A perspective on paper-based microfluidics: Current status and future trends

"Paper-based microfluidics" or "lab on paper," as a burgeoning research field with its beginning in 2007, provides a novel system for fluid handling and fluid analysis for a variety of applications including health diagnostics, environmental monitoring as well as food quality testing. The reasons why paper becomes an attractive substrate for making microfluidic systems include: (1) it is a ubiquitous and extremely cheap cellulosic material; (2) it is compatible with many chemical/biochemical/medical applications; and (3) it transports liquids using capillary forces without the assistance of external forces. By building microfluidic channels on paper, liquid flow is confined within the channels, and therefore, liquid flow can be guided in a controlled manner. A variety of 2D and even 3D microfluidic channels have been created on paper, which are able to transport liquids in the predesigned pathways on paper. At the current stage of its development, paper-based microfluidic system is claimed to be low-cost, easy-to-use, disposable, and equipment-free, and therefore, is a rising technology particularly relevant to improving the healthcare and disease screening in the developing world, especially for those areas with no- or low-infrastructure and limited trained medical and health professionals. The research in paper-based microfluidics is experiencing a period of explosion; most published works have focused on: (1) inventing low-cost and simple fabrication techniques for paper-based microfluidic devices; and (2) exploring new applications of paper-based microfluidics by incorporating efficient detection methods. This paper aims to review both the fabrication techniques and applications of paper-based microfluidics reported to date. This paper also attempts to convey to the readers, from the authors' point of view the current limitations of paper-based microfluidics which require further research, and a few perspective directions this new analytical system may take in its development.     

Advances in three-dimensional rapid prototyping of microfluidic devices for biological applications

The capability of 3D printing technologies for direct production of complex 3D structures in a single step has recently attracted an ever increasing interest within the field of microfluidics. Recently, ultrafast lasers have also allowed developing new methods for production of internal microfluidic channels within the bulk of glass and polymer materials by direct internal 3D laser writing. This review critically summarizes the latest advances in the production of microfluidic 3D structures by using 3D printing technologies and direct internal 3D laser writing fabrication methods. Current applications of these rapid prototyped microfluidic platforms in biology will be also discussed. These include imaging of cells and living organisms, electrochemical detection of viruses and neurotransmitters, and studies in drug transport and induced-release of adenosine triphosphate from erythrocytes.     

Three-dimensional fit-to-flow microfluidic assembly

Three-dimensional microfluidics holds great promise for large-scale integration of versatile, digitalized, and multitasking fluidic manipulations for biological and clinical applications. Successful translation of microfluidic toolsets to these purposes faces persistent technical challenges, such as reliable system-level packaging, device assembly and alignment, and world-to-chip interface. In this paper, we extended our previously established fit-to-flow (F2F) world-to-chip interconnection scheme to a complete system-level assembly strategy that addresses the three-dimensional microfluidic integration on demand. The modular F2F assembly consists of an interfacial chip, pluggable alignment modules, and multiple monolithic layers of microfluidic channels, through which convoluted three-dimensional microfluidic networks can be easily assembled and readily sealed with the capability of reconfigurable fluid flow. The monolithic laser-micromachining process simplifies and standardizes the fabrication of single-layer pluggable polymeric modules, which can be mass-produced as the renowned Lego^® building blocks. In addition, interlocking features are implemented between the plug-and-play microfluidic chips and the complementary alignment modules through the F2F assembly, resulting in facile and secure alignment with average misalignment of 45 μm. Importantly, the 3D multilayer microfluidic assembly has a comparable sealing performance as the conventional single-layer devices, providing an average leakage pressure of 38.47 kPa. The modular reconfigurability of the system-level reversible packaging concept has been demonstrated by re-routing microfluidic flows through interchangeable modular microchannel layers.     

Automated calibration of 3D-printed microfluidic devices based on computer vision

With the development of 3D printing techniques, the application of it in microfluidic/Lab-on-a-Chip (LoC) fabrication is becoming more and more attractive. However, to achieve a satisfying printing quality of the target devices, researchers usually require quite an amount of work in calibration trials even for high-end 3D printers. To increase the calibration efficiency of the average priced printers and promote the application of 3D printing technology in the microfluidic community, this work has presented a computer vision (CV)-based method for rapid and precise 3D printing calibration with examples on cylindrical hole/post diameters of 0.2–2.4 mm and rectangular hole/post widths of 0.2–1.0 mm by a stereolithography-based 3D printer. Our method is fully automated, which contains five steps and only needs a camera at hand to provide photos for convolutional neural network recognition. The experimental results showed that our CV-based method could provide calibrated dimensions with just one print of the specific calibration ruler to meet user desire. The higher resolution of the photo provides a higher precision in calibration. Subsequently, only one more print for the target device is needed after the calibration process. Overall, this work has provided a quick and precise calibration tool for researchers to apply 3D printing in the fabrication of their microfluidic/LoC devices with average price printers. Besides, with our open source calibration software and calibration ruler design file, researchers can modify the specific setting based on customized needs and conduct calibration on any type of 3D printer.     

Assessment of the biocompatibility of three-dimensional-printed polymers using multispecies toxicity tests

Additive manufacturing was adopted in multiple fields of life sciences. It is also becoming a popular tool for rapid prototyping of microfluidic and biomedical devices. Limited studies have been performed to investigate the biological implications of using 3D printed polymers. Here we assessed the biocompatibility of seven commercially available polymers, using a battery of standardized bioassays for chemical risk assessment. Our data show that leachates from photopolymers substrata appear to be very toxic to vertebrates and several invertebrate indicator organisms. These results demonstrate significant consequences for the use of selected photopolymers in the fabrication of bio-devices.     

Implementation of tetra-poly(ethylene glycol) hydrogel with high mechanical strength into microfluidic device technology

Hydrogels have several excellent characteristics suitable for biomedical use such as softness, biological inertness and solute permeability. Hence, integrating hydrogels into microfluidic devices is a promising approach for providing additional functions such as biocompatibility and porosity, to microfluidic devices. However, the poor mechanical strength of hydrogels has severely limited device design and fabrication. A tetra-poly(ethylene glycol) (tetra-PEG) hydrogel synthesized recently has high mechanical strength and is expected to overcome such a limitation. In this research, we have comprehensively studied the implementation of tetra-PEG gel into microfluidic device technology. First, the fabrication of tetra-PEG gel/PDMS hybrid microchannels was established by developing a simple and robust bonding technique. Second, some fundamental features of tetra-PEG gel/PDMS hybrid microchannels, particularly fluid flow and mass transfer, were studied. Finally, to demonstrate the unique application of tetra-PEG-gel-integrated microfluidic devices, the generation of patterned chemical modulation with the maximum concentration gradient: 10% per 20 μm in a hydrogel was performed. The techniques developed in this study are expected to provide fundamental and beneficial methods of developing various microfluidic devices for life science and biomedical applications.     

Optimizing design and fabrication of microfluidic devices for cell cultures: An effective approach to control cell microenvironment in three dimensions

The effects of gradients of bioactive molecules on the cell microenvironment are crucial in several biological processes, such as chemotaxis, angiogenesis, and tumor progression. The elucidation of the basic mechanisms regulating cell responses to gradients requires a tight control of the spatio-temporal features of such gradients. Microfluidics integrating 3D gels are useful tools to fulfill this requirement. However, even tiny flaws in the design or in the fabrication process may severely impair microenvironmental control, thus leading to inconsistent results. Here, we report a sequence of actions aimed at the design and fabrication of a reliable and robust microfluidic device integrated with collagen gel for cell culturing in 3D, subjected to a predetermined gradient of biomolecular signals. In particular, we developed a simple and effective solution to the frequently occurring technical problems of gas bubble formation and 3D matrix collapsing or detaching from the walls. The device here proposed, in Polydimethylsiloxane, was designed to improve the stability of the cell-laden hydrogel, where bubble deprived conditioning media flow laterally to the gel. We report the correct procedure to fill the device with the cell populated gel avoiding the entrapment of gas bubbles, yet maintaining cell viability. Numerical simulations and experiments with fluorescent probes demonstrated the establishment and stability of a concentration gradient across the gel. Finally, chemotaxis experiments of human Mesenchymal Stem Cells under the effects of Bone Morphogenetic Protein-2 gradients were performed in order to demonstrate the efficacy of the system in controlling cell microenvironment. The proposed procedure is sufficiently versatile and simple to be used also for different device geometries or experimental setups.     

Polydimethylsiloxane-based conducting composites and their applications in microfluidic chip fabrication

This paper reviews the design and fabrication of polydimethylsiloxane (PDMS)-based conducting composites and their applications in microfluidic chip fabrication. Owing to their good electrical conductivity and rubberlike elastic characteristics, these composites can be used variously in soft-touch electronic packaging, planar and three-dimensional electronic circuits, and in-chip electrodes. Several microfluidic components fabricated with PDMS-based composites have been introduced, including a microfluidic mixer, a microheater, a micropump, a microdroplet controller, as well as an all-in-one microfluidic chip.     

Rapid and cost-effective fabrication of selectively permeable calcium-alginate microfluidic device using "modified" embedded template method

In this paper, we have presented a non-lithographic embedded template method for rapid and cost-effective fabrication of a selectively permeable calcium-alginate (Ca-alginate) based microfluidic device with long serpentine delay channel. To demonstrate the versatility of the presented method, we have demonstrated two different strategies to fabricate serpentine long delay channels without using any sophisticated microfabrication techniques, in formal lab atmosphere. The procedure presented here, also, enables the preparation of a multilayered microfluidic device with channels of varying dimensions, in a single device without using any sophisticated micromachining instrumentation. In addition, we have also qualitatively studied the diffusion of small and large molecules from a Ca-alginate based microfluidic device and proposed a method to effectively control the out-flow of macro biomolecules from the crosslinked Ca-alginate matrix to create a selectively permeable matrix required for various biological and biomimetic applications, as mentioned in the Introduction section of this work.     

Rapid and cost-effective fabrication of selectively permeable calcium-alginate microfluidic device using ��modified�� embedded template method

In this paper, we have presented a non-lithographic embedded template method for rapid and cost-effective fabrication of a selectively permeable calcium-alginate (Ca-alginate) based microfluidic device with long serpentine delay channel. To demonstrate the versatility of the presented method, we have demonstrated two different strategies to fabricate serpentine long delay channels without using any sophisticated microfabrication techniques, in formal lab atmosphere. The procedure presented here, also, enables the preparation of a multilayered microfluidic device with channels of varying dimensions, in a single device without using any sophisticated micromachining instrumentation. In addition, we have also qualitatively studied the diffusion of small and large molecules from a Ca-alginate based microfluidic device and proposed a method to effectively control the out-flow of macro biomolecules from the crosslinked Ca-alginate matrix to create a selectively permeable matrix required for various biological and biomimetic applications, as mentioned in the Introduction section of this work.     

Microfluidic devices for cell cultivation and proliferation

Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined.     

A nanoporous optofluidic microsystem for highly sensitive and repeatable surface enhanced Raman spectroscopy detection

We report the demonstration of an optofluidic surface enhanced Raman spectroscopy (SERS) device that leverages a nanoporous microfluidic matrix to improve the SERS detection performance by more than two orders of magnitude as compared to a typical open microfluidic channel. Although it is a growing trend to integrate optical biosensors into microfluidic channels, this basic combination has been detrimental to the sensing performance when applied to SERS. Recently, however, synergistic combinations between microfluidic functions and photonics (i.e., optofluidics) have been implemented that improve the detection performance of SERS. Conceptually, the simplest optofluidic SERS techniques reported to date utilize a single nanofluidic channel to trap nanoparticle-analyte conjugates as a method of preconcentration before detection. In this work, we leverage this paradigm while improving upon the simplicity by forming a 3D nanofluidic network with packed nanoporous silica microspheres in a microfluidic channel; this creates a concentration matrix that traps silver nanoclusters and adsorbed analytes into the SERS detection volume. With this approach, we are able to achieve a detection limit of 400 attomoles of Rhodamine 6G after only 2 min of sample loading with high chip-to-chip repeatability. Due to the high number of fluidic paths in the nanoporous channel, this approach is less prone to clogging than single nanofluidic inlets, and the loading time is decreased compared to previous reports. In addition, fabrication of this microsystem is quite simple, as nanoscale fabrication is not necessary. Finally, integrated multimode fiber optic cables eliminate the need for optical alignment, and thus the device is relevant for portable and automated applications in the field, including point-of-sample and point-of-care detection. To illustrate a relevant field-based application, we demonstrate the detection of 12 ppb of the organophosphate malathion in water using the nanofluidic SERS microsystem.     

Uniform yeast cell assembly via microfluidics

This paper reports the use of microfluidic approaches for the fabrication of yeastosomes (yeast-celloidosomes) based on self-assembly of yeast cells onto liquid-solid or liquid-gas interfaces. Precise control over fluidic flows in droplet- and bubble-forming microfluidic devices allows production of monodispersed, size-selected templates. The general strategy to organize and assemble living cells is to tune electrostatic attractions between the template (gel or gas core) and the cells via surface charging. Layer-by-Layer (LbL) polyelectrolyte deposition was employed to invert or enhance charges of solid surfaces. We demonstrated the ability to produce high-quality, monolayer-shelled yeastosome structures under proper conditions when sufficient electrostatic driving forces are present. The combination of microfluidic fabrication with cell self-assembly enables a versatile platform for designing synthetic hierarchy bio-structures.     

A simple paper-based sensor fabricated by selective wet etching of silanized filter paper using a paper mask

We developed a novel strategy for fabrication of microfluidic paper-based analytical devices (μPADs) by selective wet etching of hydrophobic filter paper using a paper mask having a specific design. The fabrication process consists of two steps. First, the hydrophilic filter paper was patterned hydrophobic by using trimethoxyoctadecylsilane (TMOS) solution as the patterning agent. Next, a paper mask penetrated with NaOH solution (containing 30% glycerol) was aligned onto the hydrophobic filter paper, allowing the etching of the silanized filter paper by the etching reagent. The masked region turned highly hydrophilic whereas the unmasked region remains highly hydrophobic. Thus, hydrophilic channels, reservoirs, and detection zones were generated and delimited by the hydrophobic barriers. The effects of some factors including TMOS concentration, etching temperature, etching time, and NaOH concentration on fabrication of μPAD were studied. Being free of any expensive equipment, metal mask and expensive reagents, this rapid, simple, and cost-effective method could be used to fabricate μPAD by untrained personnel with minimum cost. A flower-shaped μPAD fabricated by this presented method was applied to the glucose assay in artificial urine samples with good performance, indicating its feasibility as a quantitative analysis device. We believe that this method would be very attractive to the development of simple microfluidic devices for point-of-care applications in clinical diagnostics, food safety, and environmental protection.     

Microfluidic electrospinning of biphasic nanofibers with Janus morphology

In this paper a method of electrospinning conducting and nonconducting biphasic Janus nanofibers using microfluidic polydimethylsiloxane (PDMS)-based manifolds is described. Key benefits of using microfluidic devices for nanofiber synthesis include rapid prototyping, ease of fabrication, and the ability to spin multiple Janus fibers in parallel through arrays of individual microchannels. Biphasic Janus nanofibers of polyvinylpyrrolidone (PVP)+polypyrrole (PPy)∕PVP nanofibers with an average diameter of 250 nm were successfully fabricated using elastomeric microfluidic devices. Fiber characterization and confirmation of the Janus morphology was subsequently carried out using a combination of scanning electron microscopy, energy dispersion spectroscopy, and transmission electron microscopy.     

System-level simulation of liquid filling in microfluidic chips

Liquid filling in microfluidic channels is a complex process that depends on a variety of geometric, operating, and material parameters such as microchannel geometry, flow velocity∕pressure, liquid surface tension, and contact angle of channel surface. Accurate analysis of the filling process can provide key insights into the filling time, air bubble trapping, and dead zone formation, and help evaluate trade-offs among the various design parameters and lead to optimal chip design. However, efficient modeling of liquid filling in complex microfluidic networks continues to be a significant challenge. High-fidelity computational methods, such as the volume of fluid method, are prohibitively expensive from a computational standpoint. Analytical models, on the other hand, are primarily applicable to idealized geometries and, hence, are unable to accurately capture chip level behavior of complex microfluidic systems. This paper presents a parametrized dynamic model for the system-level analysis of liquid filling in three-dimensional (3D) microfluidic networks. In our approach, a complex microfluidic network is deconstructed into a set of commonly used components, such as reservoirs, microchannels, and junctions. The components are then assembled according to their spatial layout and operating rationale to achieve a rapid system-level model. A dynamic model based on the transient momentum equation is developed to track the liquid front in the microchannels. The principle of mass conservation at the junction is used to link the fluidic parameters in the microchannels emanating from the junction. Assembly of these component models yields a set of differential and algebraic equations, which upon integration provides temporal information of the liquid filling process, particularly liquid front propagation (i.e., the arrival time). The models are used to simulate the transient liquid filling process in a variety of microfluidic constructs and in a multiplexer, representing a complex microfluidic network. The accuracy (relative error less than 7%) and orders-of-magnitude speedup (30 000X–4 000 000X) of our system-level models are verified by comparison against 3D high-fidelity numerical studies. Our findings clearly establish the utility of our models and simulation methodology for fast, reliable analysis of liquid filling to guide the design optimization of complex microfluidic networks.     

Biomimetic micro∕nanostructured functional surfaces for microfluidic and tissue engineering applications

This paper reviews our work on the application of ultrafast pulsed laser micro∕nanoprocessing for the three-dimensional (3D) biomimetic modification of materials surfaces. It is shown that the artificial surfaces obtained by femtosecond-laser processing of Si in reactive gas atmosphere exhibit roughness at both micro- and nanoscales that mimics the hierarchical morphology of natural surfaces. Along with the spatial control of the topology, defining surface chemistry provides materials exhibiting notable wetting characteristics which are potentially useful for open microfluidic applications. Depending on the functional coating deposited on the laser patterned 3D structures, we can achieve artificial surfaces that are (a) of extremely low surface energy, thus water-repellent and self-cleaned, and (b) responsive, i.e., showing the ability to change their surface energy in response to different external stimuli such as light, electric field, and pH. Moreover, the behavior of different kinds of cells cultured on laser engineered substrates of various wettabilities was investigated. Experiments showed that it is possible to preferentially tune cell adhesion and growth through choosing proper combinations of surface topography and chemistry. It is concluded that the laser textured 3D micro∕nano-Si surfaces with controllability of roughness ratio and surface chemistry can advantageously serve as a novel means to elucidate the 3D cell-scaffold interactions for tissue engineering applications.     

Optofluidic in situ maskless lithography of charge selective nanoporous hydrogel for DNA preconcentration

An optofluidic maskless photopolymerization process was developed for in situ negatively charged nanoporous hydrogel [poly-AMPS (2-acrylamido-2-methyl-1-propanesulfonic acid)] fabrication. The optofluidic maskless lithography system, which combines a high power UV source and digital mirror device, enables fast polymerization of arbitrary shaped hydrogels in a microfluidic device. The poly-AMPS hydrogel structures were positioned near the intersections of two microchannels, and were used as a cation-selective filter for biological sample preconcentration. Preconcentration dynamics as well as the fabricated polymer shape were analyzed in three-dimensions using fluorescein sample and a confocal microscope. Finally, single-stranded DNA preconcentration was demonstrated for polymerase chain reaction-free signal enhancement.     

Three dimensional microfluidics with embedded microball lenses for parallel and high throughput multicolor fluorescence detection

We report a 3D microfluidic device with 32 detection channels and 64 sheath flow channels and embedded microball lens array for high throughput multicolor fluorescence detection. A throughput of 358 400 cells/s has been accomplished. This device is realized by utilizing solid immersion micro ball lens arrays for high sensitivity and parallel fluorescence detection. High refractive index micro ball lenses (n = 2.1) are embedded underneath PDMS channels close to cell detection zones in channels. This design permits patterning high N.A. micro ball lenses in a compact fashion for parallel fluorescence detection on a small footprint device. This device also utilizes 3D microfluidic fabrication to address fluid routing issues in two-dimensional parallel sheath focusing and allows simultaneous pumping of 32 sample channels and 64 sheath flow channels with only two inlets.     

Flip channel: A microfluidic device for uniform-sized embryoid body formation and differentiation

This paper reports a two-layered polydimethylsiloxane microfluidic device—Flip channel, capable of forming uniform-sized embryoid bodies (EBs) and performing stem cell differentiation within the same device after flipping the microfluidic channel. The size of EBs can be well controlled by designing the device geometries, and EBs with multiple sizes can be formed within a single device to study EB size-dependent stem cell differentiation. During operation of the device, cells are positioned in the designed positions. As a result, observation and monitoring specific population of cells can be achieved for further analysis. In addition, after flipping the microfluidic channel, stem cell differentiation from the EBs can be performed on an unconfined flat surface that is desired for various differentiation processes. In the experiments, murine embryonic stem cells (ES-D3) are cultured and formed EBs inside the developed device. The size of EBs is well controlled inside the device, and the neural differentiation is performed on the formed EBs after flipping the channel. The EB size-dependent stem cell differentiation is studied using the device to demonstrate its functions. The device provides a useful tool to study stem cell differentiation without complicated device fabrication and tedious cell handling under better-controlled microenvironments.