Bio‐microfluidics: Biomaterials and Biomimetic Designs

Bio‐microfluidics applies biomaterials and biologically inspired structural designs (biomimetics) to microfluidic devices. Microfluidics, the techniques for constraining fluids on the micrometer and sub‐micrometer scale, offer applications ranging from lab‐on‐a‐chip to optofluidics. Despite this wealth of applications, the design of typical microfluidic devices imparts relatively simple, laminar behavior on fluids and is realized using materials and techniques from silicon planar fabrication. On the other hand, highly complex microfluidic behavior is commonplace in nature, where fluids with nonlinear rheology flow through chaotic vasculature composed from a range of biopolymers. In this Review, the current state of bio‐microfluidic materials, designs and applications are examined. Biopolymers enable bio‐microfluidic devices with versatile functionalization chemistries, flexibility in fabrication, and biocompatibility in vitro and in vivo. Polymeric materials such as alginate, collagen, chitosan, and silk are being explored as bulk and film materials for bio‐microfluidics. Hydrogels offer options for mechanically functional devices for microfluidic systems such as self‐regulating valves, microlens arrays and drug release systems, vital for integrated bio‐microfluidic devices. These devices including growth factor gradients to study cell responses, blood analysis, biomimetic capillary designs, and blood vessel tissue culture systems, as some recent examples of inroads in the field that should lead the way in a new generation of microfluidic devices for bio‐related needs and applications. Perhaps one of the most intriguing directions for the future will be fully implantable microfluidic devices that will also integrate with existing vasculature and slowly degrade to fully recapitulate native tissue structure and function, yet serve critical interim functions, such as tissue maintenance, drug release, mechanical support, and cell delivery.     

Deconstructed Microfluidic Bone Marrow On‐A‐Chip to Study Normal and Malignant Hemopoietic Cell–Niche Interactions

Human hematopoietic niches are complex specialized microenvironments that maintain and regulate hematopoietic stem and progenitor cells (HSPC). Thus far, most of the studies performed investigating alterations of HSPC‐niche dynamic interactions are conducted in animal models. Herein, organ microengineering with microfluidics is combined to develop a human bone marrow (BM)‐on‐a‐chip with an integrated recirculating perfusion system that consolidates a variety of important parameters such as 3D architecture, cell–cell/cell–matrix interactions, and circulation, allowing a better mimicry of in vivo conditions. The complex BM environment is deconvoluted to 4 major distinct, but integrated, tissue‐engineered 3D niche constructs housed within a single, closed, recirculating microfluidic device system, and equipped with cell tracking technology. It is shown that this technology successfully enables the identification and quantification of preferential interactions—homing and retention—of circulating normal and malignant HSPC with distinct niches.     

Light‐Directed Liquid Manipulation in Flexible Bilayer Microtubes

Flexible microfluidic systems have potential in wearable and implantable medical applications. Directional liquid transportation in these systems typically requires mechanical pumps, gas tanks, and magnetic actuators. Herein, an alternative strategy is presented for light‐directed liquid manipulation in flexible bilayer microtubes, which are composed of a commercially available supporting layer and the photodeformable layer of a newly designed azobenzene‐containing linear liquid crystal copolymer. Upon moderate visible light irradiation, various liquid slugs confined in the flexible microtubes are driven in the preset direction over a long distance due to photodeformation‐induced asymmetric capillary forces. Several light‐driven prototypes of parallel array, closed‐loop channel, and multiple micropump are established by the flexible bilayer microtubes to achieve liquid manipulation. Furthermore, an example of a wearable device attached to a finger for light‐directed liquid motion is demonstrated in different gestures. These unique photocontrollable flexible microtubes offer a novel concept of wearable microfluidics.     

High Throughput‐Per‐Footprint Inertial Focusing

Matching the scale of microfluidic flow systems with that of microelectronic chips for realizing monolithically integrated systems still needs to be accomplished. However, this is appealing only if such re‐scaling does not compromise the fluidic throughput. This is related to the fact that the cost of microelectronic circuits primarily depends on the layout footprint, while the performance of many microfluidic systems, like flow cytometers, is measured by the throughput. The simple operation of inertial particle focusing makes it a promising technique for use in such integrated flow cytometer applications, however, microfluidic footprints demonstrated so far preclude monolithic integration. Here, the scaling limits of throughput‐per‐footprint (TPFP) in using inertial focusing are explored by studying the interplay between theory, the effect of channel Reynolds numbers up to 1500 on focusing, the entry length for the laminar flow to develop, and pressure resistance of the microchannels. Inertial particle focusing is demonstrated with a TPFP up to 0.3 L/(min cm²) in high aspect‐ratio rectangular microfluidic channels that are readily fabricated with a post‐CMOS integratable process, suggesting at least a 100‐fold improvement compared to previously demonstrated techniques. Not only can this be an enabling technology for realizing cost‐effective monolithically integrated flow cytometry devices, but the methodology represented here can also open perspectives for miniaturization of many biomedical microfluidic applications requiring monolithic integration with microelectronics without compromising the throughput.     

Microfluidic Single‐Cell Omics Analysis

The commonly existing cellular heterogeneity plays a critical role in biological processes such as embryonic development, cell differentiation, and disease progress. Single‐cell omics‐based heterogeneous studies have great significance for identifying different cell populations, discovering new cell types, revealing informative cell features, and uncovering significant interrelationships between cells. Recently, microfluidics has evolved to be a powerful technology for single‐cell omics analysis due to its merits of throughput, sensitivity, and accuracy. Herein, the recent advances of microfluidic single‐cell omics analysis, including different microfluidic platform designs, lysis strategies, and omics analysis techniques, are reviewed. Representative applications of microfluidic single‐cell omics analysis in complex biological studies are then summarized. Finally, a few perspectives on the future challenges and development trends of microfluidic‐assisted single‐cell omics analysis are discussed.     

On‐Site Formation of Emulsions by Controlled Air Plugs

Air plugs are usually undesirable in microfluidic systems because of their detrimental effect on the system's stability and integrity. By controlling the wetting properties as well as the topographical geometry of the microchannel, it is reported herein that air plugs can be generated in pre‐defined locations to function as a unique valve, allowing for the on‐site formation of various emulsions including single‐component droplets, composite droplets with droplet‐to‐droplet concentration gradient, blood droplets, paired droplets, as well as bubble arrays without the need for precious flow control, a difficult task with conventional droplet microfluidics. Moreover, the self‐generated air valve can be readily deactivated (turned off) by the introduction of an oil phase, allowing for the on‐demand release of as‐formed droplets for downstream applications. It is proposed that the simple, yet versatile nature of this technique can act as an important method for droplet microfluidics and, in particular, is ideal for the development of affordable lab‐on‐a‐chip systems without suffering from scalability and manufacturing challenges that typically confound the conventional droplet microfluidics.     

Anisotropic Crystalline Protein Nanolayers as Multi‐Functional Biointerface for Patterned Co‐Cultures of Adherent and Non‐Adherent Cells in Microfluidic Devices

The spatial arrangement of cells in their microenvironment is known to significantly influence cellular behavior, thus making the control of cellular organization an important parameter of in vitro co‐culture models. However, recent advances in micropatterning co‐culture methods within biochips do not address the simultaneous cultivation of anchorage‐dependent and non‐adherent cells. To address this methodological gap we combine S‐layer technology with microfluidics to pattern co‐cultures to study the cell‐to‐cell and cell‐to‐surface interactions under physiologically relevant conditions. We exploit the unique self‐assembly properties of SbpA and SbsB S‐layers to create an anisotropic protein nanobiointerface on‐chip with spatially‐defined cytophilic (adhesive) and cytophobic (repulsive) properties. While microfluidics control physical parameters such as shear force and flow velocities, our anisotropic protein nanobiointerface regulates the biological aspects of the co‐culture method including biocompatibility, biostability, and affinity to non‐adherent cells. The reliability and reproducibility of our microfluidic co‐culture strategy based on laminar flow patterned protein nanolayers is envisioned to advance in vitro models for biomedical research.     

Desktop‐Stereolithography 3D‐Printing of a Poly(dimethylsiloxane)‐Based Material with Sylgard‐184 Properties

The advantageous physiochemical properties of poly(dimethylsiloxane) (PDMS) have made it an extremely useful material for prototyping in various technological, scientific, and clinical areas. However, PDMS molding is a manual procedure and requires tedious assembly steps, especially for 3D designs, thereby limiting its access and usability. On the other hand, automated digital manufacturing processes such as stereolithography (SL) enable true 3D design and fabrication. Here the formulation, characterization, and SL application of a 3D‐printable PDMS resin (3DP‐PDMS) based on commercially available PDMS‐methacrylate macromers, a high‐efficiency photoinitiator and a high‐absorbance photosensitizer, is reported. Using a desktop SL‐printer, optically transparent submillimeter structures and microfluidic channels are demonstrated. An optimized blend of PDMS‐methacrylate macromers is also used to SL‐print structures with mechanical properties similar to conventional thermally cured PDMS (Sylgard‐184). Furthermore, it is shown that SL‐printed 3DP‐PDMS substrates can be rendered suitable for mammalian cell culture. The 3DP‐PDMS resin enables assembly‐free, automated, digital manufacturing of PDMS, which should facilitate the prototyping of devices for microfluidics, organ‐on‐chip platforms, soft robotics, flexible electronics, and sensors, among others.     

In Situ Two‐Step Photoreduced SERS Materials for On‐Chip Single‐Molecule Spectroscopy with High Reproducibility

A method is developed to synthesize surface‐enhanced Raman scattering (SERS) materials capable of single‐molecule detection, integrated with a microfluidic system. Using a focused laser, silver nanoparticle aggregates as SERS monitors are fabricated in a microfluidic channel through photochemical reduction. After washing out the monitor, the aggregates are irradiated again by the same laser. This key step leads to full reduction of the residual reactants, which generates numerous small silver nanoparticles on the former nanoaggregates. Consequently, the enhancement ability of the SERS monitor is greatly boosted due to the emergence of new “hot spots.” At the same time, the influence of the notorious “memory effect” in microfluidics is substantially suppressed due to the depletion of surface residues. Taking these advantages, two‐step photoreduced SERS materials are able to detect different types of molecules with the concentration down to 10^?13m . Based on a well‐accepted bianalyte approach, it is proved that the detection limit reaches the single‐molecule level. From a practical point of view, the detection reproducibility at different probing concentrations is also investigated. It is found that the effective single‐molecule SERS measurements can be raised up to ≈50%. This microfluidic SERS with high reproducibility and ultrasensitivity will find promising applications in on‐chip single‐molecule spectroscopy.     

Three-dimensional fluidic self-assembly by axis translation of two-dimensionally fabricated microcomponents in railed microfluidics

A method for high-throughput 3D self-assembly of 2D photopatterned microstructures using railed microfluidics is presented. Vertical device patterning of heterogeneous materials requires high-level integration using conventional microelectromechanical system (MEMS) technology; however, 3D railed assembly enables easy and fast self-assembly via a fluidic axis-translation process and simple material exchange in microfluidic channels. Individually photopatterned 2D microstructures are axis-translated from in-plane to out-of-plane and fluidically self-assembled, guided by side-rails in microfluidic channels to form a 3D morphology. Since the structures are fabricated in fluidic environments, there are no fixed initial points on the channel substrate allowing fluidic horizontal stacking of erected 2D structures. The guiding mechanism of railed microfluidics enables efficient fluidic handling and deterministic 3D self-assembly of heterogeneous components such as electronic components or polymeric microstructures using only fluidic force.     

Covalent Bonding of Thermoplastics to Rubbers for Printable,Reel‐to‐Reel Processing in Soft Robotics and Microfluidics

The lamination of mechanically stiff structures to elastic materials is prevalent in biological systems and popular in many emerging synthetic systems, such as soft robotics, microfluidics, stretchable electronics, and pop‐up assemblies. The disparate mechanical and chemical properties of these materials have made it challenging to develop universal synthetic procedures capable of reliably adhering to these classes of materials together. Herein, a simple and scalable procedure is described that is capable of covalently laminating a variety of commodity (“off‐the‐shelf”) thermoplastic sheets to silicone rubber films. When combined with laser printing, the nonbonding sites can be “printed” onto the thermoplastic sheets, enabling the direct fabrication of microfluidic systems for actuation and liquid handling applications. The versatility of this approach in generating thin, multifunctional laminates is demonstrated through the fabrication of milliscale soft actuators and grippers with hinged articulation and microfluidic channels with built‐in optical filtering and pressure‐dependent geometries. This method of fabrication offers several advantages, including technical simplicity, process scalability, design versatility, and material diversity. The concepts and strategies presented herein are broadly applicable to the soft robotics, microfluidics, and advanced and additive manufacturing communities where hybrid rubber/plastic structures are prevalent.     

Addressable Microfluidic Polymer Chip for DNA‐Directed Immobilization of Oligonucleotide‐Tagged Compounds

A microfluidic polymer chip for the self‐assembly of DNA conjugates through DNA‐directed immobilization is developed. The chip is fabricated from two parts, one of which contains a microfluidic channel produced from poly(dimethylsiloxane) (PDMS) by replica‐casting technique using a mold prepared by photolithographic techniques. The microfluidic part is sealed by covalent bonding with a chemically activated glass slide containing a DNA oligonucleotide microarray. The dimension of the PDMS–glass microfluidic chip is equivalent to standard microscope slides (76 × 26 mm²). The DNA microarray surface inside the microfluidic channels is configured through conventional spotting, and the resulting DNA patches can be conveniently addressed with compounds containing complementary DNA tags. To demonstrate the utility of the addressable surface within the microfluidic channel, DNA‐directed immobilization (DDI) of DNA‐modified gold nanoparticles (AuNPs) and DNA‐conjugates of the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) are carried out. DDI of AuNPs is used to demonstrate site selectivity and reversibility of the surface‐modification process. In the case of the DNA–enzyme conjugates, the patterned assembly of the two enzymes allows the establishment and investigation of the coupled reaction of GOx and HRP, with particular emphasis on surface coverage and lateral flow rates. The results demonstrate that this addressable chip is well suited for the generation of fluidically coupled multi‐enzyme microreactors.     

Recent Advances in Microfluidic Platforms Applied in Cancer Metastasis: Circulating Tumor Cells' (CTCs) Isolation and Tumor‐On‐A‐Chip

Cancer remains the leading cause of death worldwide despite the enormous efforts that are made in the development of cancer biology and anticancer therapeutic treatment. Furthermore, recent studies in oncology have focused on the complex cancer metastatic process as metastatic disease contributes to more than 90% of tumor‐related death. In the metastatic process, isolation and analysis of circulating tumor cells (CTCs) play a vital role in diagnosis and prognosis of cancer patients at an early stage. To obtain relevant information on cancer metastasis and progression from CTCs, reliable approaches are required for CTC detection and isolation. Additionally, experimental platforms mimicking the tumor microenvironment in vitro give a better understanding of the metastatic microenvironment and antimetastatic drugs' screening. With the advancement of microfabrication and rapid prototyping, microfluidic techniques are now increasingly being exploited to study cancer metastasis as they allow precise control of fluids in small volume and rapid sample processing at relatively low cost and with high sensitivity. Recent advancements in microfluidic platforms utilized in various methods for CTCs' isolation and tumor models recapitulating the metastatic microenvironment (tumor‐on‐a‐chip) are comprehensively reviewed. Future perspectives on microfluidics for cancer metastasis are proposed.     

Temperature Gradients Drive Bulk Flow Within Microchannel Lined by Fluid–Fluid Interfaces

Surface tension gradients induce Marangoni flow, which may be exploited for fluid transport. At the micrometer scale, these surface‐driven flows can be quite significant. By introducing fluid–fluid interfaces along the walls of microfluidic channels, bulk fluid flows driven by temperature gradients are observed. The temperature dependence of the fluid–fluid interfacial tension appears responsible for these flows. In this report, the design concept for a biocompatible microchannel capable of being powered by solar irradiation is provided. Using microscale particle image velocimetry, a bulk flow generated by apparent surface tension gradients along the walls is observed. The direction of flow relative to the imposed temperature gradient agrees with the expected surface tension gradient. The phenomenon's ability to replace bulky peripherals, like traditional syringe pumps, on a diagnostic microfluidic device that captures and detects leukocyte subpopulations within blood is demonstrated. Such microfluidic devices may be implemented for clinical assays at the point of care without the use of electricity.     

One Cell,One Drop,One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co‐planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL‐sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in‐house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI‐H1299 lung squamous cell population resulting from loss‐of‐function eGFP and RAF1 gene knockout transfections.     

Microfluidic‐Based Approaches in Targeted Cell/Particle Separation Based on Physical Properties: Fundamentals and Applications

Cell separation is a key step in many biomedical research areas including biotechnology, cancer research, regenerative medicine, and drug discovery. While conventional cell sorting approaches have led to high‐efficiency sorting by exploiting the cell's specific properties, microfluidics has shown great promise in cell separation by exploiting different physical principles and using different properties of the cells. In particular, label‐free cell separation techniques are highly recommended to minimize cell damage and avoid costly and labor‐intensive steps of labeling molecular signatures of cells. In general, microfluidic‐based cell sorting approaches can separate cells using “intrinsic” (e.g., fluid dynamic forces) versus “extrinsic” external forces (e.g., magnetic, electric field, etc.) and by using different properties of cells including size, density, deformability, shape, as well as electrical, magnetic, and compressibility/acoustic properties to select target cells from a heterogeneous cell population. In this work, principles and applications of the most commonly used label‐free microfluidic‐based cell separation methods are described. In particular, applications of microfluidic methods for the separation of circulating tumor cells, blood cells, immune cells, stem cells, and other biological cells are summarized. Computational approaches complementing such microfluidic methods are also explained. Finally, challenges and perspectives to further develop microfluidic‐based cell separation methods are discussed.     

Fracture‐Based Fabrication of Normally Closed,Adjustable, and Fully Reversible Microscale Fluidic Channels

Adjustable fluidic structures play an important role in microfluidic systems. Fracture of multilayered materials under applied tension has been previously demonstrated as a convenient, simple, and inexpensive approach to fabricate nanoscale adjustable structures; here, it is demonstrated how to extend this concept to the microscale. This is achieved by a novel pairing of materials that leverages fracture mechanics to limit crack formation to a specified region, allowing to create size‐controllable and adjustable microfluidic structures. This technique can be used to fabricate “normally closed” microfluidic channels that are completely reversible, a feature that is challenging to achieve in conventional systems without careful engineering controls. The adjustable microfluidic channels are then applied to mechanically lyse single cells, and subsequently manipulate the released nuclear chromatin, creating new possibilities for epigenetic analysis of single cells. This simple, versatile, and robust technology provides an easily accessible pathway to construct adjustable microfluidic structures, which will be useful in developing complex assays and experiments even in resource‐limited settings.     

In-Plane Integration of Polymer Microfluidic Channels With Optical Waveguides– A Preliminary Investigation

The next major challenges for lab-on-a-chip (LoC) technology are 1) the integration of microfluidics with optical detection technologies and 2) the large-scale production of devices at a low cost. In this paper the fabrication and characterisation of a simple optical LoC platform comprising integrated multimode waveguides and microfluidic channels based on a photo-patternable acrylate based polymer is reported. The polymer can be patterned into both waveguides and microfluidic channels using photolithography. Devices are therefore both quick and cost-effective to fabricate, resulting in chips that are potentially disposable. The devices are designed to be highly sensitive, using an in-plane direct excitation configuration in which waveguides intersect the microfluidic channel orthogonally. The waveguides are used both to guide the excitation light and to collect the fluorescence signal from the analyte. The potential of the device to be used for fluorescence measurements is demonstrated using an aqueous solution of sodium fluorescein. A detection limit of 7 nM is achieved. The possibilities offered by such a device design, in providing a cost-effective and disposable measurement system based on the integration of optical waveguides with LoC technology is discussed.     

Microfluidics: Nanostructuring of Titania Thin Films by a Combination of Microfluidics and Block‐Copolymer‐Based Sol–Gel Templating (Small 7/2011)

Sol–gel templating of titania thin films with the amphiphilic diblock copolymer poly(dimethyl siloxane)‐block‐methyl methacrylate poly(ethylene oxide) is combined with microfluidic technology to control the structure formation. Due to the laminar flow conditions in the microfluidic cell, a better control of the local composition of the reactive fluid is achieved. The resulting titania films exhibit mesopores and macropores, as determined with scanning electron microscopy, X‐ray reflectivity, and grazing incidence small angle X‐ray scattering. The titania morphology has three features that are beneficial for application in photovoltaics: 1) a large surface‐to‐volume ratio important for charge generation with disordered hexagonally arranged mesopores of 25 nm size and a film porosity of up to 0.79, 2) enhanced light scattering that enables the absorption of more light, and 3) a dense titania layer with a thickness of about 6 nm at the substrate (bottom electrode) to prevent short circuits. An optical characterization complements the structural investigation.