Abstract
Laboratory Automation and High-Throughput Analysis
High-Resolution Dose-Response Screening Using Droplet-Based Microfluidics
Screening of chemical libraries is a critical early step in drug discovery. Normally, after being identified in a primary screen, promising compounds go through a further dose-response analysis with 7 to 10 nonduplicated data points per compound. More data points are difficult to obtain because of time and cost constraints. With such a limitation, screening is highly sensitive to the quality of the data, resulting in a high number of false positives or false negatives and the possibility of a single outlier causing a poor fit of the data. In addition, less than 10 data points are not sufficient to allow identification of subtle, complex pharmacology such as partial agonism or antagonism.
A study published by Miller et al. describes a droplet-based microfluidic system capable of generating high-quality dose-response data that contain as many as 10 000 data points, a whopping 1000 times higher than in conventional drug screening. The design of the device features (1) a high-performance liquid chromatography (HPLC) auto sampler that injects pulses of compounds in a capillary with a continuously flowing stream of buffer, where the Taylor-Aris dispersion gradually smoothes the initially rectangular concentration profile into a smooth Gaussian profile, and (2) a microfluidic droplet device that packs a few tens of picoliters of compound solution from the HPLC column and enzyme solution into a water-in-oil droplet. Because of the concentration gradient caused by Taylor-Aris dispersion, each droplet contains compounds with slightly different concentrations. Because of containment of droplets, the concentration of the enzyme and the compound remain constant within droplets, and the internal circulatory flow of droplets as they advance in the channel enhances the mixing between the enzyme and compound, making it an ideal environment for compound-enzyme interaction. The reaction can be monitored online from the fluorescence of the enzymatic reaction product as droplets pass through a laser-induced fluorescence detection point. Thanks to the large number of data points, highly precise and reproducible IC50 values are achieved. In addition, the high resolution of the data makes it possible to get a clear picture of complex dose-response relationships.
The system was tested in a screening of a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. A number of novel inhibitors are identified, with the most potent being sodium cefsulodine, which has a C50 of 27 ± 0.83 µM. (Miller, O. J., et al., Proc. Natl Acad. Sci. U. S. A.
Drug Screening in a Quantitative and Highly Parallel Manner with Soft Matter Nanoreactors
In drug screening, it is critical to rapidly examine multiple enzymatic reactions. Over the years, the microfluidic community has created ways to mix ultra-small volumes of reactants in parallel for this purpose. For example, droplet-based microfluidics can produce microdroplets with sizes on the order to 10–12 L and confine the reactants within that volume for mixing. This sample volume, however, may still be considered too large. Reactants within such a sample volume may not mix fast enough by diffusion to allow the observation of the fast enzyme kinetics. Therefore, active stirring of the sample volume, such as promoting the internal circulatory flow in moving droplets, is needed for mixing. In addition, it could be complicated to manipulate a large number of microscale droplet containers for parallel screening.
Christensen et al. introduce a new concept of mixing sub-attoliter volumes in a quantitative and highly parallel manner with nanoscale soft matter nanoreactors. These nanoscale soft matter nanoreactors are made of nanovessicles of self-assembled amphiphilic materials. The authors produce two types of sub-attoliter-sized nanovessicles containing the enzymes (target reactors) and substrates (cargo reactors). By functionalizing target vectors and cargo vectors with different charges, fusion can be promoted between two types of vectors to a single vesicle, which results in mixing of enzymes and substrates in a single contained space. The size of the vesicle is so small that rapid mixing can occur by diffusion only, and no external agitation is required. This has created a way to massive parallel mixing of components. By controlling the incubation time and the concentration of cargo reactors, the number of multiple fusion events can be adjusted, allowing the same target vector to obtain several consecutive mixing events with multiple target vectors in a controlled fashion. (Christensen, S. M., et al., Nat. Nanotech.
Semiautomated Device for Batch Extraction of Metabolites from Tissue Samples
Metabonomics is the systematic study of metabolite fingerprints for living organisms. The metabonome represents the collection of all metabolites in a biological entity, and the profiling of metabolites can provide valuable information about the physiology of cells. In recent years, metabonomics has become a mainstream research tool for investigating metabolism. The metabonomic analysis of tissue samples typically involves three sample preparation steps: homogenization, metabolite extraction, and sample filtration. Each of these steps affects the overall yield and precision of the final measurement results. Currently, there is lack of tools that would allow the automation of sample preparation. This has created problems for metabonomic analysis, especially for nuclear magnetic resonance (NMR) analysis, which requires large sample sizes and large solvent volumes.
To address this issue, Ellinger et al. present a semiautomated device for batch extraction of metabolites from tissue samples. The device, named the semiautomated metabolite batch extraction device (SAMBED), includes six components: a milling chamber, a vibrational shaker, a solvent reservoir, a homogenization platform, a filtration chamber, and a filtration platform. Tissue samples are first homogenized by the vibrational shaker in the milling chamber. The extraction solvent can be added directly into the milling chamber, and the raw extracts are then transferred to the downstream filter chambers, where small-molecule metabolites are separated from the cellular debris and macromolecules by ultra-filtration. A parallel extraction test is conducted to demonstrate the improvement of tissue extraction efficiency using the SAMBED. The SAMBED can cut the extraction time by more than 75% and deliver a better extraction quality. (Ellinger, J. J., et al., Anal. Chem.
Microfluidic Chip Technology
Hydrodynamic Tweezers
The ability to trap cells and microparticles is critical for a wide variety of biological applications such as cell separation, filtering, and single-cell analysis. Several particle-trapping mechanisms have been demonstrated, including the widely used optical tweezers as well as optoelectronic tweezers, magnetic tweezers, dielectrophoresis tweezers, plasmonic tweezers, and surface acoustic tweezers.
Lieu et al. introduce interesting hydrodynamic tweezers based on low Re steady streaming. The device configuration is extremely simple: a microchannel with certain micro-obstructions lying at the center of the fluid channel and a pair of piezoelectric disks bonded on the channel substrate. The acoustic waves generated from the piezoelectric disks causes low Re steady streaming within the fluid near the micro-obstruction structures. The micro-eddies created by streaming can be used to trap microparticles near the core of eddies. The authors conduct a thorough study of the role of various microfabricated device geometries on the flow and trapping traits of low Re steady streaming. Nine different microgeometries—namely, circle, circle protrusion, circle cavity, square, square protrusion, square cavity, diamond, triangle protrusion, and triangle cavity—are studied. Imaging of micro-eddies reveals the effect of device geometry on eddy number, shape, structure, and strength. Flow simulation and analysis are performed to understand the relation between particle-trapping position/strength and the geometry/oscillation frequency of the streaming. Through this study, a computational design methodology has been established to guide the future design of particle-trapping geometry based on low Re steady streaming. The ability of such a simple device to trap and position cell-size microparticles demonstrates the potential of this technology as a promising candidate for laboratory applications that require manipulation of cell positions. (Lieu, H. V., et al., Anal. Chem.
Review: Commercialization of Microfluidic Point-of-Care Diagnostic Devices
Microfluidics has created a lot of excitement because of its potential to revolutionize the health care industry with miniature, fully integrated point-of-care diagnostics devices. Despite the tremendous progress in the field of microfluidics in the past 20 years, it is surprising that few microfluidic devices have been successfully launched into the market. Chin et al. review the commercialization of microfluidic point-of-care diagnostic devices and examine why microfluidics has not yet fully lived up to expectations. The authors draw from their own experience in a startup point-of-care diagnostics company to identify challenges to commercialization and stress the importance of achieving a balance between the real-world impact of integrated devices versus the design of a novel single microfluidic component. (Chin, C. D., et al., Lab Chip
Microfluidic Tissue Analog Reveals Fluid Forces Control Endothelial Sprouting
Angiogenesis is the physiological process of blood vessel expansion and plays a critical role in wound healing and tumor growth. Understanding this process is of extreme importance in the field of regenerative medicine, tissue engineering, and oncology. It is known that angiogenesis is the coordinated growth and migration of endothelia cells (ECs), which causes ECs to sprout from healthy vascular channels into the surrounding avascular regions. It is well known that this process is heavily influenced by vascular endothelial growth factor (VEGF). Other factors, however, such as fluid forces (e.g., shear stress) exerted by blood and plasma, also could be involved in this process.
Previously, it was not clear whether VEGF cooperates with fluid forces to mediate angiogenesis. Song and Munn report a microfluidic tissue analogue of angiogenic sprouting and shed some light on the understanding of this complicated process. To understand how fluid flow and VEGF work together to modulate endothelia sprouting, the authors present a microfluidic device that features two individual channels filled with human umbilical vein endothelial cells (HUVECs). These two channels are connected with a collagen-filled channel that acts as a vascular region. Sprouting of endothelial cells through the collagen gel can be studied under various conditions using this setup. The shear stress on the HUVECs within each of two HUVEC channels, transverse flow in the collagen channel on the sprouting endothelia cells, and the concentration gradient between two HUVEC channels can be individually manipulated, allowing identification of factors involved in the endothelial sprouting process. With this approach, critical understanding of how fluid shear stress, direction of interstitial fluid flow, and the gradient of VEGF cooperate to modulate angiogenic sprouting is achieved. The results reveal the importance of multiple signals during the angiogenesis and the fact that fluid forces are important mediators of vascular homeostasis and morphogenesis. (Song, J. W., and Munn, L. L., Proc. Natl Acad. Sci. U. S. A.
High-Throughput Cell Separation
Simple and efficient cell separation methods are fundamentally important in biological and chemical analyses. To date, many methods capable of particle separation have been demonstrated based on cell density and sizes. For example, in centrifugal separation, centrifugal force generated by spinning a sample separates heavier cells from lighter cells in a density gradient. In addition, continuous on-chip separation schemes employ hydrodynamic methods in which properly designed channels (i.e., asymmetric obstacles inside the channel) direct cells of different sizes to follow different streamlines to be separated. Two studies show how cell separation can be further improved by looking beyond particle density and size and by using some previously untapped sources of specificity such as shape, deformability, and cell adhesion.
Cell Sorting by Shape and Deformability
In one study published by Beech et al., the authors demonstrate how shape and deformability can be used to develop a fast and efficient method for cell sorting. In this work, the separation is performed in a deterministic lateral displacement (DLD) device. The DLD devices consist of arrays of posts. When interacting with posts, particles of different sizes move in different fashions. Smaller particles tend to move along the streamlines and larger particles tend to deviate from the streamline and move along a certain direction defined by the design of the post pattern. For rigid spherical particles, the separation of particles is mainly determined by particle size. However, the authors discover that when biological cells are soft and not always perfectly spherical, the situation can be very different. Take red blood cells as an example—they are deformable and are disk shaped. Therefore, when they interact with posts in the DLD device, the effective particle size of red blood cells can be affected by their orientation and the extent that they deform.
There are simple methods for changing the effective particle sizes for cells such as red blood cells in DLD devices. For example, changing the depth of the DLD device affects the orientation of the cells, and changing the fluid shear results in a change in cell deformation, both of which cause effective particle size to change. The authors test their theory using three red blood cell types in devices with different heights and at different shear rates. Promising preliminary results are demonstrated. This work provides a possibility for researchers to tailor the DLD systems to achieve better cell separation efficiency based on cell shape and deformability. (Beech, J. P., et al., Lab Chip
Cell Sorting by Deterministic Cell Rolling
In another study, Choi et al. introduce a different cell separation method based on “deterministic cell rolling.” In this work, instead of trying to separate cells based on their physical properties, such as density, size, morphology, and deformability, the authors propose an alternative method that leverages transient interaction between receptor-ligand pairs at the cell surface. The separation is based on two hydrodynamic phenomena: hydrophoresis and cell rolling. Hydrophoresis uses slant ridges on a microchannel floor to “focus” cells (i.e., force cells to converge into the same stream). Hydrophoresis is a purely hydrodynamic process and works for cells that do not interact with the surface of slant ridges. When the surface of slant ridges is coated with ligand molecules such as P-selectin, certain cells with surface receptors, such as leukemia cells, can interact with the slant ridge surface, causing them to deviate from the path defined by the hydrophoresis. As a result, these cells can be separated from the rest of cell population. The authors test cell separation efficiency using a mixture (mixing ratio of 2:3) of HL60 and K562 cells with the slant ridge surface coated with P-selectin molecules (which only interact with the HL60 cells). After separation, cell purities of 95% and 94.3% are achieved for HL60 and K562 cells, respectively. This novel cell separation technique represents a new method of affinity cell separation and can benefit various applications such as point-of-care diagnostics and cell-based therapeutics. (Choi, S., et al., Lab Chip
Biosensing and Chemical Sensing Technology
Nonenzymatic Electrochemical Detection of Glucose
Glucose sensing is of critical importance for the diagnosis, treatment, and management of diabetes. Today most glucose sensor platforms are based on electrochemical measurement with enzymes such as glucose oxidase (GOx). The enzyme is the key for the high selectivity and sensitivity to glucose. As with all biomaterials, however, drawbacks of enzymes, such as chemical and thermal stability and reusability, have been the major challenges of the enzyme-based glucose sensors. In the past few years, there has been great interest in developing nonenzymatic glucose-sensing systems based on highly electrocatalytic electrode materials. For example, nanostructured metals (Pt, Au, Pd, Ni, Cu) and metal oxide (CuO, Co2O4, NiO) have been explored. These metals or metal dioxides can directly electrocatalyze glucose.
Nie et al. report a nonenzymatic glucose biosensor based on Ni nanoparticle/straight multiwalled carbon nanotube (SMWNT) nanohybrids. Ni nanoparticles anchored on the SMWNT create an efficient electrical network to promote the electron transfer rate and to improve the sensitivity of the sensor. SMWNTs provide benefits, such as being easily dispersed in sample preparation to facilitate the Ni nanoparticle binding to their surface. The test shows that a nanohybrid-based electrode delivers significant improvement in terms of electrocatalytic activities toward the oxidation of glucose. The sensor exhibits a linear response for a glucose concentration range between 1 µM and 1 mM, with the detection limit of 500 nM. In addition, the test reveals a decent specificity for glucose against interfering species such as ascorbic acid, uric acid, dopamine, galactose, and xylose. The results indicate that the nonenzymatic electrochemical sensor can provide a possible alternative to the traditional enzyme-based electrochemical sensor. (Nie, H., et al., Biosen. Bioelectron.
Quantification of Transcription Factor Binding in Cell Extracts Using an Electrochemical, Structure-Switching Biosensor
Measurement of transcription factor (TF) expression is critical for understanding the cellular development and disease state. Traditionally, TF expression has been studied using methods such as enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative PCR, which are cumbersome and time-consuming. Bonham et al. introduce a novel approach for quantitative detection of TF using a structure-switching electrochemical-sensing platform. Their method is based on the use of a specially designed redox modified DNA probe, which can switch between nonbinding and binding confirmations. The probe has a sequence that can bind to the TF. The binding of TF to this sequence causes the conformational change of the probe, bringing the methylene blue redox tag on the DNA probe closer to the electrode surface, and subsequently increases its electron transfer rate. The presence of the target TF, therefore, can be detected by the electric current signal increase. The capability of this new TF-sensing method is demonstrated with a common eukaryotic transcription factor TATA binding protein (TBP). A specific and quantitative detection of TBP in a complex media (crude nuclear extracts) is achieved, using a sample volume as small as 10 µL. This method provides a new technical route for TF express analysis. (Bonham, A. J., et al., J. Am. Chem. Soc.
Wearable Sensor for Heavy Metal Ion Detection
Detection of heavy metal ions has a strong implication in environmental protection, health, and safety. Anzenbacher et al. introduce a wearable sensor system that can be deposited on various objects such as protective safety wear for monitoring various heavy metal ions. The sensor is based on a so-called attoreactor. An attoreactor is the attoliter-sized junctions of electro-spun nanofibers. The authors have developed a method to synthesize ultra-small amount of fluorescence dyes in the nanofiber junctions by loading nanofibers with different reagents. The exposure of dyes to heavy metal ions results in the quenching of the dye, and the fluorescence can be recovered upon washing with water. Such reversible reactions with heavy metal ions make it possible to use these dyes as optically encoded sensors. By using combinations of different reagents in the nanofiber, various fluorescence responses can be generated, allowing the attoreactor to identify up to 11 metal ions. A demonstration is performed by coating a mat of nanofibers (which contains attoreactors) onto a nitrile glove. The functionalized nitrile glove shows immediate fluorescence quenching when exposed to a 20-µM Co2+ ion solution, suggesting the potential of this sensor system in a wearable setup. (Anzenbacher, P., et al., Angew. Chem. Int. Ed.