Abstract
Micro Reactor Technology
Continuous Protein Production in Nanoporous, Picoliter Volume Containers
Proteins are the foundation of functionalities of biological systems. The ability to synthesize proteins based on genetic information is the key for rapid screening of protein activity for pharmaceutical studies. Traditionally, protein synthesis has been performed in vivo using live host cells, and there have been various issues involved with this approach, such as protein degradation, insolubility, cytotoxicity, and so forth.
Siuti et al. tackle this problem by developing a nanopore-based reaction container to allow protein synthesis in a well-controlled cell-free environment. The authors use a micro/nano fabrication technique and essentially create a cellular scale bioreactor that includes a nanostructured silicon membrane within a microfluidic channel. The silicon membrane is capable of confining the entire protein synthesis machinery–DNA template and cell-free extract, while in the meantime allowing the continuous exchange of energy and materials between reaction container and environment. The microfluidic channel is used to deliver and remove materials/energy from the reaction chamber surrounding the reaction container to create an optimized environment for cell-free protein synthesis. The functionality of this micro reaction container system is successfully demonstrated via the synthesis of enhanced green fluorescent protein in a period of approximately 24 hours and a per-volume yield more than twice that of conventional scale batch reactions is demonstrated. The authors believe that the successful demonstration of the cell-mimicking protein synthesis micro reaction container by controlling the reaction volume, material/energy transport, and environment creates opportunities for high-throughput protein screening tools to facility therapeutic protein development. (Siuti et al., Lab on a Chip,
Microfluidic Chip Technology
A Touch-and-Go Lipid Wrapping Technique in Microfluidic Channels for Rapid Fabrication of Multifunctional Envelope-Type Gene Delivery Nanodevices
Safe, stable, and efficient gene delivery vectors are critical for gene therapy development. Traditionally, viruses have been the primary gene delivery vehicles for introducing foreign genes into cells in gene therapy. Because of the potential risks of viral vectors, such as acute immune responses, researchers have been striving to develop nonviral gene delivery vectors. Kogure et al. introduced an alternative gene delivery vector named multifunctional envelop-type nanodevices (MEND; Kogure et al., J Control Release,
Kitazoe et al. introduce a novel touch-and-go lipid wrapping technique to address some of the fabrication issues with the traditional method. The touch-and-go method starts with coating the glass surface inside microfluidic channels with multiple lipid bilayers, followed by the injection of condensed plasmid DNA core. The positively charged condensed plasmid DNA cores land on the negatively charged lipid bilayers, roll on the film, and get “wrapped” by the peeled lipid bilayer to form MENDs. Once the wrapping process is complete, MENDs are then released from the lipid bilayer due to the charge and are carried away by the injection fluid. Two key factors in this process are the control of the liquid concentration to ensure the formation of multiple lipid bilayers with proper membrane fluidity and the control of fluid injection velocity or shear at the lipid bilayer surface, both of which affect the wrapping process and the size of MENDs. Compared with the traditional lipid film hydration process, this novel touch-and-go lipid MEND fabrication technique is much faster, simpler, and more cost-effective. The authors believe it shows great promise for rapid and convenient fabrication of gene delivery vector for applications such as personalized medicine. (Kitazoe et al., Lab on a Chip,
Microfluidics-Based Diagnostics of Infectious Diseases in the Developing World
Microfluidics and Lab-on-a-Chip technology hold the promise of providing cost-effective, easy-to-use, and rapid solution for point-of-care diagnostics for resource-limited developing nations. Although many attempts have been made toward this goal, there have been few successful examples. Chin et al. introduce a fully integrated microfluidic chip for diagnosing infectious diseases in the developing world and demonstrate how new manufacturing, fluid-handling, and signal detection procedures can help address some of the practical issues involved with microfluidic chip–based diagnostic applications.
The authors present a series of innovations in chip development. The issue of high-throughput, low-cost manufacturing of microfluidic cassettes is first addressed. Instead of using traditional rapid prototyping method such as poly(dimethylsiloxane) (PDMS) based soft lithography, the authors developed a manufacturing process using materials such as transparent polystyrene and cyclic olefin copolymer. The cassettes fabricated with new materials meet the precision and thermal/chemical/mechanical stability requirements of diagnostic assays while costing less than $0.10/chip (material cost) in a high-throughput manufacturing process. Second, the authors also address the issue with automated delivery of multiple reagents for multiple reactions. A novel sample injection method involving vacuum driven metered plugs of reagents separated by air plugs stored in a plastic tubing is introduced, making it possible to sequentially inject a series of solutions using a single syringe and tubing. Third, the authors implement a sensitive yet cost-efficient ELISA assay with silver reduction amplification. The ELISA signal is easily measured via silver film optical density using lost-cost optical components such as LEDs and photodetectors (per unit cost is approximately $6.50). These three measures ensure a low-cost yet highly effective and easy-to-use point-of-care diagnostic platform. The chip has been field tested in Rwanda on hundreds of locally collected human samples for HIV and syphilis detection. The result shows sensitivities and specificities that “rival those of reference bench-top assays.” (Chin et al., Nat. Med.,
Fast Detection of Biomolecules in Diffusion-Limited Regime Using Micromechanical Pillars
At extremely low concentrations, the detection of analyte molecules using biosensors is often limited by poor mass transport efficiency. A slow mass transport rate dramatically reduces the practical sensitivity of a sensor by reducing the number of binding events or causes the detection time to be unrealistically long, rendering the device useless. Melli et al. investigate the possibility improving the sensitivity and detection speed of biosensors for extremely low-concentration detection by optimizing the dimension and shape of the biosensing elements. The method introduced in this study is a micromechanical sensor based on vertically oriented oscillating pillars. An array of vertical silicon pillars is fabricated and passivated with a C4F8 plasma to render the surface extremely hydrophobic. The analyte solution is placed on top of the pillar array. The hydrophobicity of the pillar array prevents the wetting of the analyte solution; thus, only the tips of the pillars are exposed to the analyte molecules within the solution. The adsorption of the molecules at the tip of the pillars changes the resonance frequency of the pillars, which allows the detection of biobinding events. When compared with a traditional “flat surface” detector, the vertical pillar arrays show a significant improvement in sensitivity and speed, thanks to the pillar array setup that results in the much reduced volume from which analyte molecules must diffuse to saturate the sensor surface. (Melli et al., ACS Nano,
Rolled-up Magnetic Sensor: Nanomembrane Architecture for In-Flow Detection of Magnetic Objects
Magnetic nano- and micro-particles have long been used in various biological applications such as magnetic bioseparation, enhanced magnetic resonance imaging, magnetic particle amplification for biosensing, and magnetic cell sorting. There has been great interest in integrating magnetic sensors in high-throughput on-chip analytical systems for in-flow detection of magnetic particles. On-chip magnetic sensors based on giant magnetoresistance (GMR) have been previously reported; however, because of the limitation of the fabrication process, these sensors are planar (two-dimensional), which results in limited detection performance.
Mönch et al. introduce a novel method to fabricate three-dimensional magnetic sensors using rolled-up architectures. Rolled-up architectures are fabricated by releasing the strain of a nanomembrane on a sacrificial layer, causing the membrane to bend into a micrometer-sized tube of customized size and geometry. In this work, the authors develop a roll-up GMR sensor that also acts as a fluid channel to allow the in-flow detection of magnetic particles. The three-dimensional rolled-up GMR sensors provide several advantages compared with traditional two-dimensional sensors, such as better signal-to-noise ratios and elimination of the need to align the magnetic moment of the particles prior to detection. The potential of the GMR sensor is demonstrated via the in-flow detection of ferromagnetic CrO2 nanoparticles embedded in hydrogel shells. The authors believe this GMR sensor may enable many biodetection applications, particularly in disease diagnostics and magnetic sorting of living cells. (Mönch et al., ACS Nano,
Guiding, Distribution, and Storage of Trains of Shape-Dependent Droplets
Manipulation of micro-droplets is an important topic in microfluidics. Micro-droplets in microfluidic systems can act as self-contained vessels to allow species within the droplets to be transported, sorted, and stored. This has made possible a wide variety of important on-chip applications such as droplet-based bioreactors, optimization of protein crystallization, droplet-based cell detection and sorting, and so forth. There is great interest in developing various unit operations, such as sorting, translating, dividing, merging, and storing for micro-droplets, and successful examples have been demonstrated based on active droplet control using electric kinetics, dielectrophoresis, acoustics, electro wetting, and so on. Passive fluid dynamic manipulation of droplets is often preferred because it requires less complex chip design and fabrication and causes less potential interference with biological samples inside the droplets.
Ahn et al. report an interesting on-chip droplet manipulation method using the tracks and chambers on the ceiling and bottom of microfluidic channels, respectively. It allows the guiding, distribution, and storage of micro-droplets based on purely fluid dynamic control and droplet-structure interaction. Two PDMS slabs with guiding track and chamber structures, respectively, are aligned and assembled to form the bottom and ceiling of a microfluidic channel. The depth and width of the track/chamber structures and offset between tracks and chambers are optimized to allow guiding of droplets along the tracks and to secure capturing of the droplets in the chamber. A squeezing flow is used first to align hydrodynamically the droplets in a single-file fashion before a pushing flow is used to selectively guide droplets toward different tracks. The tracks then guide micro-droplets into a different storage region where droplets are captured and stored within chamber structures. Using this design, the authors are able to guide micro-droplets containing different chemical/biological species (i.e., fluorescent dyes with different concentrations) into different regions for storage. This simple and effective micro-droplet guiding and storage method is easily integrated with other existing micro-droplet unit operation modules to enable various droplet-based microfluidic applications. (Ahn et al., Lab on a Chip,
Tissue-on-a-Chip
Ensembles of Engineered Cardiac Tissues for Physiological and Pharmacological Study: Heart-on-a-Chip
The difficulty of replicating tissue microenviroments has long been a challenge for cardiovascular physiology and pharmacology studies. Methods developed to date have been mostly limited to the measurement of single cardiomyocytes, which is far from an ideal model to study contractility and pharmacological response of heart tissue. Grosberg et al. report the design of a “heart-on-a-chip” to address this issue. The chip is based on the muscular thin-films (MTFs) that consist of an anisotropic tissue formed with cardiomyocytes cultured on a deformable elastic thin film. The contractile stress of the heart tissue is correlated to the curvature of the film, allowing the direct calculation of heart tissue contractility. In this study, up to eight MTFs are fabricated on the same chip and simultaneously measured. The chip is used to study drug dose-response by simultaneously measuring the contractile function of multiple tissues and quantification of action potential propagation speed. This heart-on-a-chip technology provides a promising platform to study cardiovascular physiology and pharmacology on tissue scale. (Grosberg et al., Lab on a Chip,
Axon Diodes for the Reconstruction of Oriented Neuronal Networks in Microfluidic Chambers
Many different experimental models have been proposed to study the complex structure of the brain, but few are capable of simultaneously providing insight into both cellular-level events and high-level integration of neuronal network. For example, the whole-brain model retains intact anatomical structure but provides only limited access to cellular-level details; cultured neuron cells allow close examination of cell phenotype but fail to reveal highly ordered neuron connectivity in the brain. To tackle this issue, Peyrin et al. present a novel microfluidic-chip based platform that allows the reconstruction of the oriented neuronal network with several different neuron populations.
The key feature of this chip platform is the asymmetrical micro-channels that allow the penetration of axon in only one direction, promoting the formation of a unidirectional neuronal network. The authors describe this unique feature as a “diode” for axonal projection. Primary neuronal cultures (cortical neurons and striatal neurons) are seeded in two separate chambers that are connected with asymmetrical microchannels, respectively. The asymmetric microchannel allows only the projection of neuron axons from cortical neuron to strata neurons, allowing the creation of an integrated binary neuronal network that fully mimics the in vivo neuronal pathway. With this model, activation of striatal differentiation by cortical axons and the synchronization of neural activity are demonstrated. The device’s capability to construct the directional neuronal pathway with a selected neuron population leads the authors to believe the same principles can be readily reapplied to study brain development, neuronal communication, and various neurodegenerative diseases. (Peyrin et al., Lab on a Chip,
High-Throughput Analytics
Review: Rare Cell Separation and Analysis by Magnetic Sorting
In recent years, interest in “personalized medicine” has been pushing the development of rare cell separation technology. Personalized medicine allows physicians to customized patient care based on an individual’s specific molecular phenotype. To do so, however, requires the separation and analysis of rare disease-related cells from the patients. Zborowski and Chalmers summarize the development high-throughput rare cell separation and analysis based on magnetic cell separation. The review article discusses the development of magnetic separation schemes for labeled rare cells such as circulating tumor cells and hematopeietic stem cells using antibody-coated magnetic particles, as well as the separation of unlabeled cells based on intrinsic magnetic moments such as malaria parasite–infected red blood cells. (Zborowski and Chalmers, Anal. Chem.,
Single DNA Molecule Patterning for High-Throughput Epigenetic Mapping
A novel method for fabricating elongated DNA molecule array on SiO2 substrate was recently reported by Cerf et al. In this study, a PDMS stamp that contains patterned microwell structures is first fabricated via soft-lithography method. A meniscus of DNA molecule solution is then dragged over the micropatterned PDMS stamp. DNA molecules are physically trapped within the microwells when meniscus travels along the PDMS surface. Because of the hydrophobic nature of the PDMS substrate, the trapped DNA is also elongated due to the capillary force as the meniscus leaves microwells. The elongated DNA microarray is subsequently transferred onto an APTES-coated coverslip via stamping. Because of the preference of DNA to remain in more hydrophilic APTES surfaces, the DNA transfer is performed reliably, and extended configuration of stretched DNA is maintained after the transfer, making possible the DNA analysis with high spatial resolution and signal averaging. The method is simple yet highly efficient: an elongated single DNA array estimated to contain more than 250 000 individual DNA molecules is obtained. The authors use the method to successfully demonstrate the high-throughput profiling of 5-methyl cytosine distribution on single phage lambda DNA molecules. (Cerf et al., Anal. Chem.,
Advances in Automation of Optical Measurement and Detection
Review: Optofluidic Microsystems for Chemical and Biological Analysis
Optical measurement and detection have long been used to perform chemical and biological analysis. In recent years, thanks to the progress in the fields of optics and microfabrication, miniature optical components such as on-chip waveguides, lenses, and resonators have emerged and been integrated within the lab-on-a-chip devices for various on-chip applications. The integration of optics with microfluidics further gave birth to a new field named optofluidics. Optofluidics focuses on synergistic integration of optical and microfluidic components to provide enhanced function and performance for on-chip analysis. In a recent review by Fan and White, the latest progress in optofluidic architectures for chemical/biological analysis is revealed. The review covers several key analytical mechanisms including refractive index, fluorescence detection, surface-enhanced Raman spectroscopy, and optical trapping/manipulation of nano/micrometer–size particles and cells. (Fan and White, Nat. Photonics,
Fluorescence Microscopy Imaging with a Fresnel Zone Plate Array–Based Optofluidic Microscope
There has been great interest in recent years in developing the chip-scale microscope to allow the direct integration of microscopic imaging components into lab-on-a-chip–based point-of-care systems. An optofluidic microscope (OFM) developed by Yang’s research group successfully demonstrates the direct on-chip bright field microscopic imaging of Caenorhabditis elegans (Cui et al., Proc. Nat. Acad. Sci. U.S.A.,
To address this issue, Pang et al. propose an improved OFM configuration for fluorescent imaging. With the new configuration, instead of placing the linear nano-aperture array directly above the CMOS chip surface (the bottom of the microfluidic channel), the authors deposit a metal layer on the ceiling of the channel and etch a linear array of Fresnel zone plates (FZP) that also spans diagonally across the channel (similar to the arrangement of nano-aperture array in original OFM configuration). Each FZP acts as a lens to focus the uniform plane fluorescent excitation light and form a focused light spot within the microfluidic channel. Similarly, when objects travel through the channel, they are illuminated by the series of light spots created by the linear FZP array. Each light spot acts as a line scanner. The time-varied fluorescence signal associated with each spot combines with the traveling speed of objects to allow the reconstruction of a two-dimensional fluorescence image of the cells. The theoretical resolution is determined by the larger of two: size of focused light spots (0.65 µm) and separation between light spots perpendicular to the flow direction (0.5 µm). In this case, the resolution achieved in the experiment is 1.0 µm, and the fluorescent imaging of cell nuclei and cytoplasm is successfully demonstrated. (Pang et al., Lab on a Chip,
Advances in Automation of Biomarker Detection
Detection of Single Enzymatic Events in Rare or Single Cells Using Microfluidics
Identification of rare abnormal cells in the background of a large normal cell population is of particular interest for diagnostic/therapeutic purpose as well as basic research. Knudsen’s research team develops a highly sensitive (single-molecule level) method for detection of rare enzymatic cleavage-ligation events based on the isothermal rolling-circle–enhanced enzyme activity detection (REEAD) assay (Stougaard et al., ACS Nano,
Recently, the team further improved the technique by adapting a microfluidic platform. With the new platform, the entire REEAD assay system, including cells to be analyzed, sensor (reporting DNA substrate), and lysis buffer, is packaged into picoliter-size micro-droplets using a water-in-oil emulsion system. The micro-droplets are subsequently immobilized within the microfluidic droplet-trapping structures where the REEAD assays are preformed. The small reaction volumes within micro-droplets eliminate the limitation of diffusion-driven mass transport and make it possible to achieve fast reaction kinetics. The confinement of the reaction system in picoliter-size micro-droplets also avoids the loss of sensitivity by concentrating the signals. Using this setup, authors are able to report, for the first time, the multiplexed detection of individual enzymatic events in single cells. The authors envision that the technology platform may enable analysis of rare cellular events such as tumor growth and development of drug resistance. (Juul et al., ACS Nano,