Abstract
Laboratory Automation
Elastomeric Negative Acoustic Contrast Particles for Affinity Capture Assays
To detect and quantify biological species of low concentration in blood, it is often necessary to separate them from blood cells. Blood separation is often performed via centrifuge or immunomagnetic assays. However, these assays are laborious and time-consuming. Cushing et al. introduce the development of elastomeric capture microparticles (ECµPs) and subsequent use of acoustophoretic separation for affinity capture assays. A simple method for fabrication of ECµPs, via cross-linking droplets of common commercially available silicone precursors in suspension, is reported. The particle surfaces are further functionalized with biomolecular recognition reagents. ECµPs are compressible particles that exhibit negative acoustic contrast in ultrasound when suspended in aqueous media, such as diluted blood, while blood cells exhibit positive acoustic contrasts. This creates an opportunity to separate biological species bound to the surface of ECµPs from blood cells. In this study, ECµPs are functionalized with antibodies for prostate-specific antigen and immunoglobulin G (IgG). Separation of ECµPs from blood cells is achieved by flowing them through a microfluidic acoustophoretic device that generates standing acoustic waves. Due to the difference in acoustic contrast, blood cells migrate toward the acoustic pressure nodes while ECµPs migrate toward the acoustic pressure antinodes. The particles are then separated within the laminar flow and collected downstream at different exits. Separated ECµPs are further analyzed via flow cytometry, and nanomolar detection for prostate-specific antigen in aqueous buffer and picomolar detection for IgG in plasma and diluted blood samples are obtained. (Cushing et al., Anal. Chem.
An Enclosed In-Gel PCR Amplification Cassette with Multitarget, Multisample Detection for Platform Molecular Diagnostics
Polymerase chain reaction (PCR) is a commonly used laboratory method for rapid replication of DNA fragments. It has significant implications for a wide range of fields such as biological research and diagnostics. In PCR, multiple reagents, including the DNA primers, DNA polymerase, reaction buffer, and so on, need to be prepared and mixed with the template in order for DNA replication reactions to proceed. They are also needed to run multiple PCR reactions in parallel to check against multiple targets. In certain applications, such as detection and monitoring of infectious diseases, the laboratory personnel often receive an overwhelming number of samples in a short period, and the assay often needs to be performed at resource-scarce point-of-care locations. Therefore, it is certainly beneficial to automate the PCR processes. Manage et al. report a self-contained disposable gel capillary cassette for DNA amplification for point-of-care diagnosis. The cassette includes capillary reaction units that can be stored at ambient temperature for a prolonged period. The procedure for manufacturing the cassettes includes filling the capillaries with PCR/gel reaction mix, using UV light for photo-polymerization of gel, and desiccating to create a dried gel “noodle” inside the capillaries. The sample can be introduced into the cassette via capillary force. DNA amplification reactions can then occur in the capillary tubes, which are placed in a portable instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis. The authors demonstrate the performance of the PCR cassette kit using raw genital swabs and urine for simultaneous detection of four sexually transmitted diseases, and the results for multiple patients can be obtained in less than an hour. The advantages of this cassette-based PCR platform include simple operation (only introduction of the raw sample is needed), multiplex detection of multiple targets, and easy expansion for a large-scale test. The authors believe that this inexpensive disposal instrument can enable point-of-care diagnosis of patients and help to achieve better and faster medical decision making. (Manage et al., Lab Chip,
Microfluidic Western Blotting
Western blotting, also referred to as protein immunoblotting, is a commonly used technique in biochemistry and molecular biology for detecting specific proteins. It typically involves the gel electrophoresis separation, transfer of separated protein to a membrane, and binding of target proteins with antibodies. Due to its lengthy process and laborious nature, a rapid, quantitative Western blotting method is highly desirable. Hughes and Herr report an automated device named µWestern blot for a Western blotting assay using a single glass microchannel with electronic control. This device is capable of performing multistep assays needed for Western blotting. The key device is a photo-patternable (blue light) and photo-reactive (UV light) polyacrylamide gel. Upon blue light photo-patterning, the gel acts as a separation matrix for protein sizing. After brief UV exposure, it forms a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. The protein is first separated using electrophoresis in the polyacrylamide gel in a microchannel, followed by protein immobilization on the gel using UV irradiation of the entire separation channel. The in situ antibody probing of the immobilized, sized protein is performed by introducing the probes using electrophoresis along the length of the microchannel. Upon probe binding, the excess probe is electrophoretically driven out of the device, and peak intensities are determined by fluorescence micrograph analysis. To validate the assay, the authors apply the µWestern blot to analyses of various samples such as human sera (human immunodeficiency virus immunoreactivity) and cell lysate (nuclear factor–κB), and favorable results are achieved, including short durations, multiplexed analyte detection, high sensitivity, and wide dynamic range, which are attributed to favorable transport and reaction conditions on the microscale. The authors further demonstrate the possibility of a 48-plex µWestern blot in a standard microscope slide form factor. This µWestern blot device provides a rapid, specific, and high-throughput platform for proteomics applications. (Hughes and Herr, Proc. Natl. Acad. Sci. U. S. A.,
Magnetic Timing Valves for Fluid Control in Paper-Based Microfluidics
Paper-based microfluidic devices are rapidly becoming an attractive low-cost, mass-producible platform for performing analytical assays. These devices are made from paper substrate with patterned channel geometries for sample transporting and manipulation. These devices do not require pumping mechanisms because fluid transport is taken care of by the fluid wicking effect of the paper, and thus the complexity of the device is dramatically reduced. In the past several years, a wide variety of devices have been developed and significantly expanded the scope of paper microfluidics. A recent example is the magnetic timing valve for fluid control in paper-based microfluidics reported by Li et al. For biological assays, it is often required that the assay be timed. For example, in an enzyme-linked immunosorbent assay (ELISA), multiple reagent needs to be added sequentially, and after each mixing, samples need to be incubated for certain periods. Although it is convenient to transport the fluids and mix them in paper-based microfluidic device, the timing and switching can be challenging. In this work, the authors develop a new type of paper-based magnetic valve that is capable of timing and switching, enabling timed fluid control in paper-based microfluidic devices. The magnetic valves consist of an ionic resistor, which can sense the event of solution flowing through and subsequently triggering the open/close of paper cantilever valves. The authors demonstrate valves that can be timed for up to 30 min, which is sufficient for ELISA applications. Using one of their valve designs, they successfully perform an enzyme-based colorimetric reaction commonly used for signal readout of ELISAs, which requires a timed delivery of an enzyme substrate to a reaction zone. The introduction of the timed valve further enriches the platform of paper-based microfluidics and helps to further improve the functionality and user-friendliness of the paper-based microfluidic devices. (Li et al., Lab Chip,
Tissue Engineering
A Tissue-Like Printed Material
For mimicking tissue behavior, it is critical for the tissue cells to communicate and cooperate to produce the emergent properties of tissue. Previously, artificial tissues consisting of synthetic mimics of cells, such as liposomes, lacked sophisticated collective behavior because these cell mimics failed to interact and communicate like real cells. Villar et al. make an important breakthrough in producing synthetic tissue with sophisticated functionalities using 3D printing technology. With a specially designed 3D printing system, the authors have printed tens of thousands of picoliter-sized droplets. The droplets do not adhere to each other but instead are joined by single lipid bilayers. This allows the formation of a cohesive material with cooperating compartments. Sophisticated 3D structures can be designed using software and subsequently printed, and biochemical composition of the droplets can be controlled to introduce functionalities. For example, the droplet network can be functionalized with membrane proteins to allow electrical communication in a fashion that mimics nerves. Furthermore, by varying the concentration of solutions inside the droplet, the authors create a 3D tissue structure that can fold into unique geometries that even the printer cannot produce. The authors envision printing droplet-based tissue networks that can further interact with tissues to help support the functionalities of failing tissues or deliver drugs upon specific signals. (Villar et al., Science,
Control of Neural Network Patterning Using Collagen Gel Photothermal Etching
Micropatterning techniques for in vitro cultures have been shown to be of great use in studying neuronal networks by controlling the pattern of cell-cell contact and neurite outgrowth. Many studies report about guiding dissociated neurons into predefined patterns. However, most of these methods employ 2D solid substrates, while neural cells in vivo develop in a complex 3D environment. To address this challenge, Odawara et al. propose a micropatterning method using 3D substrates or scaffolds that better mimic the in vivo microenvironment. The authors introduce a 3D collagen gel photothermal etching method with an infrared laser. The laser photothermal etching allows the precise control of cell adhesion and neurite projection and makes it possible to guide the neural network formation under microscopic observation. With this method, the authors are capable of creating an isolated 3D neural network with predefined cell populations and direction of neurite elongation. Neuron cells are seeded in a thick collagen gel that better mimics the 3D environment, resulting in longer survival of cells and better neurite projection. Imaging of intercellular neuron transmission reveals stronger synaptic connectivity of patterned neural networks using the 3D patterning method. The authors believe this photothermal etching technique allows for the creation of designed 3D neural networks during cultivation and for better studying of synaptic transmission, neuron-glial signaling, pathogenesis, drug responses, and so on. (Odawara et al., Lab Chip,
Biosensing and Biodetection
A Nonenzymatic Glucose Sensor Based on the Composite of Cubic Cu Nanoparticles and Arc-Synthesized Multiwalled Carbon Nanotubes
Diabetes mellitus is a disease of major public health significance. A rapid, simple, and reliable means of monitoring blood glucose is of great importance in diagnosing, treating, and managing this disease. A wide variety of glucose biosensors are available on the market, most of which are enzyme-based electrochemical sensors based on the catalytic oxidation of glucose into gluconic acid by the enzyme glucose oxidase (GOx). These sensors typically offer great sensitivity and specificity and have been great tools for physicians and patients. However, there is an ongoing challenge and unmet need with these sensors because the enzyme, GOx, is not very stable in challenging environments. Therefore, there is great interest in developing a nonenzymatic glucose sensor based on direct oxidation of glucose on the sensor electrode. The biggest advantage of a nonenzymatic sensor is the stability, reliability, and simple fabrication. Zhao et al. present a nonenzymatic glucose sensor based on a composite of cubic Cu nanoparticles and arc-synthesized multiwalled carbon nanotubes (MWCNTs), which exhibit electrocatalytic activity to the oxidation of glucose in an alkaline condition. With this new electrode configuration, the authors demonstrate an amperometric response to glucose with a fast response time of 1 s, sensitivity of 922 µA mM−1/cm−2, a wide linearity in the 0.5- to 7.5-mM concentration range, and a detection limit if 2.0 µM. The sensor is also highly stable and specific to glucose. With these promising results, the authors believe the Cu-MWCNT electrode can be a great candidate for a future nonenzymatic glucose sensor that will be able to provide superior performance as well as great stability. (Zhao et al., Biosen. Bioelectron.,
Label-Free Biodetection Using a Smartphone
Gallegos et al. introduce a smartphone-based biodetection device. The device features a label-free photonic crystal biosensor and uses the integrated smartphone camera as a spectrometer for detection. To align the smartphone camera with the optical detection components, a custom-designed cradle is fabricated to hold the smartphone. The device uses externally provided broadband light such as daylight. The broadband light enters the device via a pinhole and is subsequently collimated and linearly polarized before passing through the photonic crystal biosensor, which resonantly reflects only a narrow band of wavelengths. This narrow band of wavelengths is further split via diffraction gratings and projected onto the CMOS image sensor on the camera to obtain a high-resolution transmission spectrum. The photonic crystal sensing element features a 1D grate surface structure fabricated via a nano-replica molding process using UV-curable polymers. The photonic crystal sensor is easily exchangeable to allow various applications. The smartphone is equipped with software that converts the camera image into a visible-light spectrum that can be used for biological analysis. The authors demonstrate the functionality of the system through detection of an immobilized protein monolayer and selective detection of concentration-dependent antibody binding to a functionalized photonic crystal. This portable, easy-to-use platform can lead to the development of an inexpensive, handheld biosensor instrument with web connectivity to enable point-of-care sensing in challenging environments. (Gallegos et al., Lab Chip,
Microtextured Substrates and Microparticles Used as In Situ Lenses for On-Chip Immunofluorescence Amplification
Immunofluorescence assays, such as ELISAs, play an important role in laboratory applications such as diagnosis, drug toxicity evaluation, food quality testing, and environmental safety monitoring. An increasing number of applications have involved implementation of immunofluorescence assays on microchips. These microchips feature small physical features, which help reduce sample consumption. However, due to small sample volume, the optical fluorescence detection can be challenging. Yang and Gijs introduce a novel method to enhance the fluorescence detection for on-chip immunoassays with 3D microtextured substrates and dielectric microparticles. The microtextured substrates feature a concave structure and are used in conjunction with dielectric spherical microparticles, which act as in situ microlenses. This unique combination allows exploitation of a large number of fluorophores and enhancement of optical signal via light focusing. In a module system, mouse IgG diluted in phosphate-buffered saline is tested and a detection limit as low as 2 ng/mL is achieved. The authors also present a detailed numerical study of the light propagation within the new system, which provides key insight into the signal amplification mechanism of the in situ microlenses and microtextured substrate. (Yang and Gijs, Anal Chem,
Nanobiotechnology
Integration of Solid-State Nanopores in Microfluidic Networks via Transfer Printing of Suspended Membranes
Solid-state nanopores have been key elements of many novel single-biomolecule studies such as single-molecule DNA sequencing, micro-RNA detection, and label-free detection of single-nucleotide polymorphisms. Solid-state nanopores are usually less than 20 nm in diameter and are typically constructed by ion or electron beam irradiation in a thin dielectric membrane. The dielectric membrane is usually suspended on a rigid silicon substrate that serves as the support and separates two reservoirs filled with an electrolyte solution. When a molecule translocates through the nanopore driven by an ionic current, a signal can be measured to provide the structural and compositional information on the single molecules. This setup creates several limitations. First, the nanopores are situated between large reservoirs, making it difficult for complex single-molecule manipulation. Second, the high noise from the silicon substrate reduces the spatial and temporal solution of the nanopore detection. Jain et al. introduce a new method for nanopore-microfluidic network integration. The dielectric membrane, after nanopore fabrication, is directly transferred from a silicon support substrate into a microfluidic network. The microfluidic network makes the nanopores addressable, and owing to significant reductions in membrane capacitance, the nanopores’ detection bandwidths and noise are significantly improved. This new method allows for large-scale integration of solid-state nanopores with microfluidic systems and helps exploit the sample manipulation capabilities of microfluidic systems to enable future nanopore applications such as multidimensional analysis and parallel sensing in 2D and 3D architectures. (Jain et al., Anal. Chem.,
DNA Sequencing Using Electrical Conductance Measurements of a DNA Polymerase
Single-molecule DNA sequencing is a vibrant research area because of its implications in life science and personalized medicine. In the past decades, much effort was focused on nanopore sequencing, which is a method for identifying the DNA sequence in real time by measuring the ionic current across a synthetic or biological nanopore as a DNA molecule is driven though the pore. Great successes have been demonstrated using nanopore technology, but many problems, such as short read lengths and high error rates, remain to be solved. Chen et al. propose an alternative approach for single-molecule DNA sequencing. Instead of using nanopores, the authors use a phi29 DNA polymerase. They show that single DNA molecules can be sequenced by monitoring the electrical conductance of a phi29 DNA polymerase as it incorporates unlabeled nucleotides into a template strand of DNA. They form a protein transistor, which consists of a phi29 DNA polymerase; an IgG bearing two gold nanoparticles; and two electrodes that are connected with two gold nanoparticles. The DNA sequencing is performed by monitoring the electrical conductivity of the DNA polymerase as the enzyme incorporates nucleotides into the newly synthesized DNA strands. The authors observe well-separated plateaus of ~3 pA in electrical conductance of the DNA polymerase during DNA replication processes. A distinct plateau pattern can be found for each of the four different nucleotides, which forms the basis for sequencing. The sequencing throughput is measured to be ~22 nucleotides per second, and the authors also demonstrate the flexibility of the system with a variety of DNA polymerases and difficult templates such as homopolymers. This novel method provides a brand-new route for single-molecule DNA sequencing. (Chen et al., Nat. Nanotechnol.,