Graphene Oxide Paper Manipulation of Micro-Reactor Drops.

Digital microfluidics, which relies on the movement of drops, is relatively immune to clogging problems, making it suited for micro-reactor applications. Here, graphene oxide paper of 100 µm thickness, fabricated by blade coating sedimented dispersions onto roughened substrates, followed by drying and mechanical exfoliation, was found to be relatively free of cracks and curling. It also exhibited high wettability and elasto-capillary characteristics. Possessing low enough stiffness, it could rapidly and totally self-wrap water drops of 20 µL volume placed 2 mm from its edge when oriented between 0 and 60° to the horizontal. This complete wrapping behavior allowed drops to be translated via movement of the paper over long distances without dislodgement notwithstanding accelerations and decelerations. An amount of 2 drops that were wrapped with separate papers, when collided with each other at speeds up to 0.64 m/s, were found to eschew coalescence. This portends the development of robust digital microfluidic approaches for micro-reactors.

Low-cost Imaging of Fluorescent DNA in Agarose Gel Electrophoresis using Raspberry Pi cameras.

Low-cost analytical solutions built around microcomputers like the Raspberry Pi help to facilitate laboratory investigations in resource limited venues. Here, three camera modules (V1.3 with and without filter, as well as NoIR) that work with this microcomputer were assessed for their suitability in imaging fluorescent DNA following agarose gel electrophoresis. Evaluation of their utility was based on signal-to-noise (SNR) and noise variance metrics that were developed. Experiments conducted with samples were subjected to Polymerase Chain Reaction (PCR), and the amplified products were separated using gel electrophoresis and stained with Midori green. Image analysis revealed the NoIR camera performed the best with SNR and noise variance values of 21.7 and 0.222 respectively. In experiments conducted using UV LED lighting to simulate ethidium bromide (EtBr) excitation, the NoIR and V1.3 with filter removed cameras showed comparable SNR values.

Fog Harvesting with Highly Wetting and Nonwetting Vertical Strips.

Highly wetting and nonwetting substrates have been widely used in fogwater collection systems for enhanced water harvesting. In this work, fog harvesting substrates comprising PVC strips of different wetting properties and widths ranging from 1-5 mm were vertically aligned and spaced apart at regular intervals to give the same solid area fraction of 0.8. Evaluation of the water collection efficiencies of the tested configurations revealed that 1 mm wide superhydrophilic strips was the most efficient, achieving double the amount of water harvested compared with 2.8 mm wide strips. This finding was attributed to the low Stokes numbers of the aerosol particle distribution of the fog which tended to result in them being brought by the flow streamlines toward the air gaps between the strips. Stagnant flow regions at the edges of each strip, revealed through potential flow calculations, then caused higher liquid imbibition and impaction there for water harvesting. It was also found that the Cassie nonwetting substrates that originally exhibited contact angles of 161° transformed to Wenzel wetting with zero contact angle within 60 min of fog interception. Optical profilometry revealed no obvious difference in surface roughness between the central region and edges of the strips, indicating that surface morphology was unlikely to be a contributing factor for enhanced water collection at the edges. The findings here indicated that highly wetting vertical strip architectures with narrow widths (1 mm) were favorable over wider strips for water harvesting provided that clogging and re-entrainment were not significant factors.

Cryoprotectant-free preservation of bacteria using semi-spherical drops.

Cryopreservation is a widely used long-term preservation method to ensure the quality and vitality of microbes in laboratories and biological resource facilities. However, freeze-thaw damage and osmotic pressure changes during cryopreservation adversely impacts microbial survival. Significant expenditure of resources and expertise are required to select the right cryoprotectant and optimize its concentration for maximum survival of diverse microorganisms. This work describes a cryopreservation method that obviates the need for cryoprotectants by exploiting the unique thermal characteristics of semi-spherical drops. Here, a plurality of these drops, each 10 µl in volume, created on a highly non-wetting flat-sheet substrate with holes and frozen at -70 °C. Deriving an f (x) metric as a measure of relative viability, storage in drops in the absence of cryoprotectants was found to improve the survivability of Staphylococcus epidermidis by 1.91 times compared with the same sample stored in larger 50-µl volumes in standard 1.5-ml tubes. This also compares well with a value of 2.33 obtained with standard preservation with cryoprotectant. The drop method allows high throughput aliquoting of the bacterial culture into multiple discrete drops using multichannel pipettes or automated liquid handlers and the edges of the holes provides a pinning action that holds the drop stably against gravitational roll-offs. It also allows samples to be removed in discrete small volumes, thus, reducing the number of freeze thaw cycles and associated cell damage. The flat-sheet architecture of the substrate reduces the amount of plastic waste generated and augments green laboratory practices.

Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation.

High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13-24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.

Liquid marble clearance and restoration via gas bubble insertion and bursting.

There is hitherto a lack of a simple way to disrupt the coating of particles from liquid marbles in order to introduce additional reagents. Here, a 40 µL liquid marble, created on a superhydrophobic substrate with a 2 mm hole, forms an overhead and overhanging liquid component from which a single gas bubble of up to 28 µL volume could be introduced via the latter. This caused a localized clearing of the particle shell at the apical region of the overhead component because the particles could not be energetically sustained at the thin film region of the bubble. The subsequent dispensation of 5 µL of an external liquid directly onto the shell-free apex of the liquid marble allowed the coalescence of the two liquid bodies, bubble rupture, and restoration of complete particle shell encapsulation. The addition of the liquid via the overhanging component was alternatively found incapable of increasing the size of the overhead drop component. The localized bubble-actuated transient shell clearance at the apex of the liquid marble to allow the addition of reagents shown here portends new vistas for liquid marbles to be used in biomedical applications.

Inflight Polymerase Chain Reaction of samples with drones.

A system devised to conduct Polymerase Chain Reaction (PCR) in-flight on drones that uses the spatial displacement of capillary tubes on thermal blocks kept at 94 °C, 58 °C and 72 °C corresponding to cycling temperatures for denaturation, annealing and extension is demonstrated here. The use of acetal as the thermal block material reduced heat loss and the input power (within 18.5 W) needed to maintain the required temperatures. Tests showed that concentrations of samples down to 1.16 × 106 DNA copies/µL could be significantly and consistently detected above the background emission of the fluorescence signal intensity.

Polymerase chain reaction thermal cycling using the programmed tilt displacements of capillary tubes.

A thermal cycling method, whereby capillary tubes holding polymerase chain reactions are subjected to programmed tilt displacements so that they are moved using gravity over three spatial regions (I, II, and III) kept at different constant temperatures to facilitate deoxyribonucleic acid (DNA) denaturation, annealing, and extension, is described. At tilt speeds in excess of 0.2 rad/s, the standard deviation of static coefficient of friction values was below 0.03, indicating in sync movement of multiple capillary tubes over the holding platform. The travel time during the acceleration phase and under constant velocity between adjacent regions (I to II and II to III) and distant regions (III to I) was 0.03 s and 0.31 s, respectively. The deviations in temperature did not exceed 0.05 °C from the average at the prescribed denaturing, annealing, and extension temperatures applied. DNA amplification was determined by optical readings, the fluorescence signal was found to increase twofold after 30 thermal cycles, and 1.16 × 106 DNA copies/µl could be detected. The approach also overcomes problems associated with thermal inertia, sample adhesion, sample blockage, and handling of the reaction vessels encountered in the other thermal cycling schemes used.

Cryopreservation without dry ice-induced acidification during sample transport.

Dry ice (solid CO2) remains highly useful when temperature-sensitive biological samples need to be cryogenically transported. CO2 released during the sublimation of dry ice can diffuse through gas permeable receptacle material or any defective seals resulting in potential sample acidification and compromised integrity. In addition, the quality of cryopreservation can be undermined once the dry ice is exhausted. The dry ice carrier design described here has been demonstrated to prevent sublimated CO2 from reaching the samples while maintaining storage temperature below -60 °C for 19 h. It is also equipped with microcontroller-based temperature monitoring for traceability and CO2 gas monitoring for safety.

Syringe infusion pump with absolute piston displacement control.

A vast majority of syringe pumps operate on stepper motors, which limits their effectiveness for precision fluid delivery using estimation algorithms. Such a system also hampers the ability to ascertain if the infusion or aspiration instruction has been correctly carried out in the event of power interruptions. To address this issue, a linear servo based actuator system is described to provide absolute indications of the plunger position. System performance in terms of linearity and reliability of plunger translation were verified using a camera tracking system with syringe capacities ranging from 3 to 50 ml and at syringe plunger speeds ranging from 1 to 6.6 mm/s when distilled water was used as the medium. In investigations involving more viscous liquids, the system revealed similarly linear characteristics with 50% glycerol-water (v/v), but cyclical stick-slip behavior with Freund's adjuvant.

Living specimens under field immobilization and smartphone microscopic observation.

In this work a single glycerol-water mixture, determined to be most apt at 30% (wt/vol), was used to immobilize Drosophila samples as well as to create a liquid lens for smartphone magnification viewing. This provides the advantage of being able to observe immobilized insects directly in the field rather than in the laboratory. In order to avoid having bubbles in the liquid lens and immobilizing medium that hinder visibility, an approach was developed where a stable pendant drop is moved in tandem with the dispensing tip by gravity and stopped abruptly so that sufficient momentum is transferred to the drop for its dislodgement. With 30% glycerol-water (wt/vol) mixtures, applying a minimal stroke of 10 mm with longitudinal impact delivered a momentum of 0.1464 N/s that allowed transfer of a preselected liquid volume for the processes.

A Dynamic Graphene Oxide Network Enables Spray Printing of Colloidal Gels for High-Performance Micro-Supercapacitors.

Properly controlling the rheological properties of nanoparticle inks is crucial to their printability. Here, it is reported that colloidal gels containing a dynamic network of graphene oxide (GO) sheets can display unusual rheological properties after high-rate shearing. When mixed with polyaniline nanofiber dispersions, the GO network not only facilitates the gelation process but also serves as an effective energy-transmission network to allow fast structural recovery after the gel is deformed by high-rate shearing. This extraordinary fast recovery phenomenon has made it possible to use the conventional air-brush spray technique to print the gel with high-throughput and high fidelity on nonplanar flexible surfaces. The as-printed micro-supercapacitors exhibit an areal capacitance 4-6 times higher than traditionally spray-printed ones. This work highlights the hidden potential of 2D materials as functional yet highly efficient rheological enhancers to facilitate industrial processing of nanomaterial-based devices.

Antibody drop based handling with near-superhydrophobic mesh substrates overcomes condensation sticking.

Superhydrophobic substrates facilitate low adhesion for biological liquid handling but are hampered by wetting state changes due to condensation. Here, meshed near-superhydrophobic substrates, that are easier to produce than two-tiered architecture substrates, are shown to provide good immunity to wetting state changes while imbuing high positional resistance to roll-off by tilting when tested with 5 and 10 µL volume drops (18° and 13° respectively) of human IgG antibodies in aqueous solution at both room temperature and 4 °C. Pneumatic actuation was applied to elicit horizontal drop movement over the near-SH surface without any fragmentation, wherein higher velocities can be achieved when smaller drops and higher air pressures are used (0.385 m/s at 33 mbar with the 5 µL drop). A non-contact mode of translating a protein drop towards a highly viscous oil-based adjuvant dispensed from a steel tip allowed for both drops to be combined and aspirated back up into the tip such that subsequent repeated cycles of pendant drop formation and upward aspiration allows for effective mixing to achieve a stable emulsion. The findings here advance the development of devices that enable improved antigen-adjuvant preparation by reducing the amount of reagents required and product losses from surface adsorption.

Note: Biochemical samples centrifuged in-flight on drones.

The ability to conduct en-route centrifugation of samples improves quality and timeliness in the pre-analytical phase. This is demonstrated here on a quadcopter whereby the propellers were adapted to house and apply centrifugal forces to sample-containing capillary tubes instead of incorporating a centrifuge. Tests revealed the ability of the method to separate non-homogenized milk into a cream portion and a skim milk portion, and human whole blood into plasma, buffy coat, and red blood cell components.

Augmented reality experimentation on oxygen gas generation from hydrogen peroxide and bleach reaction.

The appreciation and understanding of gas generation through processes is vital in biochemical education. In this work, an augmented reality tool is reported to depict the redox reaction between hydrogen peroxide and sodium hypochlorite solutions, two ubiquitous oxidizing agents, to create oxygen, a combustible gas. As it operates out of smartphones or tablets, students are able to conduct the exercise collaboratively, respond in a manner similar to an actual physical experiment, and able to depict the oxygen volume changes in relation to the volume of hydrogen peroxide of different concentrations used. The tool offers to help students acquire bench skills by limiting handing risks and to mitigate possible student anxiety on handling chemical materials and implements in the laboratory. The feedback received from Year 11 and 12 high school student participants in an outreach exercise indicate the overall effectiveness of this tool. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):245-252, 2018.

Drone inflight mixing of biochemical samples.

Autonomous systems for sample transport to the laboratory for analysis can be improved in terms of timeliness, cost and error mitigation in the pre-analytical testing phase. Drones have been reported for outdoor sample transport but incorporating devices on them to attain homogenous mixing of reagents during flight to enhance sample processing timeliness is limited by payload issues. It is shown here that flipping maneuvers conducted with quadcopters are able to facilitate complete and gentle mixing. This capability incorporated during automated sample transport serves to address an important factor contributing to pre-analytical variability which ultimately impacts on test result reliability.

Variability in Microplate Surface Properties and Its Impact on ELISA.

BACKGROUND: Microplate-based immunoassays are widely used in clinical and research settings to measure a broad range of biomarkers present in complex matrices. Assay variability within and between microplates can give rise to false-negative and false-positive results leading to incorrect conclusions. To date, the contribution of microplates to this variability remains poorly characterized and described. This study provides new insights into variability in immunoassays attributable to surface characteristics of commercial microplates. METHODS: Well-to-well assay variation in γ-treated and nontreated 96-well opaque microplates suitable for chemiluminescence assays was determined by use of a validated sandwich ELISA. Microplate surface characteristics were assessed by sessile drop contact angle measurements, scanning electron microscopy, energy dispersive x-ray spectroscopy, and atomic force microscopy. RESULTS: All microplate types tested exhibited vendor-specific assay response profiles; and "rogue" plates with very high intraassay variation and deviant mean assay responses were found. Within-plate, location-dependent bias in assay responses and variability in well contact angle were also observed. We demonstrate substantial differences in well-surface properties with putative effects on protein-coating reproducibility and hence consistency in immunoassay responses. A surface "cleaning" effect on manufacturing residues was attributed to γ-irradiation, and treated microplates manifest increased polar functionalities, surface roughness, and assay responses. CONCLUSIONS: Our data suggest that tighter control of variability in surface roughness, wettability, chemistry, and level of residual contaminants during microplate preparation is warranted to improve consistency of ELISA assay read out.

Simultaneous Multidrop Creation with Superhydrophobic Wells for Field Environmental Sensing of Nanoparticles.

Facile creation of multiple drops at appropriate volumes on surfaces without the use of sophisticated instrumentation facilitates downstream evaporative preconcentration of liquid samples for analytical purposes. In this work, a superhydrophobic (SH) substrate comprising wells with a perforated mesh base was developed for simultaneous drop creation in a quick and convenient manner. In contrast to the method of pouring liquid directly over the SH wells, consistent liquid filling was readily achieved by a simple immersion approach. This method works well even for challenging situations where well diameters are smaller than 3.4 mm. Despite the poor liquid-retention properties of SH surfaces, inverting the wells did not result in liquid detachment under gravitational force, indicating strong pinning effects afforded by the well architecture. The perforated base of the well allowed the liquid to be completely removed from the well by compressed air. High-speed camera image processing was used to study the evolution of drop contact angle and displacement with time. It was found that the liquid body was able to undergo strong oscillations. Optical spectroscopy was used to confirm the ability of evaporative preconcentration of silver nanoparticles.

Millimeter-Sized Hole Damming.

Valves used to control liquid filling and draining processes from storage typically need to be actuated. Here, we show that similar flow enabling and restricting operations can be achieved through millimeter scale holes that function according to the amount of hydrostatic pressure applied without any other intervention. This phenomena is exhibited using receptacles where the base is made of either a hydrophilic or superhydrophobic substrate with hole sizes ranging from 1.0-2.0 mm. The construction is such that the drainage flow velocities are of the same order in both substrates and follow Torricelli's law trends. Nevertheless, the primary mechanisms responsible for resisting the onset of flow in each substrate are different; nonbreaching of the advancing contact angle threshold in the former, and stable maintenance of an elastic-like deformation of the liquid-gas interface that is connected to the surrounding plastron in the latter. These differences are demonstrated using an upward jet of water delivered to the orifice, where a discharging flow from the hydrophilic base occurred before the threshold hydrostatic pressure condition was attained, while liquid from the jet is subsumed into the liquid body of the receptacle with the superhydrophobic base without any leakage. These findings portend advantages in simplicity and robustness for a myriad of liquid-related processes.