Microfluidic ELISA on non-passivated PDMS chip using magnetic bead transfer inside dual networks of channels

Achieving efficient passivation of micro-channels against non-specific adsorption of biomolecules is a critical aspect in the development of microfluidic ELISA systems. Usual surface treatments such as pre-coating of the channels with serum albumin, exposure to oxygen plasma, polyethylene glycol grafting however exhibit a lack of long-term stability, with procedures that can be time-consuming, complex or associated with costly materials and instruments. In this paper, we present a new fluidic design combined with an original strategy of manipulating magnetic beads in order to reduce assay noise in bead-based microfluidic ELISA without the need for prior channel pre-treatment. The novelty of the system relies on the physical separation of the immune complex formation phase and the enzymatic reaction phase into two independent networks of channels. These networks are linked by fluidic bridges, whose openings are controlled by pressure valves, and through which the beads are magnetically transferred. A standard curve for the quantification of a model antibody was obtained within 30 minutes. A detection limit of 100 pg mL(-1) (660 fM) and good linearity of the signal up to 4 ng mL(-1) were observed.     

A new floating electrode structure for generating homogeneous electrical fields in microfluidic channels

In this article a new parallel electrode structure in a microfluidic channel is described that makes use of a floating electrode to get a homogeneous electrical field. Compared to existing parallel electrode structures, the new structure has an easier production process and there is no need for an electrical connection to both sides of the microfluidic chip. With the new chip design, polystyrene beads suspended in background electrolyte have been detected using electrical impedance measurements. The results of electrical impedance changes caused by beads passing the electrodes are compared with results in a similar planar electrode configuration. It is shown that in the new configuration the coefficient of variation of the impedance changes is lower compared to the planar configuration (0.39 versus 0.56) and less dependent on the position of the beads passage in the channel as a result of the homogeneous electrical field. To our knowledge this is the first time that a floating electrode is used for the realization of a parallel electrode structure. The proposed production method for parallel electrodes in microfluidic channels can easily be applied to other applications.     

Rapid isolation and detection of cancer cells by utilizing integrated microfluidic systems

The present study reports a new three-dimensional (3D) microfluidic platform capable of rapid isolation and detection of cancer cells from a large sample volume (e.g. ~1 mL) by utilizing magnetic microbead-based technologies. Several modules, including a 3D microfluidic incubator for the magnetic beads to capture cancer cells, a microfluidic control module for sample transportation and a nucleic acid amplification module for genetic identification, are integrated into this microsystem. With the incorporation of surface-modified magnetic beads, target cancer cells can be specifically recognized and conjugated onto the surface of the antibody-coated magnetic microbeads by utilizing a swirling effect generated by the new 3D microfluidic incubator, followed by isolating and purifying the magnetic complexes via the incorporation of an external magnet and a microfluidic control module, which washes away any unbound waste solution. Experimental results show that over 90% of the target cancer cells can be isolated from a large volume of bio-samples within 10 min in the 3D microfluidic incubator. In addition, the expressed genes associated with ovarian and lung cancer cells can also be successfully amplified by using the on-chip nucleic acid amplification module. More importantly, the detection limit of the developed system is found to be 5 × 10(1) cells mL(-1) for the target cancer cells, indicating that this proposed microfluidic system may be adapted for clinical use for the early detection of cancer cells. Consequently, the proposed 3D microfluidic system incorporated with immunomagnetic beads may provide a promising automated platform for the rapid isolation and detection of cancer cells with a high sensitivity.     

Rapid purification of cell encapsulated hydrogel beads from oil phase to aqueous phase in a microfluidic device

In this paper, we demonstrate a new type of microfluidic chip that can realize continuous-flow purification of hydrogel beads from a carrier oil into aqueous solution by using a laminar-like oil/water interface. The microfluidic chip is composed by two functional components: (1) a flow-focusing bead generation module that can control size and shape of beads, (2) a bead extraction module capable of purifying hydrogel beads from oil into aqueous solution. This module is featured with large branch channels on one side and small ones on the opposite side. Water is continuously infused into the bead extraction module through the large branch channels, resulting in a laminar-like oil/water interface between the branch junctions. Simulation and experimental data show that the efficiency of oil depletion is determined by the relative flow rates between infused water and carrier oil. By using such a microfluidic device, viable cells (HCT116, colon cancer cell line) can be encapsulated in the hydrogel beads and purified into a cell culture media. Significantly improved cell viability was achieved compared to that observed by conventional bead purification approaches. This facile microfluidic chip could be a promising candidate for sample treatment in lab-on-a-chip applications.     

Numerical simulation of polymerization in interdigital multilamination micromixers

Free radical polymerization in microfluidic devices modeled with the help of numerical simulations is discussed. The simulation method used allows the simultaneous solvation of partial differential equations resulting from the hydrodynamics, thermal and mass transfer (convection, diffusion and chemical reaction). Three microfluidic devices are modeled, two interdigital multilamination micromixers respectively with a large and short focusing section, and a simple T-junction followed by a microtube reactor together considered as a bilamination micromixer with a large focusing section. The simulations show that in spite of the heat released by the polymerization reaction, the thermal transfer in such microfluidic devices is high enough to ensure isothermal conditions. Moreover, for low radial Peclet number, microfluidic devices with a large focusing section can achieve better control over the polymerization than a laboratory scale reactor as the polydispersity index obtained is very close to the theoretical limiting value. As the characteristic dimension of the microfluidic device increases, i.e. for high radial Peclet number, the reactive medium cannot be fully homogenized by the diffusion transport before leaving the system resulting in a high polydispersity index and a loss in the control of the polymerization.     

Mixing characteristics of a bubble mixing microfluidic chip for genomic DNA extraction based on magnetophoresis: CFD simulation and experiment

Mixing a small amount of magnetic beads and regents with large volume samples evenly in microcavities of a microfluidic chip is always the key step for the application of microfluidic technology in the field of magnetophoresis analysis. This article proposes a microfluidic chip for DNA extraction by magnetophoresis, which relies on bubble rising to generate turbulence and microvortices of various sizes to mix magnetic beads with samples uniformly. The construction and working principle of the microfluidic chip are introduced. CFD simulations are conducted when magnetic beads and samples are irritated by the generation of gas bubbles with the variation of supply pressures. The whole mixing process in the microfluidic chip is observed through a high-speed camera and a microfluidic system when the gas bubbles are generated continuously. The influence of supply pressure on the mixing characteristics of the microfluidic chip is investigated and discussed with both simulation and experiments. Compared with magnetic mixing, bubble mixing can avoid the magnetic beads gather phenomenon caused by magnetic forces and provide a rapid and high efficient solution to realize mixing small amount of regents in large volume samples in a certain order without complex moving structures and operations in a chip. Two applications of mixing with the proposed microfluidic chip are also carried out and discussed.     

Monitoring spatial distribution of ethanol in microfluidic channels by using a thin layer of cholesteric liquid crystal

Monitoring spatial distribution of chemicals in microfluidic devices by using traditional sensors is a challenging task. In this paper, we report utilization of a thin layer of cholesteric liquid crystal for monitoring ethanol inside microfluidic channels. This thin layer can be either a polymer dispersed cholesteric liquid crystal (PDCLC) layer or a free cholesteric liquid crystal (CLC) layer separated from the microfluidic device by using a thin film of PDMS. They both show visible colorimetric responses to 4% of ethanol solution inside the microfluidic channels. Moreover, the spatial distribution of ethanol inside the microfluidic channel can be reflected as a color map on the CLC sensing layers. By using this device, we successfully detected ethanol produced from fermentation taking place inside the microfluidic channel. These microfluidic channels with embedded PDCLC or embedded CLC offer a new sensing solution for monitoring volatile organic compounds in microfluidic devices.     

Microfluidic tectonics platform: A colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system

A fabrication platform for realizing integrated microfluidic devices is discussed. The platform allows for creating specific microsystems for multistep assays in an ad hoc manner as the components that perform the assay steps can be created at any location inside the device via in situ fabrication. The platform was utilized to create a prototype microsystem for detecting botulinum neurotoxin directly from whole blood. Process steps such as sample preparation by filtration, mixing and incubation with reagents was carried out on the device. Various microfluidic components such as channel network, valves and porous filter were fabricated from prepolymer mixture consisting of monomer, cross-linker and a photoinitiator. For detection of the toxoid, biotinylated antibodies were immobilized on streptavidin-functionalized agarose gel beads. The gel beads were introduced into the device and were used as readouts. Enzymatic reaction between alkaline phosphatase (on secondary antibody) and substrate produced an insoluble, colored precipitate that coated the beads thus making the readout visible to the naked eye. Clinically relevant amounts of the toxin can be detected from whole blood using the portable enzyme-linked immunosorbent assay (ELISA) system. Multiple layers can be realized for effective space utilization and creating a three-dimensional (3-D) chaotic mixer. In addition, external materials such as membranes can be incorporated into the device as components. Individual components that were necessary to perform these steps were characterized, and their mutual compatibility is also discussed.     

Diffusion of Oligonucleotides from within Iron‐Cross‐Linked,Polyelectrolyte‐Modified Alginate Beads: A Model System for Drug Release

An analytical model to describe diffusion of oligonucleotides from stable hydrogel beads is developed and experimentally verified. The synthesized alginate beads are Fe³⁺‐cross‐linked and polyelectrolyte‐doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross‐linking, that is, the absence of a surface‐layer barrier in this case. Furthermore, the results suggest a new simple approach to measuring the diffusion coefficient of mobile oligonucleotide molecules inside hydrogels. The considered alginate beads provide a model for a well‐defined component in drug‐release systems and for the oligonucleotide‐release transduction steps in drug‐delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate, which induces full oligonucleotide release with nondiffusional kinetics.     

Construction of microscale structures in enclosed microfluidic networks by using a magnetic beads based method

A large number of microscale structures have been used to elaborate flowing control or complex biological and chemical reaction on microfluidic chips. However, it is still inconvenient to fabricate microstructures with different heights (or depths) on the same substrate. These kinds of microstructures can be fabricated by using the photolithography and wet-etching method step by step, but involves time-consuming design and fabrication process, as well as complicated alignment of different masters. In addition, few existing methods can be used to perform fabrication within enclosed microfluidic networks. It is also difficult to change or remove existing microstructures within these networks. In this study, a magnetic-beads-based approach is presented to build microstructures in enclosed microfluidic networks. Electromagnetic field generated by microfabricated conducting wires (coils) is used to manipulate and trap magnetic beads on the bottom surface of a microchannel. These trapped beads are accumulated to form a microscale pile with desired shape, which can adjust liquid flow, dock cells, modify surface, and do some other things as those fabricated microstructures. Once the electromagnetic field is changed, trapped beads may form new shapes or be removed by a liquid flow. Besides being used in microfabrication, this magnetic-beads-based method can be used for novel microfluidic manipulation. It has been validated by forming microscale dam structure for cell docking and modified surface for cell patterning, as well as guiding the growth of neurons.     

基于微流控芯片的微阵列分析

微阵列芯片具有高通量、微量化和自动化等特点,已经在很多领域得到广泛应用。但是微阵列芯片仍然具有不足之处,如所需设备昂贵、分析时间较长、灵敏度不高、多样品平行分析能力不足等。微流控芯片微米级的通道具有相对较大的比表面积和较短的扩散距离,能够显著加快分析速度、提高检测效率、增强分析性能,并且能够加工大量的平行通道用于多样品分析。目前已经有大量文献报道将微流控芯片和微阵列芯片相结合,发展了独特的杂交方式并在实验和理论上分别证明了两者相结合的优势,本文综述了将微流控芯片技术应用于微阵列分析的研究进展,着重介绍了在微流控芯片上进行微阵列分析时的杂交方式、促进杂交的措施以及杂交过程的数学建模,同时也介绍了其他分析步骤方面的进展。最后分析了目前微流控芯片技术在进行微阵列杂交应用方面的不足及其原因,并指出这两项技术相结合的优势和未来。     

Dynamic microarray system with gentle retrieval mechanism for cell-encapsulating hydrogel beads

This paper describes a selective retrieval method for arrayed monodisperse hydrogel beads containing cells. We implemented modifications such as: (i) the incorporation of cavities as nucleation sites, (ii) indirect retrieval using bubble powered jets and (iii) the use of low boiling point fluid in our device to realize a gentle optical-based retrieval method. Parametric studies confirmed that these modifications dramatically reduced both the intensity and duration of applied laser for bubble formation. We also demonstrated for the first time the formation of a bead-based dynamic cell microarray by introducing cell-encapsulating alginate beads into our dynamic microfluidic system, and successfully retrieved an alginate bead from a fluidic trap. Tests with trypan blue revealed that membrane integrity of the encapsulated cells was not compromised by the retrieval process.     

Hydrophilic strips for preventing air bubble formation in a microfluidic chamber

In a microfluidic chamber, unwanted formation of air bubbles is a critical problem. Here, we present a hydrophilic strip array that prevents air bubble formation in a microfluidic chamber. The array is located on the top surface of the chamber, which has a large variation in width, and consists of a repeated arrangement of super‐ and moderately hydrophilic strips. This repeated arrangement allows a flat meniscus (i.e. liquid front) to form when various solutions consisting of a single stream or two parallel streams with different hydrophilicities move through the chamber. The flat meniscus produced by the array completely prevents the formation of bubbles. Without the array in the chamber, the meniscus shape is highly convex, and bubbles frequently form in the chamber. This hydrophilic strip array will facilitate the use of a microfluidic chamber with a large variation in width for various microfluidic applications.     

In vitro angiogenesis assay for the study of cell-encapsulation therapy

Cell encapsulation within alginate beads has potential as a sustained release system for delivering therapeutic agents in vivo while protecting encapsulated cells from the immune system. There is, however, no in vitro model for cell-encapsulation therapy that provides a suitable platform for quantitative assessment of physiological responses to secreted factors. Here we introduce a new microfluidic system specifically designed to evaluate and quantify the pro-angiogenic potential of factors secreted from human fetal lung fibroblasts encapsulated in beads on an intact endothelial cell monolayer. We confirmed that cell-encapsulating beads induced an angiogenic response in vitro, demonstrated by a strong correlation between the encapsulated cell density in the beads and the length of the vascular lumen formed in vitro. Conditions established by in vitro tests were then further shown to exert a pro-angiogenic response in vivo using a subcutaneous mouse model, forming an extensive network of functional luminal structures perfused with red blood cells.     

T cell chemotaxis in a simple microfluidic device

This paper describes the use of a simple microfluidic device for studying T cell chemotaxis. The microfluidic device is fabricated in poly(dimethylsiloxane) (PDMS) using soft-lithography and consists of a "Y" type fluidic channel. Solutions are infused into the device by syringe pumps and generate a concentration gradient in the channel by diffusion. We show that the experimentally measured gradient profiles agree nicely with theoretical predictions and the gradient is stable in the observation region for cell migration. Using this device, we demonstrate robust chemotaxis of human T cells in response to single and competing gradients of chemokine CCL19 and CXCL12. Because of the simplicity of the device, it can flexibly control gradient generation in space and time, and would allow generation of multiple gradient conditions in a single chip for highly parallel chemotaxis experimentation. Visualization of T cell chemotaxis has previously been limited to studies in 3D matrices or under agarose assays, which do not allow precise control or variation in conditions. Acknowledging the importance of lymphocyte homing in the adaptive immune response, the ability to study T cell chemotaxis in microfluidic devices offers a new approach for investigating lymphocyte migration and chemotaxis in vitro.     

Droplet microfluidics with magnetic beads: a new tool to investigate drug–protein interactions

In this study, we give the proof of concept for a method to determine binding constants of compounds in solution. By implementing a technique based on magnetic beads with a microfluidic device for segmented flow generation, we demonstrate, for individual droplets, fast, robust and complete separation of the magnetic beads. The beads are used as a carrier for one binding partner and hence, any bound molecule is separated likewise, while the segmentation into small microdroplets ensures fast mixing, and opens future prospects for droplet-wise analysis of drug candidate libraries. We employ the method for characterization of drug–protein binding, here warfarin to human serum albumin. The approach lays the basis for a microfluidic droplet-based screening device aimed at investigating the interactions of drugs with specific targets including enzymes and cells. Furthermore, the continuous method could be employed for various applications, such as binding assays, kinetic studies, and single cell analysis, in which rapid removal of a reactive component is required.     

The dielectrophoretic levitation and separation of latex beads in microchips.

A linear travelling wave dielectrophoretic (twDEP) microchip was fabricated and used to investigate both the levitation and the twDEP motion of latex beads as a function of applied potential and frequency, suspending medium conductivity, bead size, and surface characteristics. The surface conductance of the latex beads was characterised by measurement of the dielectrophoretic (DEP) crossover frequency. Collection of sample prior to initiation of twDEP was achieved using positive DEP forces generated by an integrated pair of parallel electrodes positioned in front of the twDEP array within the microfluidic channel. The principle of linear twDEP separation is shown using latex beads and rabbit heart cells.     

Synthesis of agar microparticles using temperature-controlled microfluidic devices for Cordyceps militaris cultivation

A temperature-controlled microfluidic approach was developed for fabricating monodispersed agar beads with the potential to be a brand-new strategy for cultivating Cordyceps militaris. The proposed microfluidic system features a circulating water bath with precise temperature control (temperature deviation ?T<0.1°C). This device holds the promise of allowing us to develop a temperature-controlled system, characterized as simple, low cost, and easy to set up and use. The size-controllable agar beads were achieved by utilizing microfluidic emulsification in the cross-junction channel under temperature-controlled conditions. The flow conditions of the dispersed/continuous phases were adjusted to generate various sizes of agar beads. Our results show that the microparticles produced are as small as 176 μm with a 95% particle size distribution within 5 μm. The prepared agar microparticles performed well as a substrate for the cultivation of C. militaris. The proposed method could also be applied for encapsulating biomaterials, enzymes, drugs, catalysts, and nanoparticles into agar beads for biomedical applications.     

Microfluidic biofunctionalisation protocols to form multi‐valent interactions for cell rolling and phenotype modification investigations

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro‐fluidic protocols. Many available processes either require expensive and time‐consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi‐valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin–biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC‐I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC‐I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC‐I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC‐I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.     

Nanofluidic system for the studies of single DNA molecules

Here, we describe a simple and low-cost lithographic technique to fabricate size-controllable nanopillar arrays inside the microfluidic channels for the studies of single DNA molecules. In this approach, nanosphere lithography has been employed to grow a single layer of well-ordered close-packed colloidal crystals inside the microfluidic channels. The size of the polymeric colloidal nanoparticles could be trimmed by oxygen plasma treatment. These size-trimmed colloidal nanoparticles were then used as the etching mask in a deep etching process. As a result, well-ordered size-controllable nanopillar arrays could be fabricated inside the microfluidic channels. The gap distance between the nanopillars could be tuned between 20 and 80 nm allowing the formation of nanofluidic system where the behavior of a single lambda-phage DNA molecule has been investigated. It was found that the lambda-phage DNA molecule could be fully stretched in the nanofluidic system formed by nanopillars with 50 nm gap distance at a field of 50 V/cm.