Abstract
Smartphone Diagnostics
Detection and Spatial Mapping of Mercury Contamination in Water Samples Using a Smart-Phone
Detection of environmental contamination such as trace-level toxic heavy metal ions mostly relies on bulky and costly analytical instruments. However, a considerable global need exists for portable, rapid, specific, sensitive, and cost-effective detection techniques that can be used in resource-limited and field settings. Wei et al. introduce a smartphone-based handheld platform that allows the quantification of mercury(II) ions in water samples with parts per billion (ppb) level of sensitivity.
For this task, the authors create an integrated optomechanical attachment to the built-in camera module of a smartphone to digitally quantify mercury concentration using a plasmonic gold nanoparticle (Au NP) and aptamer-based colorimetric transmission assay that is implemented in disposable test tubes. This smartphone attachment weighs <40 g. With this device, Mercury(II) ion concentration in water samples can be quantified with a two-color ratiometric method employing light-emitting diodes (LEDs) at 523 and 625 nm. A custom-developed smart application is used to process each acquired transmission image on the same phone to achieve a limit of detection of 3.5 ppb.
Using this smartphone-based detection platform, the authors generate a mercury contamination map by measuring water samples at over 50 locations in California, taken from city tap water sources, rivers, lakes, and beaches. The authors believe with its cost-effective design, field portability, and wireless data connectivity, this sensitive and specific heavy metal detection platform running on cellphones could be rather useful for distributed sensing, tracking, and sharing of water contamination information as a function of both space and time (Wei, Q., et al. ACS Nano,
Development of the Smartphone-Based Colorimetry for Multi-Analyte Sensing Arrays
Hong and Chang report development of a smartphone app (application) that digitizes the colors of a colorimetric sensor array. A colorimetric sensor array consists of multiple paper-based sensors and reports the detection results in terms of color change. Evaluation of the color changes is normally done by the naked eye, which may cause uncertainties due to personal subjectivity and the surrounding conditions.
In this report, the authors take advantage of the spectrometric functions of the smartphone cameras. The work focuses on development of a practical app for immediate point-of-care (POC) multianalyte sensing without additional devices. First, the individual positions of the sensors are automatically identified by the smartphone; second, the colors measured at each sensor are digitized based on a correction algorithm; and third, the corrected colors are converted to concentration values by preloaded calibration curves. Through these sequential processes, the sensor array taken in a smartphone snapshot undergoes laboratory-level spectrometric analysis. The combined advantages of inexpensive and convenient paper-based colorimetry and the ubiquitous smartphone result in a ready-to-go POC diagnostic device (Hong, J. I., and Chang, B.-Y. Lab Chip,
Cholesterol Testing on a Smartphone
Home self-diagnostic tools for blood cholesterol monitoring have been around for over a decade, but their widespread adoption has been limited by the relatively high cost of acquiring a quantitative test strip reader, complicated procedure for operating the device, and inability to easily store and process results. To address these challenges, Oncescu et al. have developed a smartphone accessory and software application that allows for the quantification of cholesterol levels in blood.
Through a series of human trials, the authors demonstrate that the system can accurately quantify total cholesterol levels in blood within 60 s by imaging standard test strips. In addition, the authors demonstrate how the diagnostic accessory is optimized to improve measurement sensitivity and reproducibility across different individual smartphones. With the widespread adoption of smartphones and increasingly sophisticated image-processing technology, accessories such as the one presented in this work will allow cholesterol monitoring to become more accurate and widespread, greatly improving preventive care for cardiovascular disease (Oncescu, V., et al. Lab Chip,
Lab Automation
The LabTube: A Novel Microfluidic Platform for Assay Automation in Laboratory Centrifuges
Assay automation is the key for successful transformation of modern biotechnology into routine workflows. Yet, it requires considerable investment in processing devices and auxiliary infrastructure, which is not cost-efficient for laboratories with low or medium sample throughput or point-of-care testing.
To close this gap, Kloke et al. present the LabTube platform, which is based on assay-specific disposable cartridges for processing in laboratory centrifuges. LabTube cartridges comprise interfaces for sample loading and downstream applications and fluidic unit operations for release of prestored reagents, mixing, and solid-phase extraction. Process control is achieved by a centrifugally actuated ballpen mechanism.
To demonstrate the workflow and functionality of the LabTube platform, the authors show two LabTube automated sample preparation assays from laboratory routines: DNA extractions from whole blood and purification of His-tagged proteins. Equal DNA and protein yields are observed compared with manual reference runs, while LabTube automation could significantly reduce the hands-on time to 1 min per extraction (Kloke, A., et al. Lab Chip,
A Versatile-Deployable Bacterial Detection System for Food and Environmental Safety Based on LabTube-Automated DNA Purification, LabReader-Integrated Amplification, Readout and Analysis
Contamination of foods is a public health hazard that episodically causes thousands of deaths and sickens millions worldwide. To ensure food safety and quality, rapid, low-cost, and easy-to-use detection methods are desirable. With this in mind, Hoehl et al. introduce the LabSystem for integrated, automated DNA purification, amplification, and detection.
The LabSystem consists of a disposable, centrifugally driven DNA purification platform (LabTube) and a low-cost UV/vis-reader (LabReader). For demonstration of the LabSystem in the context of food safety, purification of Escherichia coli (nonpathogenic E. coli and pathogenic verotoxin-producing E. coli [VTEC]) in water and milk and the product-spoiler Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice is integrated and optimized in the LabTube. Inside the LabReader, the purified DNA is amplified, read out, and analyzed using both qualitative isothermal loop-mediated DNA amplification (LAMP) and quantitative real-time PCR. For the LAMP-LabSystem, the combined detection limits for purification and amplification of externally lysed VTEC and A. acidoterrestris are 102–103 cell equivalents. In the PCR-LabSystem for E. coli cells, the quantification limit is 102 cell equivalents, including LabTube-integrated lysis.
The demonstrated LabSystem requires only a laboratory centrifuge (to operate the disposable, fully closed LabTube) and a low-cost LabReader for DNA amplification, readout, and analysis. Compared with commercial DNA amplification devices, the LabReader improves sensitivity and specificity by the simultaneous readout of four wavelengths and the continuous readout during temperature cycling. The use of a detachable eluate tube as an interface affords semi-automation of the LabSystem, which does not require specialized training. It reduces the hands-on time from about 50 to 3 min with only two handling steps: sample input and transfer of the detachable detection tube (Hoehl, M. M., et al. Analyst,
World-to-Digital-Microfluidic Interface Enabling Extraction and Purification of RNA from Human Whole Blood
Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. However, handling of “real-world” samples has been a challenge because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration.
To address this challenge, Jebrail et al. share a novel “world-to-DMF” interface in which an integrated companion module drives the large-volume sample through a 10-µL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, the authors use this system for extraction of RNA from human whole-blood lysates (110–380 µL) and further purification in microscale volumes (5–15 µL) on the DMF device itself.
Processing by the system is >2-fold faster and consumes 12-fold less reagents, yet produces RNA yields and quality fully comparable to conventional preparations and supporting quantitative reverse transcription PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. The authors believe this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications (Jebrail, M. J., et al. Anal. Chem.,
High-Throughput Screening
Membrane-on-a-Chip: Microstructured Silicon/Silicon-Dioxide Chips for High-Throughput Screening of Membrane Transport and Viral Membrane Fusion
Screening of transport processes across biological membranes is hindered by the challenges of establishing fragile supported lipid bilayers and the difficulty of determining at which side of the membrane reactants reside.
Kusters et al. report a method for the generation of suspended lipid bilayers with physiologically relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation is investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with a fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers (Kusters, L., et al. ACS Nano,
Microfluidic High-Throughput Culturing of Single Cells for Selection Based on Extracellular Metabolite Production or Consumption
Phenotyping single cells based on the products they secrete or consume is a key bottleneck in many biotechnology applications, such as combinatorial metabolic engineering for the overproduction of secreted metabolites. Wang et al. report a flexible high-throughput approach that uses microfluidics to compartmentalize individual cells for growth and analysis in monodisperse nanoliter aqueous droplets surrounded by an immiscible fluorinated oil phase.
The authors use this system to identify xylose-overconsuming Saccharomyces cerevisiae cells from a population containing one such cell per 104 cells and to screen a genomic library to identify multiple copies of the xylose isomerase gene as a genomic change contributing to high xylose consumption, a trait important for lignocellulosic feedstock utilization. The authors also enrich L-lactate–producing E. coli clones 5800× from a population containing one L-lactate producer per 104 D-lactate producers. The authors believe this approach has broad applications for single-cell analyses, such as in strain selection for the overproduction of fuels, chemicals, and pharmaceuticals (Wang, B. L., et al. Nat. Biotech.,
Micro-Scaffold Array Chip for Upgrading Cell-Based High-Throughput Drug Testing to 3D Using Benchtop Equipment
Cell-based high-throughput drug screening accelerates the pace of drug discovery that is routinely operated on planar high-density multiwell plates with sophisticated robotic liquid-dispensing systems for cell seeding and drug administration. Considerable efforts have been made to upgrade in vitro cellular models from 2D to a more biomimetic 3D configuration. For instance, in anticancer drug screening, tumor spheroids are increasingly applied as a gold-standard 3D model exhibiting cellular behaviors and drug responses distinguishable from the 2D counterpart. However, translation of spheroids to high-throughput drug screening is challenging because preformation of spheroids and subsequent translocation to multiwell plates for drug testing are usually uncontrollable and time/reagent consuming, and cell loss is inevitable during medium exchange for drug testing.
Li et al. report an off-the-shelf micro-scaffold array chip that enables high-throughput 3D cell culture, drug administration, and quantitative in situ assays entirely on the same chip. The sponge-like micro-scaffolds function both as absorbents to realize parallel auto-loading of cells or drugs and as barriers to prevent cell loss during medium exchange via centrifugation. Rapid manual loading of cell suspensions or drugs into the 96 isolated micro-scaffolds on the chip is achieved in the timescale of several seconds, and meanwhile, total medium consumption reduces to the order of microliters. Proof-of-concept demonstration of drug cytotoxicity testing is performed on multiple cancer cells using common benchtop equipment, making it accessible to most biomedical laboratories with basic cell culture setups. Higher cellular drug resistance is constantly obtained with this platform compared with the planar cultures, which is partially attributed to the malignant phenotype of cancer cells yielded by enhanced cell-matrix interactions in the micro-scaffolds. Interestingly, the high drug resistance of 3D cultured cells in the micro-scaffold is shown to be density independent in contrast to the density-dependent drug response for 2D cultured cells, indicating intrinsic differences between the two culture models. This platform is expected to facilitate upgrade of the current cell-based high-throughput drug testing to the 3D level and be widely applicable across various disciplines (Li, X., et al. Lab Chip,
Paper-Based Microfluidics
Wire, Mesh, and Fiber Electrodes for Paper-Based Electroanalytical Devices
Fosdick et al. report the use of microwire and mesh working electrodes in paper analytical devices fabricated by origami paper folding (oPADs). The new finding of this work is that Au wires and carbon fibers having diameters ranging from micrometers to tens of micrometers can be incorporated into oPADs and that their electrochemical characteristics are consistent with the results of finite element simulations. These electrodes are fully compatible with both hollow channels and paper channels filled with cellulose fibers, and they are easier to incorporate than typical screen-printed carbon electrodes. The results also demonstrate that the Au electrodes can be cleaned prior to device fabrication using aggressive treatments, and they can be easily surface modified using standard thiol-based chemistry (Fosdick, S. E., et al. Anal. Chem.,
Multilayer Paper-Based Device for Colorimetric and Electrochemical Quantification of Metals
The release of metals and metal-containing compounds into the environment is a growing concern in developed and developing countries, as human exposure to metals is associated with adverse health effects in virtually every organ system. Unfortunately, quantifying metals in the environment is expensive; analysis costs using certified laboratories typically exceed $100/sample, making the routine analysis of toxic metals cost-prohibitive for applications such as occupational exposure or environmental protection.
Rattanarat et al. report a simple, inexpensive technology with the potential to render toxic metals detection accessible for both the developing and developed world that combines colorimetric and electrochemical microfluidic paper-based analytical devices (mPAD) in a 3D configuration. Unlike previous mPADs designed for measuring metals, the device reported here separates colorimetric detection on one layer from electrochemical detection on a different layer. Separate detection layers allow different chemistries to be applied to a single sample on the same device. To demonstrate the effectiveness of this approach, colorimetric detection is shown for Ni, Fe, Cu, and Cr and electrochemical detection for Pb and Cd. Detection limits as low as 0.12 µg (Cr) are achieved on the colorimetric layer while detection limits as low as 0.25 ng (Cd and Pb) are achieved on the electrochemical layer. Selectivity for the target analytes is demonstrated for common interferences. As an example of the device utility, particulate metals collected on air sampling filters are analyzed. Levels measured with the mPAD match known values for the certified reference samples of collected particulate matter (Rattanarat, P., et al. Anal. Chem.,
Rational Selection of Substrates to Improve Color Intensity and Uniformity on Microfluidic Paper-Based Analytical Devices
Evans et al. report a systematic investigation on the effect of paper type on the analytical performance of a series of microfluidic paper-based analytical devices (µPADs) fabricated using a CO2 laser engraver. Samples include three different grades of Whatman chromatography paper and three grades of Whatman filter paper. According to the data collected and the characterization performed, different papers offer a wide range of flow rates, thickness, and pore sizes. After optimizing the channel widths on the µPAD, the focus of this study is directed toward the color intensity and color uniformity formed during a colorimetric enzymatic reaction. According to the results described, the type of paper and the volume of reagents dispensed in each detection zone can determine the color intensity and uniformity. This study provides rational guidelines for the selection of paper substrates for the fabrication of µPADs (Evans, E., et al. Analyst,
Lab on a Disk
Fully Integrated Lab-on-a-Disc for Nucleic Acid Analysis of Food-Borne Pathogens
Kim et al. describe a micro total analysis system for molecular analysis of Salmonella, a major food-borne pathogen. The authors develop a centrifugal microfluidic device, which integrates the three main steps of pathogen detection—DNA extraction, isothermal recombinase polymerase amplification (RPA), and detection—onto a single disk. A single laser diode is used for wireless control of valve actuation, cell lysis, and noncontact heating in the isothermal amplification step, thereby yielding a compact and miniaturized system.
To achieve high detection sensitivity, rare cells in large volumes of phosphate-buffered saline (PBS) and milk samples are enriched before loading onto the disk by using antibody-coated magnetic beads. The entire procedure, from DNA extraction through to detection, is completed within 30 min in a fully automated fashion. The final detection is carried out using lateral flow strips by direct visual observation; detection limit is 10 cfu/mL and 102 cfu/mL in PBS and milk, respectively. This device allows rapid molecular diagnostic analysis and does not require specially trained personnel or expensive equipment. The authors expect that it would have an array of potential applications, including the detection of food-borne pathogens, environmental monitoring, and molecular diagnostics in resource-limited settings (Kim, T.-H., et al. Anal. Chem.,
Fully Automated Circulating Tumor Cell Isolation Platform with Large-Volume Capacity Based on Lab-on-a-Disc
Full automation with high purity for circulating tumor cell (CTC) isolation has been regarded as a key goal to make CTC analysis a bench-to-bedside technology. Park et al. report a novel centrifugal microfluidic platform that can isolate the rare cells from a large volume of whole blood. To isolate CTCs from whole blood, the authors introduce a disk device having the biggest sample capacity as well as manipulating blood cells for the first time.
The fully automated disk platform can handle 5 mL of blood by designing the blood chamber having a triangular obstacle structure (TOS) with lateral direction. To guarantee high purity that enables molecular analysis with the rare cells, CTCs are bound to the microbeads covered with anti-EpCAM to discriminate density between CTCs and blood cells, and the CTCs, being heavier than blood cells, are settled only under a density gradient medium (DGM) layer.
To understand the movement of CTCs under centrifugal force, the authors perform computational fluid dynamics simulation and find that their major trajectories are the boundary walls of the DGM chamber, thereby optimizing the chamber design. After whole blood is inserted into the blood chamber of the disk platform, size- and density-amplified cancer cells are isolated within 78 min, with minimal contamination as much as approximately 12 leukocytes per milliliter.
As a model of molecular analysis toward personalized cancer treatment, the authors perform epidermal growth factor receptor (EGFR) mutation analysis with HCC827 lung cancer cells, and the isolated cells are then successfully detected for the mutation by PCR clamping and direct sequencing (Park, J.-M., et al. Anal. Chem.,