Programmable assembly of a metabolic pathway enzyme in a pre-packaged reusable bioMEMS device

We report a biofunctionalization strategy for the assembly of catalytically active enzymes within a completely packaged bioMEMS device, through the programmed generation of electrical signals at spatially and temporally defined sites. The enzyme of a bacterial metabolic pathway, S-adenosylhomocysteine nucleosidase (Pfs), is genetically fused with a pentatyrosine "pro-tag" at its C-terminus. Signal responsive assembly is based on covalent conjugation of Pfs to the aminopolysaccharide, chitosan, upon biochemical activation of the pro-tag, followed by electrodeposition of the enzyme-chitosan conjugate onto readily addressable sites in microfluidic channels. Compared to traditional physical entrapment and surface immobilization approaches in microfluidic environments, our signal-guided electrochemical assembly is unique in that the enzymes are assembled under mild aqueous conditions with spatial and temporal programmability and orientational control. Significantly, the chitosan-mediated enzyme assembly can be reversed, making the bioMEMS reusable for repeated assembly and catalytic activity. Additionally, the assembled enzymes retain catalytic activity over multiple days, demonstrating enhanced enzyme stability. We envision that this assembly strategy can be applied to rebuild metabolic pathways in microfluidic environments for antimicrobial drug discovery.     

Spontaneous generation of emulsion droplets by autonomous fluid-pumping using the gas permeability of poly(dimethylsiloxane) (PDMS)

Conventional droplet-based microfluidic systems require expensive, bulky external apparatuses, such as electric power supplies and pressure-driven pumps for fluid transportation. This study demonstrates an alternative way to produce emulsion droplets by autonomous fluid-handling based on the gas permeability of poly(dimethylsiloxane) (PDMS). Furthermore, basic concepts of fluid-handling are expanded to control the direction of the microfluid in the microfluidic device. The alternative pumping energy resulting from the high gas permeability of PDMS is used to generate water-in-oil (W/O) emulsions, which require no additional structures apart from microchannels. We can produce emulsion droplets by simple loading of the oil and aqueous solutions into the inlet reservoirs. During the operation of the microfluidic device, changes in droplet size, volumetric flow rate, and droplet generation frequency were quantitatively analyzed. As a result, we found that changes in the wetting properties of the microchannel greatly influence the volumetric flow rate and droplet generation frequency. This alternative microfluidic approach for preparing emulsion droplets in a simple and efficient manner is designed to improve the availability of emulsion droplets for point of care bioanalytical applications, in situ synthesis of materials, and on-site sample preparation tools.     

Fluorinated liquid-enabled protein handling and surfactant-aided crystallization for fully in situ digital microfluidic MALDI-MS analysis

A droplet (digital) microfluidic device has been developed that enables complete protein sample preparation for MALDI-MS analysis. Protein solution dispensing, disulfide bond reduction and alkylation, tryptic digestion, sample crystallization, and mass spectrometric analysis are all performed on a single device without the need for any ex situ sample purification. Fluorinated solvents are used as an alternative to surfactants to facilitate droplet movement and limit protein adsorption onto the device surface. The fluorinated solvent is removed by evaporation and so does not interfere with the MALDI-MS analysis. Adding a small amount of perfluorooctanoic acid to the MALDI matrix solution improves the yield, quality and consistency of the protein-matrix co-crystals, reducing the need for extensive 'sweet spot' searching and improving the spectral signal-to-noise ratio. These innovations are demonstrated in the complete processing and MALDI-MS analysis of lysozyme and cytochrome c. Because all of the sample processing steps and analysis can be performed on a single digital microfluidic device without the need for ex situ sample handling, higher throughput can be obtained in proteomics applications. More generally, the results presented here suggest that fluorinated liquids could also be used to minimize protein adsorption and improve crystallization in other types of lab-on-a-chip devices and applications.     

Novel one-pot route to monodisperse thermosensitive hollow microcapsules in a microfluidic system

We present a simple one-pot synthetic approach for the preparation of monodisperse thermo-sensitive poly(N-isopropylacrylamide) (PNIPAM) microcapsules in a microfluidic system. Based on the mechanism of shear force-driven break-off, aqueous droplets of monomer solution are continuously generated in an immiscible continuous phase containing photoinitiators. Under UV irradiation, activated initiators are diffused into the interface between the continuous phase and the aqueous droplets, which trigger polymerization of NIPAM monomers. The PNIPAM microcapsules produced are hollow microcapsules with a thin shell membrane, high monodispersity, and fast response to environmental temperature. In addition, the size of microcapsules produced can be manipulated by the flow rate of the continuous phase or aqueous phase and different concentrations of surfactant to control interfacial tension between continuous phase and aqueous phase. Furthermore, the versatility of this approach enables the preparation of monodisperse microcapsules having the capability to encapsulate various materials such as proteins and nanoparticles under mild conditions. The in situ microfluidic synthetic method provides a novel approach for the preparation of monodisperse hollow microcapsules via a one-pot route.     

Flexible generation of gradient electrospinning nanofibers using a microfluidic assisted approach

The nanofiber surface modified with physical or chemical gradients is very useful in a wide range of areas including tissue engineering, regenerative medicine, drug screening, and biomaterial chemistry. In this work, we presented a novel and straightforward microfluidic assisted approach to produce electrospinning nanofibers containing gradients in different compositions, nanoparticles and biomolecule concentrations. The series of gradient nanofibers were mainly produced by using a two inlet microfluidic device in combination with an electrospinning nozzle on a 3-D controllable platform, which exhibited different functions and properties. The controlled nanofibers with incorporated biomolecule gradient were used for guiding the spatial differentiation in mesenchymal stem cells (MSCs). This established approach is very simple, and flexible to operate, which might find enormous potential for biology and tissue engineering applications.     

Electrochemical biosensors based on Ti3C2Tx MXene: future perspectives for on-site analysis

MXenes are recently developed two-dimensional layered materials composed of early transition metal carbides and/or nitrides that provide unique characteristics for biosensor applications. This review presents the recent progress made on the usage and applications of MXenes in the field of electrochemical biosensors, including microfluidic biosensors and wearable microfluidic biosensors, and highlights the challenges with possible solutions and future needs. The multilayered configuration and high conductivity make these materials as an immobilization matrix for the biomolecule immobilization with activity retention and to be explored in the fabrication of electrochemical sensors, respectively. First, how the MXene nanocomposite as an electrode modifier affects the sensing performance of the electrochemical biosensors based on enzymes, aptamer/DNA, and immunoassays is well described. Second, recent developments in MXene nanocomposites as wearable biosensing platforms for the biomolecule detection are highlighted. This review pointed out the future concerns and directions for the use of MXene nanocomposites to fabricate advanced electrochemical biosensors with high sensitivity and selectivity. Specifically, possibilities for developing microfluidic electrochemical sensors and wearable electrochemical microfluidic sensors with integrated biomolecule detection are emphasized.     

Direct imaging of aligned neurofilament networks assembled using in situ dialysis in microchannels

We report a technique to produce aligned neurofilament networks for direct imaging and diffraction studies using in situ dialysis in a microfluidic device. The alignment is achieved by assembling neurofilaments from protein subunits confined within microchannels. Resulting network structure was probed by polarized optical microscopy and atomic force microscopy, which confirmed a high degree of protein alignment inside the microchannels. This technique can be expanded to facilitate structural studies of a wide range of filamentous proteins and their hierarchical assemblies under varying assembly conditions.     

Electrochemical fabrication of conducting polymer nanowires in an integrated microfluidic system

In this paper, we introduce a new approach for the in situ electrochemical fabrication of an individually addressable array of conducting polymer nanowires (CPNWs) positioned within an integrated microfluidic device and also demonstrate that such an integrated device can be used as a chemical sensor immediately after its construction.     

Simple approach to study biomolecule adsorption in polymeric microfluidic channels

Herein a simple analytical method is presented for the characterization of biomolecule adsorption on cyclo olefin polymer (COP, trade name: Zeonor^®) substrates which are widely used in microfluidic lab-on-a-chip devices. These Zeonor^® substrates do not possess native functional groups for specific reactions with biomolecules. Therefore, depending on the application, such substrates must be functionalized by surface chemistry methods to either enhance or suppress biomolecular adsorption. This work demonstrates a microfluidic method for evaluating the adsorption of antibodies and oligonucleotides surfaces. The method uses centrifugal microfluidic flow-through chips and can easily be implemented using common equipment such as a spin coater. The working principle is very simple. The user adds 40 L of the solution containing the sample to the starting side of a microfluidic channel, where it is moved through by centrifugal force. Some molecules are adsorbed in the channel. The sample is then collected at the other end in a small reservoir and the biomolecule concentration is measured. As a pilot application, we characterized the adsorption of goat anti-human IgG and a 20-mer DNA on Zeonor^®, and on three types of functionalized Zeonor: 3-aminopropyltriethoxysilane (APTES) modified surface with mainly positive charge, negatively charged surface with immobilized bovine serum albumin (BSA), and neutral, hydrogel-like film with polyethylene glycol (PEG) characteristics. This simple analytical approach adds to the fundamental understanding of the interaction forces in real, microfluidic systems. This method provides a straightforward and rapid way to screen surface compositions and chemistry, and relate these to their effects on the sensitivity and resistance to non-specific binding of bioassays using them. In an additional set of experiments, the surface area of the channels in this universal microfluidic chip was increased by precision milling of microscale trenches. This modified surface was then coated with APTES and tested for its potential to serve as a unique protein dilution feature.     

Enhancing protease activity assay in droplet-based microfluidics using a biomolecule concentrator

We introduce an integrated microfluidic device consisting of a biomolecule concentrator and a microdroplet generator, which enhances the limited sensitivity of low-abundance enzyme assays by concentrating biomolecules before encapsulating them into droplet microreactors. We used this platform to detect ultralow levels of matrix metalloproteinases (MMPs) from diluted cellular supernatant and showed that it significantly (~10-fold) reduced the time required to complete the assay and the sample volume used.     

Collagen microsphere production on a chip

We have developed an integrated microfluidic material processing chip and demonstrated the rapid production of collagen microspheres encapsulating cells with high uniformity and cell viability. The chip integrated three material processing steps. Monodisperse microdroplets were generated at a microfluidic T junction between aqueous and mineral oil flows. The flow was heated immediately to 37 °C to initiate collagen fiber assembly within a gelation channel. Gelled microspheres were extracted from the mineral oil phase into cell culture media within an extraction chamber. Collagen gelation immediately after microdroplet generation significantly reduced coalescence among microdroplets that led to non-uniform microsphere production. The microfluidic extraction approach led to higher microsphere recovery and cell viability than when a conventional centrifugation extraction approach was employed. These results indicate that chip-based material processing is a promising approach for cell-ECM microenvironment generation for applications such as tissue engineering and stem cell delivery.     

Microfluidic production of biopolymer microcapsules with controlled morphology

We report a microfluidic approach to generating capsules of biopolymer hydrogels. Droplets of an aqueous solution of a biopolymer were emulsified in an organic phase comprising a cross-linking agent. Polymer gelation was achieved in situ (on a microfluidic chip) by diffusion-controlled ionic cross-linking of the biopolymer, following the transfer of the cross-linking agent from the continuous phase to the droplets. Gelation was quenched by collecting particles in a large pool of cross-linking agent-free liquid. The structure of microgels (from capsules to gradient microgels to particles with a uniform structure) was controlled by varying the time of residence of droplets on the microfluidic chip and the concentration of the cross-linking agent in the continuous phase. We demonstrated the encapsulation of a controlled number of polystyrene beads in the microgel capsules. The described approach was applied to the preparation of capsules of several polysaccharides such as alginate, kappa-carrageenan, and carboxymethylcellulose.     

Direct synthesis and integration of functional nanostructures in microfluidic devices

Integration of functional nanostructures within a microfluidic device can synergize the advantages of both unique properties of nanomaterials and diverse functionalities of microfluidics. In this paper, we report a novel and simple method for the in situ synthesis and integration of ZnO nanowires by controlled hydrothermal reaction within microfluidic devices. By modulating synthesis parameters such as the seed preparation, synthesis time, and heating locations, the morphology and location of synthesized nanowires can be easily controlled. The applications of such nanostructure-integrated microfluidics for particle trapping and chemiresistive pH sensing were demonstrated.     

Simple and cheap microfluidic devices for the preparation of monodisperse emulsions

Droplet microfluidics, which can generate monodisperse droplets or bubbles in unlimited numbers, at high speed and with complex structures, have been extensively investigated in chemical and biological fields. However, most current methods for fabricating microfluidic devices, such as glass etching, soft lithography in polydimethylsiloxane (PDMS) or assembly of glass capillaries, are usually either expensive or complicated. Here we report the fabrication of simple and cheap microfluidic devices based on patterned coverslips and microscope glass slides. The advantages of our approach for fabricating microfluidic devices lie in a simple process, inexpensive processing equipment and economical laboratory supplies. The fabricated microfluidic devices feature a flexible design of microchannels, easy spatial patterning of surface wettability, and good chemical compatibility and optical properties. We demonstrate their utilities for generation of monodisperse single and double emulsions with highly controllable flexibility.     

Cell culture chip using low-shear mass transport

We have developed a flow cell that allows culturing adherent cells as well as suspended cells in a stable, homogeneous, and low-shear force environment. The device features continuous medium supply and waste exchange. In this paper, a simple and fast protocol for device design, fabrication, and assembly (sealing) based on a poly(dimethylsiloxane) (PMDS)/glass slide hybrid structure is described. The cell culture system performance was monitored, and the effective shear force inside the culture well was also determined. By manipulating the device dimensions and volumetric flow rate, shear stress was controlled during experiments. Cell adhesion, growth, proliferation, and death over long-term culture periods were observed by microscopy. The growth of both endothelial and suspension cells in this device exhibited comparable characteristics to those of traditional approaches. The low-shear culture device significantly reduced shear stress encountered in microfluidic systems, allowing both adherent and suspended cells to be grown in a simple device.     

Digital microfluidics using soft lithography

Although microfluidic chips have demonstrated basic functionality for single applications, performing varied and complex experiments on a single device is still technically challenging. While many groups have implemented control software to drive the pumps, valves, and electrodes used to manipulate fluids in microfluidic devices, a new level of programmability is needed for end users to orchestrate their own unique experiments on a given device. This paper presents an approach for programmable and scalable control of discrete fluid samples in a polydimethylsiloxane (PDMS) microfluidic system using multiphase flows. An immiscible fluid phase is utilized to separate aqueous samples from one another, and a novel "microfluidic latch" is used to precisely align a sample after it has been transported a long distance through the flow channels. To demonstrate the scalability of the approach, this paper introduces a "general-purpose" microfluidic chip containing a rotary mixer and addressable storage cells. The system is general purpose in that all operations on the chip operate in terms of unit-sized aqueous samples; using the underlying mechanisms for sample transport and storage, additional sensors and actuators can be integrated in a scalable manner. A novel high-level software library allows users to specify experiments in terms of variables (i.e., fluids) and operations (i.e., mixes) without the need for detailed knowledge about the underlying device architecture. This research represents a first step to provide a programmable interface to the microfluidic realm, with the aim of enabling a new level of scalability and flexibility for lab-on-a-chip experiments.     

Analysis of pressure-driven air bubble elimination in a microfluidic device

We report an analysis of pressure-driven bubble elimination for a gas-permeable microfluidic device. In this study, we described bubble elimination in a microfluidic device employing a gas permeation model and calculated the removal efficiency of bubbles. The correction factor for the simplified model was estimated with respect to the applied pressure. Based on the established model, the required time to remove a trapped bubble with a certain area was shown to be within an error of 11.58% by comparison with experimental results. Exploiting the model equation, we were able to completely remove the air bubbles appearing during the process of filling a microfluidic device with an aqueous solution.     

A Robust Microfluidic Device for the Synthesis and Crystal Growth of Organometallic Polymers with Highly Organized Structures

A simple and robust microfluidic device was developed to synthesize organometallic polymers with highly organized structures. The device is compatible with organic solvents. Reactants are loaded into pairs of reservoirs connected by a 15 cm long microchannel prefilled with solvents, thus allowing long‐term counter diffusion for self‐assembly of organometallic polymers. The process can be monitored, and the resulting crystalline polymers are harvested without damage. The device was used to synthesize three insoluble silver acetylides as single crystals of X‐ray diffraction quality. Importantly, for the first time, the single‐crystal structure of silver phenylacetylide was determined. The reported approach may have wide applications, such as crystallization of membrane proteins, synthesis and crystal growth of organic, inorganic, and polymeric coordination compounds, whose single crystals cannot be obtained using traditional methods.     

Investigating the effects of precursor concentration and gelling parameters on droplet-based generation of Ca-Alginate microgels: identifying new stable modes of droplet formation

Droplet-based microfluidics is an attractive approach for producing microgels due to its high potential to control the size and shape of the particles and precisely entrap the substances within the hydrogel matrix. However, the microfluidic generation of monodisperse microgels with desired structures is still challenging. Indeed, the rheological and interfacial properties of the immiscible fluids, as well as the adopted gelling strategy, play important roles in microfluidic methods. Herein, sodium alginate droplets with different concentrations are generated via a microfluidic device with a flow-focusing unit. Besides, a combined in situ and ex situ strategy is optimized to crosslink sodium alginate droplets in the presence of calcium ions. The effects of alginate concentration and junction width in the flow focusing unit are investigated on droplet size and droplet formation regimes. It is observed that by increasing the alginate concentration, the dripping regime of droplet formation may be transformed to one of the binary dripping or quasijetting regimes. In the binary dripping regime, two successive different-sized droplets are generated in each period of droplet formation, which leads to low monodispersity in the collected droplets. However, the droplets produced in the quasijetting regime are interestingly monodisperse and also smaller than those of the dripping and binary dripping regimes. The breakup dynamics of the alginate thread is also analyzed with a computational fluid dynamics (CFD) code. This analysis discloses that the viscous stresses, as well as the viscous dissipation, have important roles in controlling the stable modes of droplet formation.