Assessing TEG6S reliability between devices and across multiple time points: A prospective thromboelastography validation study.

The TEG6S is a novel haemostasis analyser utilising resonance technology. It offers potentially greater coagulation information and ease of use, however has not been independently validated in a clinical setting. We aimed to determine if the TEG6S is reliable between devices and across time points. We performed a prospective observational study with ethical approval. For interdevice reliability, we performed simultaneous analysis on two TEG6S devices on 25 adult ICU patients. For time point reliability, we performed repeated sampling across five different time points on 15 adult participants. Blood was collected with informed consent, or as standard care, before four-channel citrated kaolin analysis. We observed almost perfect interdevice reliability across all TEG parameters. The Lin's concordance correlation coefficients (95% CI, major axis regression slope, intercept) were R-time: 0.96 (0.92-0.99, 0.88, 0.57); K-time: 0.93 (0.87-0.98, 1.07, 0.00); Alpha Angle: 0.87 (0.78-0.96, 1.20, -14.10); Maximum Amplitude: 0.99 (0.98-0.99, 1.02, -1.38); Clot Lysis: 0.89 (0.82-0.97, 1.20, 0.07). Additionally, we observed moderate-to-high reliability across time points. Demonstrating almost perfect agreement across different devices and moderate-to-high reliability across multiple time points, suggests the TEG6S platform can be used with haemostatic accuracy and generalisability. This has potentially significant implications for clinical practice and multi-site research programs.

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.

Versatile wetting measurement of microplate wells.

A method to measure the contact angle, which is indicative of wetting, using small liquid volumes dispensed directly on microplate wells is described and demonstrated. Experiments with enhanced green protein samples of volumes 4.4-6 µl showed no measured variance in the contact angle. Experiments with phosphate buffer solution with varied concentrations of a non-ionic detergent (Tween 20) dissolved, however, revealed smaller contact angles with increased detergent concentration. It is experimentally shown that drops can be located up to 7° from the lowest position of the well without affecting the accuracy of contact angle measurements. Numerical simulations confirm the ability of the drops to manifest the correct contact angle despite the lack of axis-symmetry in their shape while residing on a circular surface. This method offers a convenient means to determine the wetting characteristics of different liquid samples in different microplates.

Liquid-body resonance while contacting a rotating superhydrophobic surface.

We advance a scheme in which a liquid body on a stationary tip in contact with a rotating superhydrophobic surface is able to maintain resonance primarily from stick-slip events. With tip-to-surface spacing in the range 2.73 ≤ h < 2.45 mm for a volume of 10 µL, the liquid body was found to exhibit resonance independent of the speed of the drum. The mechanics were found to be due to a surface-tension-controlled vibration mode based on the natural frequency values determined. With spacing in the range 2.45 ≤ h < 2.15 mm imposed for a volume of 10 µL, the contact length of the liquid body was found to vary with rotation of the SH drum. This was due to the stick-slip events being able to generate higher energy fluctuations causing the liquid-solid contact areas to vary since the almost oblate spheroid shape of the liquid body had intrinsically higher surface energies. This resulted in the natural frequency perturbations being frequency- and amplitude-modulated over a lower frequency carrier. These findings have positive implications for microfluidic sensing.

Controlled transport of captive bubbles on plastrons.

Captive bubbles that reside on superhydrophobic surfaces with plastrons move uncontrollably when tilted. A system based on creating moveable local apexes on flexible superhydrophobic foils is shown to allow controlled transport. Simulations done reveal that specific bubble transport speeds are needed to form concentration gradients suited for aerotaxis study and sensing.

Concentrating nanoparticles in environmental monitoring.

There are significant challenges in assessing the toxicity of nanoparticles in the environment in which effective methods for detection are crucial. An inexpensive method that uses superhydrophobic well with an evaporating droplet followed by a simple squeeze flow is described here and found to provide practical high nanoparticle collection from samples for detection. The process could be hastened by placing a radiant heater close to the droplet if temperature rises in the sample can be tolerated.

Plastron-Mediated Growth of Captive Bubbles on Superhydrophobic Surfaces.

Captive bubbles on a superhydrophobic (SH) surface have been shown to increase in volume via injection of air through the surrounding plastron. The experimental contact diameter against volume trends were found to follow that predicted by the Surface Evolver simulation generally but corresponded with the simulated data at contact angle (CA) = 158° when the volume was 20 µL but that at CA = 170° when the volume was increased to 180 µL. In this regime, there was a simultaneous outward movement of the contact line as well as a small reduction in the slope that the liquid-air interface makes with the horizontal as air was injected. At volumes higher than 180 µL, air injection caused the diameter to reduce progressively until detachment. The inward movement of the contact line in this regime allowed the bubble body to undergo shape deformations to stay attached onto the substrate with larger volumes (300 µL) than predicted (220 µL at CA = 170°) using simulation. In experiments to investigate the effect of translating the SH surface, movement of captive bubbles was possible with 280 µL volume but not with 80 µL volume. This pointed to the possibility of transporting gas-phase samples on SH surfaces using larger captive bubble volumes.

Drop transfer between superhydrophobic wells using air logic control.

Superhydrophobic surfaces aid biochemical analysis by limiting sample loss. A system based on wells here tolerated tilting up to 20° and allowed air logic transfer with evidence of mixing. Conditions for intact transfer on 15 to 60 µL drops using compressed air pressure operation were also mapped.

Scribed transparency microplates mounted on a modified standard microplate.

The immense cost effectiveness of using transparencies as analyte handling implements in microplate instrumentation offers the possibility of application even in resource-limited laboratories. In this work, a standard microplate was adapted to serve as the permanent base for disposable scribed transparencies. The approach is shown to ameliorate evaporation, which can affect assay accuracy when analytes need to be incubated for some time. It also offers assurance against fluorescence measurement errors due to the cross-talk of samples from adjacent wells.

Transparency microplates under impact.

Transparency microplates enable biochemical analysis in resource-limited laboratories. During the process of transfer, the analytes tittered into the wells may undergo spillage from one well to another due to lateral impact. Sidelong impact tests conducted found the absence of non-linear effects (e.g., viscoelastic behavior) but high energy loss. Finite element simulations conducted showed that the rectangular plate holding the transparencies could undergo z-axis deflections when a normal component of the force was present despite constraints being used. High speed camera sequences confirmed this and also showed the asymmetrical z-axis deflection to cause the contact line closer to impact to displace first when the advancing condition was exceeded. Capillary waves were found to travel toward the contact line at the opposite end, where if the advancing contact angle condition was exceeded, also resulted in spreading. The presence of surface scribing was found to limit contact line movement better. With water drops dispensed on scribed transparencies, immunity from momentum change of up to 9.07 kgm/s on impact was possible for volumes of 40 µL. In the case of glycerol drops immunity from momentum change of up to 9.07 kgm/s on impact extended to volumes of 90 µL. The improved immunity of glycerol was attributed to its heightened dampening characteristics and its higher attenuation of capillary waves. Overall, scribed transparency microplates were able to better withstand spillage from accidental impact. Accidental impact was also found not to cause any detrimental effects on the fluorescence properties of enhanced green fluorescent protein samples tested.

Squeezed flow preconcentration for probe tip biosensors.

The preconcentration of analytes improves sensing using probe tips. In this work, we report a method based on creating a squeeze flow between a cylinder and circular coverslip to preconcentrate material at the liquid-gas interface while allowing a probe tip to be readily inserted there. In verification tests using enhanced green fluorescent protein, this capacity is proven. We estimated a 9.7 times increase in probability for fluorophores to be picked up at the tip using inference from fluorescence intensity distributions found. The method is expeditious, simple, and inexpensive, and it does not require any electrical energy source to operate.

MRT letter: Micro- to nanoscale sample collection for high throughput microscopy.

In high throughput microscopy, it is often assumed that the objects under investigation are fixed spatially. In addition, it is also presumed that the objects are sufficiently populated, otherwise there will be need to search through vast tracks of field of views before any recording can be done. The ability to collect objects at one location in the hydrated state is thus desirable and this is a challenge when the density of target objects in a sample is very low. In this work, we report that the generation of a squeezing flow from a circular coverslip compressing on suspensions is able to collect particulate (microbeads, fluorescent nanobeads and live algal cells) and non-particulate (EGFP) objects at the rim region of the coverslip. With a coverslip of 13 mm diameter, volumes between 2 µL and 4 µL were found to completely fill the coverslip without breaching the rims. Sample compression speeds between 100 µm/s and 1000 µm/s did not have any effect on object collection outcomes. In effect, the simple placement of coverslips on top the drop of sample by hand without a motorized translator was found to produce similar collection outcomes. Quantitative measurements confirmed that all the objects investigated were displaced and relocated at the rim regions to a very high degree.

Microplates based on liquid bridges between glass rods.

Microplating that (i) does not necessitate complex or precise machinery to dispense small liquid volumes, (ii) enables fluorescent optical diagnosis, and (iii) permits simple analyte mixing mechanically is desirable. We advance here a novel approach that employs the formation of a liquid bridge held in place by capillary forces between glass rod tubes located parallel to each other. Experimental investigations made on liquid filling characteristics show conformance to theoretical notions. Analytical development showed the presence of regions of minimal uncertainty in the cross-sectional area of the liquid body arising from variations in the contact angle which permit consistent fluorescence measurements. Cyclical translation of the rods relative to each other, which cause rupture and reattachment of the liquid bridge, was found to engender good mixing. Strong linear trends were found in fluorescence signals relative to EGFP fluorophore concentration using standard and optical fiber (which offer targeted) excitation illumination. The open nature of liquid handling in the approach reported here and the positive results obtained portend the ability for development as integrated lab-on-a-chip devices.

Surface-scribed transparency-based microplates.

Transparency sheets, which are normally associated with use on overhead projectors, offer lowered costs and high amenability for optical diagnostics in microplate instrumentation. An alternative microplate design in which circles are scribed on the surface of the transparency to create the boundaries to hold the drop in place is investigated here. The 3D profile of the scribed regions obtained optically showed strong likelihood of affecting three-phase contact line interactions. During dispensation, the contact angle (≈95°) was larger than the drop advancing state (≈80°) due to a period of nonadhesion, where the contact angle later reduced to the drop advancing state followed by increase in the liquid area coverage on the substrate. It was established that 50 µL was needed to fill the well fully, and the maximum volume retainable before breaching was 190 µL. While the tilt angle needed for displacement reduced significantly from 50 to 95 µL, this was markedly better than nonscribed surfaces, where tilt angles always had to be kept to within 30°. It was found that there was greater ability to fill the well with smaller volumes with dispensation at the center. This was attributed to the growing contact line not meeting the scribed edge in parallel if liquid was dispensed closer to it, wherein pinning reduction in some directions permitted liquid travel along the scribed edge to undergo contact angle hysteresis. Fluorescence measurements conducted showed no performance compromise when using scribed transparency microplates over standard microplates.

Microplate well coverage mixing using superhydrophobic contact.

Two important challenges in microplate instrumentation are to achieve full well sample coverage and complete mixing. An effective approach of using superhydrophobic rods to accomplish these challenges is reported here. Experiments conducted showed that analytes above 50µl could be made to completely cover the bottom of 96-well standard and transparency microplates. Complete mixing was accomplished by moving the rod parallel to the well bottom while contacting the liquid. The approach is simple and controlled, and it minimizes the problems of spillage and cross-contamination. It works with analytes with varied volumes and of different viscosities present in each well of the microplate.

Transparency-based microplates for fluorescence quantification.

Microplates for use in resource-limited laboratories should ideally not require processes that involve substantial large-scale production in order to be viable. We describe and demonstrate here an approach of using a silicone sheet with holes, conveniently cut out precisely using an inexpensive cutting plotter to correspond with regions where liquid is to be dispensed, and attaching it to a transparency to create very thin well arrays. With this, the contact angle hysteresis behavior of liquid could be harnessed to produce taller drop shapes so that the fiber probe used could read in the emitted light more effectively. Experimentation conducted revealed fluorescence measurements that were significantly more sensitive than standard microplates, notwithstanding that smaller volumes of liquid were needed. This was achieved using both the fiber optic and imaging evaluation modes. The two methods investigated, one with a lid placed and one without, showed the latter to produce marginally more sensitive readings as opposed to improved immunity from the environment with the former. These favorable measurement characteristics were found to be achievable with an estimated production cost of AU $0.40 and fabrication times of 3.5 min (96 wells) and 6.5 min (384 wells) per plate.

Surface tension drawing of liquid from microplate capillary wells.

Pressure differentials are routinely used to actuate flow in capillaries. We advance here an alternative means of flow generation that capitalizes on the extension of a liquid bridge achieved by the drawing of a rod through the action of surface tension. This meets the exigencies of creating controllable flow using simpler and more compact means. We found the ability to generate controllable flow to be strongly affected by the liquid bridge sustaining features, and that the use of rod diameters larger than the capillary was more conducive. The extensional flow resulting from the rupture of the liquid bridge was also found to have a strong circulation component which facilitated mixing. The approach here is highly amenable for use in capillary well microplates which have significant advantages over standard microplates. The features of this approach offer usage possibilities in biochemical applications in the field, such as in the leukocyte cell adhesion and hemagglutination tests of blood samples.

Using the meniscus in a capillary for small volume contact angle measurement in biochemical applications.

The contact angle is a sensitive parameter often used to define wettability. With the increasing movement toward smaller liquid volumes in many biochemical applications, a key challenge lies in how to perform measurements in the retainer holding the reagent for rapid evaluation and limited material loss. Here, we report a simple and robust method to determine the contact angle of small volumes using the microscopic imaging of a capillary meniscus that requires only the radius and meniscus height information. An error analysis of the measurement finds the method to be highly accurate. We also uncovered that illumination delivered from the liquid end of the capillary lights up the interface, thereby facilitating the measurement.

Absorbance and fluorometric sensing with capillary wells microplates.

Detection and readout from small volume assays in microplates are a challenge. The capillary wells microplate approach [Ng et al., Appl. Phys. Lett. 93, 174105 (2008)] offers strong advantages in small liquid volume management. An adapted design is described and shown here to be able to detect, in a nonimaging manner, fluorescence and absorbance assays minus the error often associated with meniscus forming at the air-liquid interface. The presence of bubbles in liquid samples residing in microplate wells can cause inaccuracies. Pipetting errors, if not adequately managed, can result in misleading data and wrong interpretations of assay results; particularly in the context of high throughput screening. We show that the adapted design is also able to detect for bubbles and pipetting errors during actual assay runs to ensure accuracy in screening.