devices Naik et al. 2021;Yan et al. 2009), solar cells (Chu et al. 2021;Hoth et al. 2007), LCD fabrication (Alaman et al. 2016;Gupta et al. 2019), and in many other disciplines. Compared with the various commercial inkjet technologies, such as thermal , acoustic (Wang et al. 2019), and pneumatic (Brand et al. 2019), the piezoelectric inkjet (PIJ) printing employs the electric potential to activate the mechanical vibration of a lead zirconium titanate (PZT) membrane. ...
The piezoelectric inkjet (PIJ) printing technique, as a typical drop-on-demand (DOD) inkjet process, employs the electric potential for activating the mechanical vibration of a lead zirconium titanate (PZT) membrane. As a result, the constant flow of the fluidic ink within the solid channel is attained, resulting in the formation of a droplet in the nozzle. This droplet will be subsequently deposited on a substrate, which renders the PIJ method one of the most indispensable tools for various applications in MEMS, cell printing, LCD fabrication, etc. However, by considering a specific driving waveform, an air bubble will be generated and trapped within the solid channel after the ink droplet is ejected from the nozzle, which would inevitably affect the subsequent printing process. Additionally, there are scarce reports in the literature that have dealt with this issue in depth. Along these lines, in this work, a conservative level set method in conjunction with the inverse piezoelectric effect and the fluid–structure interaction is proposed for analyzing the PIJ printing process. On top of that, benchmark effectiveness against the experimental tests is introduced, in which an air bubble can be observed to be generated and further be trapped within the nozzle channel. The evolution of air bubble formation and trapping process was then visually analyzed in depth by considering the ink–solid–air interaction in the form of numerical investigation, which has never been reported in the literature according to our best knowledge. In addition, a variety of both numerical and experimental results have been provided to illustrate the coalescence of the trapped air bubbles from smaller bubbles to a large bubble and to demonstrate how exactly the trapped air bubbles affect the print quality. Furthermore, the influence of the driving waveform on the evolution of the trapped air bubble was explored, which could be of great advantage for the better control of the printing process for various PIJ printheads.
Protein-based bioactive coatings have emerged as a versatile and promising strategy for enhancing the performance and biocompatibility of diverse biomedical materials and devices. Through surface modification, these coatings confer novel biofunctional attributes, rendering the material highly bioactive. Their widespread adoption across various domains in recent years underscores their importance. This review systematically elucidates the behavior of protein-based bioactive coatings in organisms and expounds on their underlying mechanisms. Furthermore, it highlights notable advancements in artificial synthesis methodologies and their functional applications in vitro. A focal point is the delineation of assembly strategies employed in crafting protein-based bioactive coatings, which provides a guide for their expansion and sustained implementation. Finally, the current trends, challenges, and future directions of protein-based bioactive coatings are discussed.
Microfluidic systems have been widely explored based on microfluidic technology, and it has been widely used for biomedical screening. The key parts are the fabrication of the base scaffold, the construction of the matrix environment in the 3D system, and the application mechanism. In recent years, a variety of new materials have emerged, meanwhile, some new technologies have been developed. In this review, we highlight the properties of high throughput and the biomedical application of the microfluidic chip and focus on the recent progress of the fabrication and application mechanism. The emergence of various biocompatible materials has provided more available raw materials for microfluidic chips. The material is not confined to polydimethylsiloxane (PDMS) and the extracellular microenvironment is not limited by a natural matrix. The mechanism is also developed in diverse ways, including its special physical structure and external field effects, such as dielectrophoresis, magnetophoresis, and acoustophoresis. Furthermore, the cell/organ-based microfluidic system provides a new platform for drug screening due to imitating the anatomic and physiologic properties in vivo. Although microfluidic technology is currently mostly in the laboratory stage, it has great potential for commercial applications in the future.
Wearable devices integrated with various electronic modules, biological sensors, and chemical sensors have drawn large public attention. Due to their inherent advantages of superior stretchability, elaborate microstructure, high integration of multiple functions, and low cost, microfluidics are an excellent candidate and have already been widely used in wearable devices. Well‐designed microfluidic devices can realize excellent multiple functions in wearable devices, including sample collecting, handling and storage, sample analysis, signal converting and amplification, mechanic sensing, and power supplying. Moreover, the microfluidic wearable devices with further integration of wireless modules have exhibited potential applications in healthcare monitoring, clinical assessment, and human and intelligent device interaction. This review focuses on the latest advances on multifunctional wearable devices based on microfluidics, primarily including general functions and designs of microfluidic wearable devices, and their specific applications in physiological signal monitoring, clinical diagnosis and therapeutics, and healthcare.
A Y-shaped microfluidic separator with an array of hard-magnetic elements integrated in a non-magnetic substrate is designed to realize continuous separation of magnetic beads under an external bias field. By fixing the magnetization directions of the hard-magnetic elements parallel to the wall of microchannel, the spatial distribution of the magnetic field can be adjusted by the geometrical size of elements, the gap between two neighboring elements, and the direction of the external bias field. In this work, magnetic beads comprising of multiple magnetic/superparamagnetic nanoparticles (10 nm Fe5Si3, Fe3Si or Fe3O4) in polymer matrixes are used. Herein, Kelvin force on the magnetic bead is described by treating each magnetized superparamagnetic nanoparticle as an equivalent magnetic dipole. With the closed-form Kelvin force, a two-way model that takes bead-fluid interaction into account is adopted to investigate the trajectories of magnetic beads in our Y-shaped microfluidic system. Using the two-way model, the influence of the beads’ size, the direction of the external bias field, the magnetic bead concentration and the bead-fluid interaction on the trajectories of magnetic beads is also investigated. 100% collection or separation efficiency can be realized just by adjusting inlet velocities. In addition, it is theoretically demonstrated that two kinds of magnetic beads with small size difference (down to 100 nm) can be successfully separated by using our Y-shaped microfluidic separator.
Acoustofluidics: From lab to clinic Research into using sound waves to separate biological particles from fluid moving through tiny channels is rapidly advancing, but more is needed to develop real-life clinical applications. Tony Jun Huang of Duke University in the US and colleagues reviewed the latest studies in the rapidly growing field of ‘acoustofluidic separation’. While progress has been made in using the technology to separate micro-sized objects like cells and bacteria from fluids, few breakthroughs have been achieved for separating nanoparticles, like lipids and viruses. Also, advances are needed to speed up the separation process. Still, the field shows much promise for the development of label-free, point-of-care devices for cancer diagnosis, placental health monitoring, and isolating bacteria from water or food. Focusing on product development and technological improvements will enable this technology to find real-world applications, the researchers conclude.
A novel method to drive and manipulate fluid in a contactless way in a microelectrode-microfluidic system is demonstrated by combining the Lorentz and magnetic field gradient forces. The method is based on the redox-reaction [Fe(CN)6]3−/[Fe(CN)6]4− performed in a magnetic field oriented perpendicular to the ionic current that crosses the gap between two arrays of oppositely polarized microelectrodes, generating a magnetohydrodynamic flow. Additionally, a movable magnetized CoFe micro-strip is placed at different positions beneath the gap. In this region, the magnetic flux density is changed locally and a strong magnetic field gradient is formed. The redox-reaction changes the magnetic susceptibility of the electrolyte near the electrodes, and the resulting magnetic field gradient exerts a force on the fluid, which leads to a deflection of the Lorentz force-driven main flow.
Particle Image Velocity measurements and numerical simulations demonstrate that by combining the two magnetic forces, the flow is not only redirected, but also a local change of concentration of paramagnetic species is realized.
Wearable sweat sensors rely either on electronics for electrochemical detection or on colorimetry for visual readout. Non-ideal form factors represent disadvantages of the former, while semiquantitative operation and narrow scope of measurable biomarkers characterize the latter. Here, we introduce a battery-free, wireless electronic sensing platform inspired by biofuel cells that integrates chronometric microfluidic platforms with embedded colorimetric assays. The resulting sensors combine advantages of electronic and microfluidic functionality in a platform that is significantly lighter, cheaper, and smaller than alternatives. A demonstration device simultaneously monitors sweat rate/loss, pH, lactate, glucose, and chloride. Systematic studies of the electronics, microfluidics, and integration schemes establish the key design considerations and performance attributes. Two-day human trials that compare concentrations of glucose and lactate in sweat and blood suggest a potential basis for noninvasive, semi-quantitative tracking of physiological status.
Various materials are fabricated to form specific structures/patterns at the micro‐/nanoscale, which exhibit additional functions and performance. Recent liquid‐mediated fabrication methods utilizing bottom‐up approaches benefit from micro‐/nanofluidic technologies that provide a high controllability for manipulating fluids containing various solutes, suspensions, and building blocks at the microscale and/or nanoscale. Here, the state‐of‐the‐art micro‐/nanofluidic approaches are discussed, which facilitate the liquid‐mediated patterning of various hybrid‐scale material structures, thereby showing many additional advantages in cost, labor, resolution, and throughput. Such systems are categorized here according to three representative forms defined by the degree of the free‐fluid–fluid interface: free, semiconfined, and fully confined forms. The micro‐/nanofluidic methods for each form are discussed, followed by recent examples of their applications. To close, the remaining issues and potential applications are summarized.
Microfluidic systems have advanced beyond natural and life science applications and lab‐on‐a‐chip uses. A growing trend of employing microfluidic technologies for environmental detection has emerged thanks to the precision, time‐effectiveness, and cost‐effectiveness of advanced microfluidic systems. This paper reviews state‐of‐the‐art microfluidic technologies for environmental applications, such as on‐site environmental monitoring and detection. Microdevices are extensively used in collecting environmental samples as a means to facilitate detection and quantification of targeted components with minimal quantities of samples. Likewise, microfluidic‐inspired approaches for separation and treatment of contaminated water and air, such as the removal of heavy metals and waterborne pathogens from wastewater and carbon capture are also investigated. The latest advances in microfluidic technologies for environmental applications are reviewed, covering microdevices for detecting environmental samples of interest to droplet microfluidic synthesis of functional materials for environmental remediation, including water treatment and carbon capture and storage. A critical analysis of the current status and challenges and opinions on the future development of the rapidly developed technologies are also presented.
Inertial microfluidics has become a popular topic in microfluidics research for its good performance in particle manipulation and its advantages of simple structure, high throughput, and freedom from an external field. Compared with traditional microfluidic devices, the flow field in inertial microfluidics is between Stokes state and turbulence, whereas the flow is still regarded as laminar. However, many mechanical effects induced by the inertial effect are difficult to observe in traditional microfluidics, making particle motion analysis in inertial microfluidics more complicated. In recent years, the inertial migration effect in straight and curved channels has been explored theoretically and experimentally to realize on-chip manipulation with extensive applications from the ordinary manipulation of particles to biochemical analysis. In this review, the latest theoretical achievements and force analyses of inertial microfluidics and its development process are introduced, and its applications in circulating tumor cells, exosomes, DNA, and other biological particles are summarized. Finally, the future development of inertial microfluidics is discussed. Owing to its special advantages in particle manipulation, inertial microfluidics will play a more important role in integrated biochips and biomolecule analysis.
Since the pioneering work of Ashkin and coworkers, back in 1970, optical manipulation gained an increasing interest among the scientific community. Indeed, the advantages and the possibilities of this technique are unsubtle, allowing for the manipulation of small particles with a broad spectrum of dimensions (nanometers to micrometers size), with no physical contact and without affecting the sample viability. Thus, optical manipulation rapidly found a large set of applications in different fields, such as cell biology, biophysics, and genetics. Moreover, large benefits followed the combination of optical manipulation and microfluidic channels, adding to optical manipulation the advantages of microfluidics, such as a continuous sample replacement and therefore high throughput and automatic sample processing. In this work, we will discuss the state of the art of these optofluidic devices, where optical manipulation is used in combination with microfluidic devices. We will distinguish on the optical method implemented and three main categories will be presented and explored: (i) a single highly focused beam used to manipulate the sample, (ii) one or more diverging beams imping on the sample, or (iii) evanescent wave based manipulation.
We present an acoustic microfluidic mixing approach via acousto-mechanically induced micro-vortices sustained by localized ultrahigh frequency (UHF) acoustic fields. A micro-fabricated solid-mounted thin-film piezoelectric resonator (SMR) with a frequency of 1.54 GHz has been integrated into microfluidic systems. Experimental and simulation results show that UHF-SMR triggers strong acoustic field gradients to produce efficient and highly localized acoustic streaming vortices, providing a powerful source for microfluidic mixing. Homogeneous mixing with 87% mixing efficiency at a Peclet number of 35520 within 1 ms has been achieved. The proposed strategy shows a great potential for microfluidic mixing and enhanced molecule transportation in minimized analytical systems.
3D printing has the potential to significantly change the field of microfluidics. The ability to fabricate a complete microfluidic device in a single step from a computer model has obvious attractions, but it is the ability to create truly three dimensional structures that will provide new microfluidic capability that is chal- lenging, if not impossible to make with existing approaches. This critical review covers the current state of 3D printing for microfluidics, focusing on the four most frequently used printing approaches: inkjet (i3DP), stereolithography (SLA), two photon polymerisation (2PP) and extrusion printing (focusing on fused deposi- tion modeling). It discusses current achievements and limitations, and opportunities for advancement to reach 3D printing's full potential.
Background:
For bioanalytical systems sensitivity and biomolecule activity are critical issues. The immobilization of proteins into multilayer systems by the layer-by-layer deposition has become one of the favorite methods with this respect. Moreover, the combination of nanoparticles with biomolecules on electrodes is a matter of particular interest since several examples with high activities and direct electron transfer have been found. Our study describes the investigation on silica nanoparticles and the redox protein cytochrome c for the construction of electro-active multilayer architectures, and the electron transfer within such systems. The novelty of this work is the construction of such artificial architectures with a non-conducting building block. Furthermore a detailed study of the size influence of silica nanoparticles is performed with regard to formation and electrochemical behavior of these systems.
Results:
We report on interprotein electron transfer (IET) reaction cascades of cytochrome c (cyt c) immobilized by the use of modified silica nanoparticles (SiNPs) to act as an artificial matrix. The layer-by-layer deposition technique has been used for the formation of silica particles/cytochrome c multilayer assemblies on electrodes. The silica particles are characterized by dynamic light scattering (DLS), Fourier transformed infrared spectroscopy (FT-IR), Zeta-potential and transmission electron microscopy (TEM). The modified particles have been studied with respect to act as an artificial network for cytochrome c and to allow efficient interprotein electron transfer reactions. We demonstrate that it is possible to form electro-active assemblies with these non-conducting particles. The electrochemical response is increasing linearly with the number of layers deposited, reaching a cyt c surface concentration of about 80 pmol/cm2 with a 5 layer architecture. The interprotein electron transfer through the layer system and the influence of particle size are discussed.
Conclusions:
This study demonstrates the ability to construct fully electro-active cyt c multilayer assemblies by using carboxy-modified silica nanoparticles. Thus it can be shown that functional, artificial systems can be build up following natural examples of protein arrangements. The absence of any conductive properties in the second building block clearly demonstrates that mechanisms for electron transfer through such protein multilayer assemblies is based on interprotein electron exchange, rather than on wiring of the protein to the electrode.The construction strategy of this multilayer system provides a new controllable route to immobilize proteins in multiple layers featuring direct electrochemistry without mediating shuttle molecules and controlling the electro-active amount by the number of deposition steps.
Passive microfluidic channel geometries for control of droplet fission, fusion and sorting are designed, fabricated, and tested. In droplet fission, the inlet width of the bifurcating junction is used to control the range of breakable droplet sizes and the relative resistances of the daughter channels were used to control the volume of the daughter droplets. Droplet fission is shown to produce concentration differences in the daughter droplets generated from a primary drop with an incompletely mixed chemical gradient, and for droplets in each of the bifurcated channels, droplets were found to be monodispersed with a less than 2% variation in size. Droplet fusion is demonstrated using a flow rectifying design that can fuse multiple droplets of same or different sizes generated at various frequencies. Droplet sorting is achieved using a bifurcating flow design that allows droplets to be separated base on their sizes by controlling the widths of the daughter channels. Using this sorting design, submicron satellite droplets are separated from the larger droplets.
Acoustic fields have shown wide utility for micromanipulation, though their implementation in microfluidic devices often requires accurate alignment or highly precise channel dimensions, including in typical standing surface acoustic wave (SSAW) devices and resonant channels. In this work we investigate an approach that permits continuous microscale focusing based on diffractive acoustics, a phenomenon where a time-averaged spatially varying acoustic pressure landscape is produced by bounding a surface acoustic wave (SAW) transducer with a microchannel. By virtue of diffractive effects, this acoustic field is formed with the application of only a single travelling wave. As the field is dictated by the interplay between a propagating substrate-bound wave and a channel geometry, the pressure distribution will be identical for a given channel orientation regardless of its translation on a SAW substrate, and where small variations in channel size have no substantive effect on the pressure field magnitude or overall particle migration. Moreover, in the case of a channel with dimensions on the order of the diffractive fringe pattern spacing, the number of focusing positions will be identical for all channel orientations, with acoustic radiation forces pushing suspended particles to the channel edges. We explore this highly robust particle manipulation technique, determining two distinct sets of streaming and acoustic radiation dominant concentration positions, and show the continuous focusing of polystyrene 1 μm and 0.5 μm diameter particles and fluorescently labeled E. coli bacteria cells at flow rates exceeding those of previous microfluidic implementations for micron and submicron sized particles.
Intracellular delivery is essential to therapeutic applications such as genome engineering and disease diagnosis. Current methods lack simple, non-invasive strategies, and are often hindered by long incubation time or high toxicity. Hydrodynamic approaches offer rapid and controllable delivery of small molecules, but thus far have not been demonstrated for delivering functional proteins. In this work, we developed a robust hydrodynamic approach based on gigahertz (GHz) acoustics to achieve rapid and non-invasive cytosolic delivery of biologically active proteins. With this method, GHz-based acoustic devices trigger oscillations through a liquid medium (acoustic streaming) generating shear stress on the cell membrane and inducing transient nanoporation. This mechanical effect enhances membrane permeability and enables cytosolic access to cationic proteins without disturbing their bioactivity. We evaluated the versatility of this approach through delivery of cationic fluorescent proteins to a range of cell lines, all of which displayed equally efficient delivery speed (≤ 20 minutes). Delivery of multiple enzymatically active proteins with functionality related to apoptosis or genetic recombination further demonstrated the relevance of this method.
Herein, thin-layer potentiometry combined with ion-exchange membranes as barriers for charged interferences is demonstrated for the analytical detection of creatinine (CRE) in undiluted human urine. Briefly, CRE diffuses through an anion-exchange membrane (AEM) from a sample contained in one fluidic compartment to a second reservoir, containing the enzyme CRE deiminase. There, CRE reacts with the enzyme, and the formation of ammonium is dynamically monitored by potentiom-etric ammonium-selective electrodes. This analytical concept is integrated into a lab-on-a-chip microfluidic cell that allows for a high sample throughput and the operation under stop-flow mode, which allows CRE to passively diffuse across the AEM. Conveniently, positively charged species (i.e. potassium, sodium and ammonium, among others) are repelled by the AEM and never reach the ammonium-selective electrodes; thus, possible interference in the response can be avoided. As a result, the dynamic potential response of the electrodes is entirely ascribed to the stoichiometric formation of ammonium. The new CRE biosensor exhibits a Nernstian slope, within a linear range of response from 1 to 50 mM CRE concentration. As expected, the response time (1560 min) primarily depends on the CRE diffusion across the AEM. CRE analysis in urine samples displayed excellent results, without requiring sample pre-treatment (before the introduction of the sample in the microfluidic chip), and with high compatibility with development into a potential point-of-care clinical tool. In an at-tempt to decrease the analysis time, the presented analytical methodology for CRE detection is translated into an all-solid-state platform, in which the enzyme is immobilized on the surface of the ammonium-selective electrode and with the AEM on top. While more work is necessary in this direction, the CRE sensor appears to be promising for CRE analysis in both urine and blood.
Inertial particle separation using passive hydrodynamic forces has obtained great attention in the microfluidics community because of its operation simplicity and high throughput sample processing. Due to the passive nature of inertial microfluidics, each inertial sorting device is typically fixed to a certain cut-off size for particle separation that is mainly dependent on the channel geometry and dimensions, which however lacks the tunability in the separation threshold to fulfill the needs in different sorting applications. In this work, we explore the use of non-Newtonian viscoelastic fluids to achieve size-tunable elasto-inertial particle focusing and sorting in a microfluidic device with reverse wavy channel structures. The balance and competition among inertial lift force, Dean drag force and the controllable elastic lift force gives rise to interesting size-based particle focusing phenomena with tunability in the equilibrium focusing positions. Seven differently sized fluorescent microspheres (0.3, 2, 3, 5, 7, 10 and 15 μm) are used to investigate the effects of flow rate, viscoelastic fluid concentration and particle size on the tunable elasto-inertial focusing behavior. With the sorting tunability, we have achieved a highly effective sorting of a particle mixture into three subpopulations based on the particle size, i.e., small, intermediate and large subpopulations. We even demonstrate the controllable tunability among three separation thresholds for elasto-inertial particle sorting without changing the geometry and dimension of the microfluidic device. The tunability of the developed elasto-inertial particle focusing and sorting can significantly broaden its application in a variety of biomedical research.
Paper has unique advantages over other materials, including low cost, flexibility, porosity, and self-driven liquid pumping, thus making it widely used in various fields in biology, chemistry, physics and materials science. Recently, many multifunctional and highly integrated membrane-based devices have been achieved with the rapid development of membrane-building materials such as paper and pseudo-paper. Therefore, the rigid boundary between paper and other membranes has become blurred; paper can be considered a flexible membrane, and membranes with appropriately flexible or porous structures can also be defined as paper. Paper can manipulate liquids and respond photoelectrically to external objects to be measured, making it suitable for (bio)chemical sensing (chromatographic analysis, electrochemical analysis and wearable sensing). This review focuses on the development of microfluidic devices built with both traditional paper and other flexible membranes, including fabrication, (bio)chemical sensing, microfluidics manipulation and multiple applications.
We report a microfluidic fluorescence activated cell-sorting (μFACS) device that employs traveling surface acoustic waves (TSAW) to sort cells at rates comparable to conventional jet-in-air FACS machines, with high purity and viability. The device combines inertial flow focusing and sheath flow to align and evenly space cells, improving the sorting accuracy and screening rate. We sort with an interdigital transducer (IDT) whose tapered geometry allows precise positioning of the TSAW for optimal cell sorting. We sort three different cell lines at several kHz, at cell velocities exceeding one meter per second, while maintaining both sorting purity and cell viability at around 90% simultaneously.
In the last two decades, microfluidic technology has emerged as a highly efficient tool for the study of various chemical and biological reactions. Recently, we reported that high-throughput detection of various concentrations of a reagent is possible using a continuous gradient microfluidic channel combined with a surface-enhanced Raman scattering (SERS) detection platform. In this continuous flow regime, however, the deposition of nanoparticle aggregates on channel surfaces induces the “memory effect,” affecting both sensitivity and reproducibility. To resolve this problem, a SERS-based gradient droplet system was developed. Herein, the serial dilution of a reagent was achieved in a stepwise manner using microfluidic concentration gradient generators. Then various concentrations of a reagent generated in different channels were simultaneously trapped into the tiny volume of droplets by injecting an oil mixture into the channel. Compared to the single-phase regime, this two-phase liquid/liquid segmented flow regime allows minimization of resident time distributions of reagents through localization of reagents in encapsulated droplets. Consequently, the sample stacking problem could be solved using this system because it greatly reduces the memory effect. We believe that this SERS-based gradient droplet system will be of significant utility in simultaneously monitoring chemical and biological reactions for various concentrations of a reagent.
High-throughput screening of cell-secreted proteins is essential for various biotechnological applications. In this article, we show a microfluidic approach to perform the analysis of cell-secreted proteins in nanoliter droplet arrays by two complementary methods, fluorescence microscopy and mass spectrometry. We analyzed the secretion of the enzyme phytase, a phosphatase used as an animal feed additive, from a low number of yeast cells. Yeast cells were encapsulated in nanoliter volumes by droplet microfluidics and deposited on spatially-defined spots on the surface of a glass slide mounted on the motorized stage of an inverted fluorescence microscope. During the following incubation for several hours to produce phytase, the droplets can be monitored by optical microscopy. After addition of a fluorogenic substrate at a defined time, the relative concentration of phytase was determined in every droplet. Moreover, we demonstrate the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to monitor the multi-step conversion of the native substrate phytic acid by phytase secreted in 7 nL droplets containing 50-100 cells. Our method can be adapted to various other protocols. As the droplets are easily accessible, compounds such as assay reagents or matrix molecules can be added to all or to selected droplets only, or part of the droplet volume could be removed. Hence, this platform is a versatile tool for questions related to cell secretome analysis.
Several biomedical analyses are performed on particular types of cells present in body samples or using functionalized microparticles. Success in such analyses depends on the ability to separate or isolate the target cells or microparticles from the rest of the sample. In conventional procedures, multiple equipment such as centrifuge, magnets, and macro-scale filters are used for such purposes, which are time-consuming, associated with human error, and require several operational steps. In the past two decades, there has been a tendency to develop microfluidic techniques, so-called lab-on-a-chip, to miniaturize and automate these procedures. The processes used for the separation and isolation of the cells and microparticles are scaled down into a small microfluidic chip, requiring very small amounts of the samples. Differences in the physical and biological properties of the target cells from the other components present in the sample are the key to the development of such microfluidic techniques. These techniques are categorized as filtration, hydrodynamic, dielectrophoretic, acoustic and magnetic based methods. Here we review the microfluidic techniques developed for sorting, separation, and isolation of cells and microparticles for biomedical applications. The mechanisms behind such techniques are thoroughly explained and the applications in which these techniques have been adopted are reviewed.
Leukocytes subpopulations contain crucial physiology information, hence precise specific leukocyte separation is very important for leukemia diagnosis and analysis. However, the conventional centrifugation and immunofluorescence-based separation methods are inaccurate and inconvenient due to the overlapping cell size and density or complex marking process. Here we report a new label-free technology for precise leukocyte subpopulations separation by synergy of acoustic and optical technologies. Surface acoustic wave (SSAW) solved the problem of gente and precise focusing cells in optical system, besides, SSAW is also used for the separation of granulocytes which have obviouse size distinction from other components. While for lymphocyte and monocyte which have overlap in size/density, optical force could distinguish them accurately based on RI difference with the covenience of acoustic pre-focusing part. The experiment conducts separation of three kinds of leukocyte subtypes with considerable throughput and purity, we obtain 99% pure lymphocytes, 98% pure monocytes, and 95% pure granulocytes. Experimental results prove the device has robust ability in leukocytes phenotypes separation, with the advantage of non-invasive, label-free and precise. In the future, this convenient hybrid method will also be a potential powerful tool for clinical auxiliary diagnosis and analysis.
Transparent semiconducting oxides (TSO) are promising candidates for the fabrication of flexible and low-cost electronic devices, as they contain only abundant materials, are nontoxic, and exhibit high carrier mobilities. The formation of rectifying Schottky-barrier contacts is a prerequisite for devices, such as rectifiers, photodetectors, and metal-semiconductor field-effect transistors, and it was found that the presence of oxygen plays an essential role during the formation of the Schottky contacts. With electrical measurements on Pt/zinc-tin-oxide (ZTO) and PtOx/ZTO Schottky-barrier contacts and depth-resolved x-ray photoelectron spectroscopy measurements we demonstrate the important role of oxygen at the interface between TSOs and the metal contact for the rectifying behavior of diodes. In the vicinity of the interface, PtOx is reduced to Pt in a two-step process. Pt(OH)4 is reduced within one day, whereas the reduction of PtO takes place over a time period of several weeks. The reduction results in improved rectification compared to Pt/ZTO, due to a filling of oxygen vacancies, which leads to a reduction of the free-carrier concentration in the vicinity of the PtOx/ZTO interface. This increases the depletion layer width and subsequently reduces the tunneling current, resulting in a higher rectification ratio. The time scale of the permanent performance improvement can be shortened significantly by applying a reverse bias to the diode. The described mechanism is most likely also present at other transparent-semiconducting-oxide–metal interfaces.
Protein-based materials provide an inherently biocompatible and sustainable platform for the generation of functional materials. Translating protein properties into protein films resistant to aqueous degradation is crucial for most applications such as tissue engineering and controlled drug delivery. Current methods to stabilize protein films use three main strategies: employing the relatively limited variety of naturally self-assembling proteins, using added cross-linkers or heat curing. While the cross-linking strategy generates functionally diverse structures, unreacted additives retained in cross-linked protein films can adversely affect their final behavior. Traditional heat curing results in hydrophobic surface and loss of protein inherent properties. We demonstrate here a scalable, additive-free, fluorous media assisted thermal treatment for the fabrication of stable, hydrophilic protein films. This approach is general in terms of protein building block, retaining much of their native structure and surface properties upon heating. We demonstrate the versatility of this strategy through fabrication of antifouling coatings on complex three-dimensional surfaces. The utility of these films as biomaterials is highlighted through the generation of highly biocompatible non-fouling surfaces and regulation of cellular adhesion through choice of protein precursor.
Nonspecific binding (NSB) is a general issue for surface based biosensors. Various approaches have been developed to prevent or remove the NSBs. However, these approaches either increased the background signals of the sensors or limited to specific transducers interface. In this work, we developed a hydrodynamic approach to selectively remove the NSBs using a microfabricated hypersonic resonator with 2.5 gigahertz (GHz) resonant frequency. The high frequency device facilitates to generate multiple controlled micro-vortices which then create cleaning forces at the solid-liquid interfaces. The competitive adhesive and cleaning forces have been investigated using the finite element method (FEM) simulation, identifying the feasibility of the vortices induced NSB removal. NSB proteins have been selectively removed experimentally both on the surface of the resonator and on other substrates which contact the vortices. Thus, the developed hydrodynamic approach is believed to be a simple and versatile tool for NSB removal and compatible to many sensor systems. The unique feature of the hypersonic resonator is that it can be used as a gravimetric sensor as well, thus a combined NSB removal and protein detection dual functional biosensor system is developed.
Droplet microfluidics generates and manipulates discrete droplets through immiscible multiphase flows inside microchannels. Due to its remarkable advantages, droplet microfluidics bears significant value in an extremely wide range of area. In this review, we provide a comprehensive and in-depth insight into droplet microfluidics, covering fundamental research from microfluidic chip fabrication and droplet generation to the applications of droplets in bio(chemical) analysis and materials generation. The purpose of this review is to convey the fundamentals of droplet microfluidics, a critical analysis on its current status and challenges, and opinions on its future development. We believe this review will promote communications among biology, chemistry, physics, and materials science.
Several gallium-based liquid metal alloys are liquid at room temperature. As 'liquid', such alloys have a low viscosity and a high surface tension while as 'metal', they have high thermal and electrical conductivities, similar to mercury. However, unlike mercury, these liquid metal alloys have low toxicity and a negligible vapor pressure, rendering them much safer. In comparison to mercury, the distinguishing feature of these alloys is the rapid formation of a self-limiting atomically thin layer of gallium oxide over their surface when exposed to oxygen. This oxide layer changes many physical and chemical properties of gallium alloys, including their interfacial and rheological properties, which can be employed and modulated for various applications in microfluidics. Injecting liquid metal into microfluidic structures has been extensively used to pattern and encapsulate highly deformable and reconfigurable electronic devices including electrodes, sensors, antennas, and interconnects. Likewise, the unique features of liquid metals have been employed for fabricating miniaturized microfluidic components including pumps, valves, heaters, and electrodes. In this review, we discuss liquid metal enabled microfluidic components, and highlight their desirable attributes including simple fabrication, facile integration, stretchability, reconfigurability, and low power consumption, with promising applications for highly integrated microfluidic systems.
In the last decade microfabrication processes including rapid prototyping techniques have advanced rapidly and achieved a fairly mature stage. These advances have encouraged and enabled the use of microfluidic devices by a wider range of users with applications in biological separations and cell and organoid cultures. Accordingly, a significant current challenge in the field is controlling biomolecular interactions at interfaces and the development of novel biomaterials to satisfy the unique needs of the biomedical applications. Poly(dimethylsiloxane) (PDMS) is one of the most widely used materials in the fabrication of microfluidic devices. The popularity of this material is the result of its low cost, simple fabrication allowing rapid prototyping, high optical transparency, and gas permeability. However, a major drawback of PDMS is its hydrophobicity and fast hydrophobic recovery after surface hydrophilization. This results in significant nonspecific adsorption of proteins as well as small hydrophobic molecules such as therapeutic drugs limiting the utility of PDMS in biomedical microfluidic circuitry. Accordingly, here, we focus on recent advances in surface molecular treatments to prevent fouling of PDMS surfaces towards improving its utility and expanding its use cases in biomedical applications.
Cellulose nanocrystals, rod-like crystalline nanoparticles, are a bio-based material that can be a great alternative to obtain films with tunable optical properties. Iridescent and light diffracting films can readily be obtained via the drying of a suspension of these cellulose nanocrystals. The characteristics of the particle deposition process together with the self-assembly in the precluding suspension has a direct effect on the optical properties of the obtained films. Particle deposition onto a substrate is affected by the flow dynamics inside sessile droplets and usually yields a ring-shaped deposition pattern commonly referred to as the coffee-ring effect. We set out to measure and describe the drying kinetics under different conditions. We found that the Marangoni flow inside the droplet was too small to counteract the capillary flow that deposits CNCs at the edges, resulting in the coffee-ring effect, irrespective of the atmospheric humidity. By varying the amount of ethanol in the atmosphere, we were able to find a balance between (1) colloidal stability in the droplet which is reduced due to ethanol diffusion into the droplet, and (2) increasing the Marangoni flow relative to capillary flow inside the droplet by changing the droplet surface tension. We could thus make iridescent films with a uniform thickness.
Isotropic microparticles prepared from a suspension that undergoes polymerization have long been used for a variety of applications. Bulk emulsification procedures produce polydisperse emulsion drops that are transformed into spherical microparticles through chemical or physical consolidation. Recent advances in droplet microfluidics have enabled the production of monodisperse emulsions that yield highly uniform microparticles, albeit only on a drop-by-drop basis. In addition, microfluidic devices have provided a variety of means for particle functionalization through shaping, compartmentalizing, and microstructuring. These functionalized particles have significant potential for practical applications as a new class of colloidal materials. This feature article describes the current state of the art in the microfluidic-based synthesis of monodisperse functional microparticles. The three main sections of this paper discuss the formation of isotropic microparticles, engineered microparticles, and hybrid microparticles. The complexities of the shape, compartment, and microstructure of these microparticles increase systematically from the isotropic to the hybrid types. Each section discusses the key idea underlying the design of the particles, their functionalities, and their applications. Finally, we outline the current limitations and future perspectives on microfluidic techniques for producing microparticles.
A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells (CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 μL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.
Flow field flow fractionation (F4) is an invaluable separation tool for large analytes, including nanoparticles and biomolecule complexes. However, sample loss due to analyte-channel membrane interaction limits extensive usage of F4 at present, which could be strongly affected by the carrier fluid composition. This work studied the impacts of carrier fluid (CF) composition on nanoparticle (NP) recovery in F4, with focus on high ionic strength conditions. Successful analysis of NPs in a biomolecules-friendly environment could expand the applicability of F4 to the developing field of nanobiotechnology. Recovery of the unfunctionalized polystyrene NPs of 199, 102, and 45nm in CFs with various pH (6.2, 7.4 and 8.2), increasing ionic strength (0-0.1M), and different types of co- and counter-ions, were investigated. Additionally, elution of the 85nm carboxylate NPs and two proteins, human serum albumin (HSA) and immunoglobulin (IgG), at high ionic strengths (0-0.15M) was investigated. Our results suggested that (1) electrostatic repulsion between the negatively charged NPs and the regenerated cellulose membrane was the main force to avoid particle adsorption on the membrane; (2) larger particles experienced higher attractive force and thus were influenced more by variation in CF composition; and (3) buffers containing weak anions or NPs with weak anion as the surface functional groups provided higher tolerance to the increase in ionic strength, owing to more anions being trapped inside the NP porous structure. Protein adsorption onto the membrane was also briefly investigated in salted CFs, using HSA and IgG. We believe our findings could help to identify the basic carrier fluid composition for higher sample recovery in F4 analysis of nanoparticles in a protein-friendly environment, which will be useful for applying F4 in bioassays and in nanotoxicology studies.
The dynamic adhesion behavior of micrometer-scale silica particles is investigated numerically for a low Reynolds number shear flow over a planar collecting wall with randomly distributed electrostatic heterogeneity at the 10-nanometer scale. The hydrodynamic forces and torques on a particle are coupled to spatially varying colloidal interactions between the particle and wall. Contact and frictional forces are included in the force and torque balances to capture particle skipping, rolling, and arrest. These dynamic adhesion signatures are consistent with experimental results and are reminiscent of motion signatures observed in cell adhesion under flowing conditions, although for the synthetic system the particle-wall interactions are controlled by colloidal forces rather than physical bonds between cells and a functionalized surface. As the fraction of the surface (Theta) covered by the cationic patches is increased from zero, particle behavior sequentially transitions from no contact with the surface to skipping, rolling, and arrest, with the threshold patch density for adhesion (Theta(crit)) always greater than zero and in quantitative agreement with experimental results. The ionic strength of the flowing solution determines the extent of the electrostatic interactions and can be used to tune selectively the dynamic adhesion behavior by modulating two competing effects. The extent of electrostatic interactions in the plane of the wall, or electrostatic zone of influence, governs the importance of spatial fluctuations in the cationic patch density and thus determines if flowing particles contact the wall. The distance these interactions extend into solution normal to the wall determines the strength of the particle-wall attraction, which governs the transition from skipping and rolling to arrest. The influence of Theta, particle size, Debye length, and shear rate is quantified through the construction of adhesion regime diagrams, which delineate the regions in parameter space that give rise to different dynamic adhesion signatures and illustrate selective adhesion based on particle size or curvature. The results of this study are suggestive of novel ways to control particle-wall interactions using randomly distributed surface heterogeneity.