Abstract
High-Throughput Imaging
QR-on-a-Chip: A Computer-Recognizable Micro-Pattern Engraved Microfluidic Device for High-Throughput Image Acquisition
Yun et al. introduce a novel method to achieve high-throughput image acquisition based on a computer-recognizable micro-pattern implemented on a microfluidic device. The authors integrate the QR code, a 2D barcode system, onto the microfluidic device to simplify imaging of multiple regions of interest (ROIs). A standard QR code pattern is created in micro-channels using arrays of cylindrical structures of polydimethylsiloxane. Using the recognition of the micro-pattern, the proposed system enables (1) device identification, which allows referencing additional information of the device, such as device imaging sequences or the ROIs, and (2) composing a coordinate system for an arbitrarily located microfluidic device with respect to the stage. With these functionalities, this method allows one-step high-throughput imaging for data acquisition in microfluidic devices without further manual exploration and locating of the desired ROIs. The authors report a significant reduction in the preparation time for image acquisitions. (Yun, K. et al. Lab Chip
Continuous-Flow C. elegans Fluorescence Expression Analysis with Real-Time Image Processing through Microfluidics
The nematode Caenorhabditis elegans is a commonly used essential model organism in neuroscience research due to its stereotyped anatomy, relevance to human biology, and capacity for genetic manipulation. Many automated chip designs based on immobilization-imaging-release approaches have been developed to address the intrinsic challenges associated with performing manual operations on C. elegans. There are several common disadvantages in these systems, such as the exertion of physical stress on the worms and limited throughput. Yan et al. report a continuous-flow, high-throughput, automated C. elegans analyzer based on droplet encapsulation and real-time image processing for analysis of fluorescence expression in worms.
To demonstrate its capabilities, two strains of C. elegans nematodes with different levels of expression of green fluorescent protein (GFP) are first mixed in a buffer solution. The worms are encapsulated in water-in-oil droplets to restrict random locomotion. The droplets are closely packed in a two-layer polydimethylsiloxane platform and are flowed through a narrow straight channel in which a region of interest is defined and continuously recorded by a frame acquisition device. Based on the number of pixels counted in the selected color range, GFP expression is analyzed to differentiate between two strains, and nearly 100% accuracy and a throughput of 0.5 s/worm are achieved. (Yan, Y. et al. Bios. Bioelec.
An Image Cytometer Based on Angular Spatial Frequency Processing and Its Validation for Rapid Detection and Quantification of Waterborne Microorganisms
Perez et al. report a novel image cytometer design for the detection of very small particulates and demonstrate its capability in water analysis. The device is a compact microscope composed of off-the-shelf components, such as a light-emitting diode (LED) source, a complementary metal–oxide–semiconductor (CMOS) image sensor, and a specific combination of optical lenses that allow Fourier transform processing of the sample volume. Waterborne microorganisms, such as Escherichia coli, Legionella pneumophila, and phytoplankton, are detected by interrogating the volume sample either in a fluorescent or label-free mode, that is, with or without fluorescein isothiocyanate molecules attached to the micro-organisms, respectively. A sensitivity of 50 cells/mL is achieved, which can be further increased to 0.2 cells/mL by preconcentrating an initial sample volume of 500 mL with an ad hoc fluidic system. The authors also show the capability of the proposed image cytometer of differentiating microbiological populations by size with a resolution of 3 µm and operating in real contaminated water. (Perez, J. M. et al. Analyst
Concurrent Detection of Cellular and Molecular Cancer Markers Using an Immunomagnetic Flow System
Huang et al. report a detection system for simultaneous measurement of cellular and molecular markers of cancer. Magnetic beads conjugated with antibodies against a specific antigen are used to capture both free molecules and whole cells overexpressing the antigen. The target-bound beads then flow through a microfluidic chamber where they are drawn to a glass surface by an external magnetic field. The cells and molecules captured on the surface are quantitatively analyzed using fluorescent microscopy.
The system is characterized by detecting free folate receptor (FR) and an FR+ cancer cell line (KB) in culture media. The system can detect as little as 10 pM of FR and capture 87% of the spiked KB cells at a volumetric throughput of 3 mL/min. The authors perform the detection of 100 KB cells and 200 pM FR spiked into healthy human blood to simulate detection of rare cells and protein biomarkers present in a cancer patient’s blood sample. The FR concentration measures 244 pM (including the intrinsic FR present in the blood), and the total number of KB cells in the sample is estimated to be 98.
The potential of this approach in clinical diagnostics is also demonstrated by detecting both FR+ cells and free FR in an ascites sample obtained from an ovarian cancer patient. Due to the system’s capability of simultaneous detection, its high throughput, and its overall simplicity, the authors expect it to be highly useful in a wide range of research settings. (Huang, W. et al. Anal. Chem.
Novel Imaging and Optical Techniques
A Gold Nanoparticle-Based Fluorescence Sensor for Highly Sensitive and Selective Detection of Thiols in Living Cells
A novel gold nanoparticle (AuNP)–based sensor for detecting thiols in aqueous solution is reported by Xu et al. Meso-(4-pyridinyl)-substituted BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes are coordinated to AuNP surfaces and effectively quench the fluorescence of organic/inorganic hybrid systems.
The fluorescent quenching mechanism is mainly ascribed to the highly efficient fluorescent resonance energy transfer and the inner filter effect. In the presence of thiols, meso-(4-pyridinyl)-substituted BODIPY chromophores are displaced and released from the AuNP surfaces and thus restore the fluorescence of BODIPY chromophores. The modulation of the fluorescence-quenching efficiency of BODIPY-AuNPs in the presence of thiols can yield a large turn-on fluorescence enhancement (40-fold) in aqueous solution. This novel AuNP-based fluorescence sensor shows desired properties such as high specificity, a relatively low detection limit (30 nM for Cys), appreciable water solubility, and rapid response time (within 2 min for Cys/Hcy). The authors also apply the sensor for monitoring and imaging of intracellular thiols within living HeLa cells. (Xu, J. et al. Bios. Bioelec.
Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging
Glycosylation is a ubiquitous posttranslational modification that is present in over 50% of the proteins in the human genome. It has an important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers for many diseases, including cancer. Neves et al. report the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal magnetic resonance imaging probe. Significant N-azidoacetylgalactosamine–dependent T1 contrast is observed in vivo 2 h after probe administration. The tumor, kidney, and liver show significant contrast, and several other tissues, including the pancreas, spleen, heart, and intestines, also show a very high contrast (>10-fold). The authors believe this approach has the potential to enable the rapid and noninvasive magnetic resonance imaging of glycosylated tissues in vivo in preclinical models of disease. (Neves, A. A. et al. Angew. Chem.
Highly Efficient Photothermal Semiconductor Nanocomposites for Photothermal Imaging of Latent Fingerprints
Optical imaging of latent fingerprints (LFPs) has been widely used in forensic and antiterrorist applications, but it suffers from interference from autofluorescence and the substrates’ background color. Cui et al. report highly efficient protothermal semiconductor nanoparticles that help address these problems.
Cu7S4 nanoparticles (NPs), with excellent photothermal properties, are synthesized using a new strategy and then fabricated into amphiphilic nanocomposites (NCs) via polymerization of allyl mercaptan coated on Cu7S4 NPs for enhanced affinities toward LFPs. The authors demonstrate a very high photothermal conversion efficiency (52.92% at 808 nm) of Cu7S4 NCs and effective imaging of LFPs left on different substrates (with various background colors), which are extremely useful for crime scene investigations. In addition, by fabricating Cu7S4-CdSe@ZnS NCs, the authors further develop a fluorescent-photothermal dual-mode imaging strategy for detection of trinitrotoluene (TNT) in LFPs. (Cui, J. et al. Anal. Chem.
Optofluidic Tunable Lenses Using Laser-Induced Thermal Gradient
Chen et al. report a new optofluidic tunable lens design using a laser-induced thermal gradient. This design uses two straight chromium strips at the bottom of a microfluidic chamber to absorb the continuous pump laser to heat up the moving benzyl alcohol solution, creating a 2D refractive index gradient in the area between the two hot strips. The refractive index gradient distribution creates liquid optical elements that can be regarded as a cascade of a series of refractive lenses and can be used to focus and manipulate light.
Computational fluid dynamics simulation shows that a stable thermal lens can be built up within 200 ms. Experiments also demonstrate the continuous tuning of focal length from initially infinite to the minimum 1.3 mm, as well as off-axis focusing by offsetting the pump laser spot. Data analyses further show the empirical dependences of the focal length on the pump laser intensity and the flow velocity. The authors believe that compared with previously reported liquid lenses, this design shows many benefits such as fast tuning speed, aberration-free focusing, and remote control, and it enables the use of homogeneous fluids for easy integration with other optofluidic systems. (Chen, Q. et al. Lab Chip
High-Throughput Assays
Acoustofluidic Fluorescence Activated Cell Sorter
Selective isolation of cell subpopulations with defined biological characteristics is crucial for many biological studies and clinical applications. Nawaz et al. present the development of an acoustofluidic fluorescence-activated cell sorting (FACS) device that simultaneously performs on-demand, high-throughput, high-resolution cell detection and sorting, integrated onto a single chip. This acoustofluidic FACS device uses the “microfluidic drifting” technique to precisely focus cells/particles three-dimensionally and achieves a flow of single-file particles/cells as they pass through a laser interrogation region. Short bursts (150 µs) of standing surface acoustic waves triggered by an electronic feedback system are then used to sort fluorescently labeled particles/cells with desired biological properties.
The authors demonstrate continuous isolation of fluorescently labeled HeLa cells from unlabeled cells at a throughput of ∼1200 events/s with a purity reaching 92.3% ± 3.39%. Furthermore, 99.18% postsort cell viability indicates that this acoustofluidic sorting technique maintains a high integrity of cells. In summary, this integrated acoustofluidic FACS device shows two-way cell sorting with high purity, biocompatibility, and biosafety. The authors believe this device has significant potential for use as a low-cost, high-performance, portable, and user-friendly FACS instrument. (Nawaz, A. H. et al. Anal. Chem.
Kilo-Scale Droplet Generation in Three-Dimensional Monolithic Elastomer Device (3D MED)
Droplet-based microfluidics is an important tool in a variety of applications, including materials synthesis and high-throughput biological assays. However, the translation of droplet microfluidics technology into commercial applications requires scale-up of droplet generation from the laboratory (<10 mL h−1) to the industrial (>1 L h−1) scale.
To address this challenge, Jeong et al. have developed a 3D monolithic elastomer device (3D MED) for mass production of monodisperse emulsion droplets. Using double-sided imprinting, 3D microchannels are formed in a single elastomer piece that has 1000 parallel-flow focusing generators (k-FFGs). Compared to previous work that parallelizes droplet generation, the 3D MED eliminates the need for alignment and bonding of multiple pieces and thus makes it possible to achieve the high flow rates and pressure necessary for the kiloscale generation of droplets.
Using this approach, the authors demonstrate mass production of water-in-oil (W/O) emulsion droplets at production rates as high as 1.5 L h−1 (>30 billion 45-µm diameter droplets per hour) with a coefficient of variation of droplet diameter of only 6.6%. The authors believe that due to the simplicity, robustness, and manufacturability of this 3D MED architecture, it is well suited to bridge the gap between the continuously growing library of promising microfluidic droplet technologies and industry applications. (Jeong, H.-H. et al., Lab Chip,
A High-Throughput Platform for Formulating and Screening Multifunctional Nanoparticles Capable of Simultaneous Delivery of Genes and Transcription Factors
Simultaneous delivery of multiple genes and proteins (e.g., transcription factors [TFs]) is an emerging issue surrounding therapeutic research due to their ability to regulate cellular circuitry. Current gene and protein delivery strategies, however, are based on slow-batch synthesis, which is ineffective, poorly controlled, and incapable of simultaneous delivery of both genes and proteins with synergistic functions. Consequently, advances in this field have been limited to in vitro studies.
By integrating microfluidic technologies with a supramolecular synthetic strategy, Liu et al. present a high-throughput approach for formulating and screening multifunctional supramolecular nanoparticles self-assembled from a collection of functional modules to achieve simultaneous delivery of one gene and TF with unprecedented efficiency both in vitro and in vivo. The authors envision that this new approach could open new avenues for immunotherapy, stem cell reprogramming, and other therapeutic applications. (Liu, Y. et al. Angew. Chem.
Novel Sensing Technologies
Smartphone-Based Point-of-Care Testing of Salivary α-Amylase for Personal Psychological Measurement
Chen et al. report a smartphone-based potentiometric biosensor for point-of-care testing of salivary α-amylase (sAA), which is one of the most sensitive indices of autonomic nervous system activity and therefore a promising noninvasive biomarker for mental health. The biosensing system includes a smartphone, a potentiometric reader, and a sensing chip with preloaded reagents. The saliva sample wicks into the reaction zone on the sensing chip. sAA reacts with the preloaded reagents and results in conversion of an electron mediator Fe(CN)63− to Fe(CN)64−. The sensing chip is then inserted into the detection zone for the potentiometric measurement. The potential measured by the smartphone-powered potentiometric reader is sent to the smartphone app via a USB port and converted into sAA concentration based on a calibration curve. Using this method, sAA in real human samples can be quantified in less than 5 min. The results are shown to be in good agreement with the reference method and correlated to psychological states of the subjects. (Chen, Q. et al. Lab Chip
Portable Kit for Identification and Detection of Drugs in Human Urine Using Surface-Enhanced Raman Spectroscopy
Han et al. report a portable kit for rapid and reliable surface-enhanced Raman scattering (SERS) detection of drugs in human urine. This kit contains two sealed reagent tubes, a packet of standardized SERS substrates, and a mini Raman device. A 3-min pretreatment protocol extracts amphetamines from urine, with an extraction efficiency of 80%. A detection limit of amphetamines in human urine of at least 0.1 ppm is achieved. The portable kit was also successfully used for detecting MA, 3,4-methylenedioxymethamphetamine (MDMA), and methcathinone (MC) in 30 volunteers’ urine samples with various clinical natures, and the dual-analyte detection of MA and MDMA implies a good capability of multiplex analysis. Ultraperformance liquid chromatography examination and the SERS recovery test show that the pretreatment procedure is sufficient to reduce the high background signals caused by complex components in urine and demonstrates the practicability and the resistance to false positives, which is a vital problem for clinical and forensic applications. The authors believe this portable kit shows great promise toward a rapid, reliable, and portable method for public safety and health care. (Han, Z. et al. Anal. Chem.
Integration of an Optical CMOS Sensor with a Microfluidic Channel Allows a Sensitive Readout for Biological Assays in Point-of-Care Tests
Dorst et al. present a microfluidic detection module that allows a sensitive readout of biological assays in point-of-care (POC) tests. The proposed detection module consists of a microfluidic flow cell with an integrated complementary metal–oxide–semiconductor (CMOS)–based single-photon counting optical sensor. The authors believe this detection module could serve as the core technology in standalone POC tests for mobile applications or in resource-limited environments.
The performance of the detection module is demonstrated in three assays: a peptide, a protein, and an antibody detection assay. The antibody detection assay with readout in the detection module shows a 7-fold improvement in sensitivity compared to that of the traditional colorimetric plate-based enzyme-linked immunosorbent assay. The protein and peptide assays also show lower limits of detection (LLODs) of 200 fM and 460 fM, respectively.
These results demonstrate that the sensitivity of the immunoassays based on this CMOS sensor is comparable with lab-based immunoassays and at least equal to or better than current mainstream POC devices. This technology offers potential for detection of low concentrations of biomarkers and can be particularly useful in clinician offices and patient homes where currently only the less sensitive lateral flow and dipstick POC tests are implemented. (Dorst, B. V. et al., Bios. Bioelec.