μL Droplet Research Articles

A simple and low-cost paper-based device for simultaneous determination of hematocrit and hemoglobin levels in point-of-care settings

Hematocrit and hemoglobin levels are common blood tests used to assess the overall health status of patients as well as the oxygen-carrying capacity of the blood. The gold-standard technologies to determine the hematocrit and hemoglobin levels are precise and fast, but they are not pragmatic in extreme point-of-care settings due to the prohibitive cost, process complexity, and sophisticated facilities. Therefore, here, we report the development of a microfluidic paper-based analytical device to determine hematocrit and hemoglobin levels simultaneously. This device exploits the area of the stain formed by a spreading drop of 20 μl of whole blood on Whatman filter paper immobilized with sodium chloride and ethylenediaminetetraacetic acid for hematocrit level, whereas for hemoglobin concentration, it utilizes the gray color intensity of a 20 μl droplet of a mixture of blood and de-ionized water. The performance of the device is verified by comparing with gold-standard results of the automated hematology analyzer, showing a high degree of correlation (R2) of 0.9651 and 0.9701 for hematocrit and hemoglobin levels, respectively. The bias and standard deviation of the differences between the two measurements for hematocrit determination are 0.002 and 1.005, respectively, while the bias and standard deviation of differences between the two methods for hemoglobin determination are 0.065 and 0.398, respectively. This device is likely to provide a simple, fast, disposable, and inexpensive tool to determine the hematocrit and hemoglobin levels in resource-constraint settings.

Magneto-responsive wettability-switchable surfaces for in-situ large distance droplets capturing-releasing-bouncing

In this paper, superhydrophobic magnetically responsive micro-plate arrays (SMMAs) with reverse switching wettability were studied for in-situ capturing, releasing, and directional bouncing of droplets over a large distance. A novel method for producing superhydrophobic pattern on one side surface of the micro-plates by laser texturing at 90 degree bending state, created water contact angle of 151°, a rolling angle of < 10°, and a maximum surface adhesion force of 35.5 μN. Reversible switching between droplets (volume 5–40 μL) capturing with high adhesion force (high surface energy) and droplet releasing with low adhesion force were achieved spontaneously. The instantaneous response of the SMMAs to magnetic field provided a guarantee for the directional bouncing of droplets in long distance. Droplets transportation up to 40 μL, directional bouncing of 5 μL droplets with a vertical bounce height of 4.5 mm and a horizontal bounce distance of 24.7 mm have been achieved. This work provides a novel approach for flexible droplets manipulation and multifunctional operation of droplets for non-contact, remote-driven and non-destructive reagent transfer applications.

Aggregation of Ag nanoparticle based on surface acoustic wave for surface-enhanced Raman spectroscopy detection of dopamine

BackgroundDopamine (DA), a vital neurotransmitter, plays a critical role in the human brain and relates to neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Numerous studies have explored detection of such biomarkers through surface-enhanced Raman spectroscopy (SERS). However, most of the studies focused on SERS detection face significant challenges with plasmonic nanostructure development. Such challenges often include time-consuming processes, complex fabrication, specialized chemical labeling, poor reproducibility, and random hotspot generation. Therefore, the need for simple and rapid nanostructure development is evident in SERS. ResultsWe propose an innovative SERS-active sensing technique for 50 nm silver nanoparticle (AgNP) clustering based on surface acoustic wave (SAW). When a 1 μL droplet of AgNP colloid is dispensed onto the SAW-propagation zone, the AgNP cluster is deposited after the droplet completely evaporates, developing plasmonic nanogaps for SERS hotspot caused by spherical AgNP aggregation. By optimizing the SAW system through the hydrophobic treatment and modulation of the operational power, the SAW-induced AgNP clustering showed densely packed AgNP within a dot-like configuration (∼2200 AgNP μm−2), effectively preventing particle welding. The characterization of 4-mercaptobenzoic acid as a probe analyte revealed that concentrations as low as 1.14 pM was detected using our SAW-SERS system under 785 nm laser excitation. Moreover, DA was detected up to 4.28 nM with a determination of 0.99 (R2). SignificanceThis technique for AgNP clustering induced by SAW provides a rapid, in situ, label-free SERS sensing method with outstanding sensitivity and linearity. A mere act of dropping can create extensive plasmonic hotspots featuring nanogap of ∼1.5 nm. The SAW-induced AgNP clustering can serve as an ultrasensitive SERS-active substrate for diverse molecular detections, including neurotransmitter detection.

MODL-26. A PROTOCOL TO ADAPT GLIOBLASTOMA IN VITRO CULTURE SYSTEMS INTO ORGANOID MODELS USING NEUROCULT™ NS-A PROLIFERATION MEDIUM

Abstract Glioblastoma (GBM) is a malignant and aggressive brain tumor that is difficult to treat due to its heterogeneity. Traditional in vitro models (i.e. monolayers and tumorspheres) do not recapitulate this heterogeneity, leading to difficulty assessing new therapeutic strategies. Culturing GBM cells as organoids may provide a better method to retain the phenotype of the parent tumor. Here, we present a protocol for generating GBM organoids using NeuroCult™ NS-A Proliferation Medium, a medium commonly used for tumorsphere cultures. Two methods were assessed for the initial stage of formation, each suitable depending on throughput and cell source. In the first method, tumorspheres were dissociated and single cells were mixed with Growth Factor Reduced Matrigel® at a 1:4 ratio to form 15 μL droplets with 1000 cells per droplet. In the second method, 9000 cells from dissociated tumorspheres were seeded per well in a 96-well U-bottom plate and cultured for 5 days to form aggregates. On day 6, the aggregates were embedded in 15 μL Matrigel® droplets. In both methods, polymerized droplets were then cultured in suspension in stationary 6-well plates for 3 days, then maintained on an orbital shaker with full-medium changes every 2 - 3 days. Organoids were cryosectioned and processed for immunofluorescence at 5 - 6 weeks. In vivo, GBM tumors display an architecture containing regions of high and low nutrient and oxygen availability around a necrotic core. GBM organoids generated using this protocol exhibited distinct areas of proliferation near the organoid rim (Ki67+) and hypoxia (CA9+) near the core, mimicking this in vivo architecture (n = 3 GBM cell lines). Organoids grew to 2 - 4 mm in diameter and expressed glioma markers including TUJ1, Nestin, SOX2, OLIG2, and GFAP. These data show that NeuroCult™ NS-A Proliferation Medium can be used to adapt traditional GBM culture systems into organoids.

Droplet freeze-thawing system based on solid surface vitrification and laser rewarming

Ultra-rapid cooling and rewarming rate is a critical technical approach to achieve ice-free cells during the freezing and melting process. A set of ultra-rapid solid surface freeze-thaw visualization system was developed based on a sapphire flim, and experiments on droplet freeze-thaw were carried out under different cryoprotectant components, volumes and laser energies. The results showed that the cooling rate of 1 μL mixed cryoprotectant [1.5 mol/L propylene glycol (PG) + 1.5 mol/L ethylene glycol (EG) + 0.5 mol/L trehalose (TRE)] could be 9.2×10 3 °C/min. The volume range of 1-8 μL droplets could be vitrified. After comparing the proportions of multiple cryoprotectants, the combination of equal proportion mixed permeability protectant and trehalose had the best vitrification freezing effect and more uniform crystallization characteristics. During the rewarming operation, the heating curve of glassy droplets containing gold nanoparticles was measured for the first time under the action of 400-1 200 W laser power, and the rewarming rate was up to the order of 10 6 °C/min. According to the droplet images of different power rewarming processes, the laser power range for ice-free rewarming with micron-level resolution was clarified to be 1 400-1 600 W. The work of this paper simultaneously realizes the ultra-high-speed temperature ramp-up, transient visual observation and temperature measurement of droplets, providing technical means for judging the ice free droplets during the freeze-thaw process. It is conducive to promoting the development of ultra-rapid freeze-thaw technology for biological cells and tissues.

Directional droplet transport behavior on gradient wettability wedge track with extreme wettability contrast

To enhance transport capacity of open-surfaces, a gradient wettability wedge track laid on superhydrophobic background (wedge track No. 2) was fabricated by using masking tape and alkaline-assisted oxidation. The effects of gradient wettability and extreme wettability contrast are emphasized and investigated. The maximum instant velocity of a 10 μL droplet on track No. 2 is ∼ 883.53 mm/s with final transport displacement x = 46.79 mm, which was 64.15 % faster and 11.67 % farther than that on a superhydrophilic wedge track (wedge track No. 1) respectively. Oscillation happens at the wide end of wedge tracks when kinetic energy remains. Oscillation time on wedge track No. 2 is longer, which means gradient wettability can provide an extra driving force. This novel wedge track highlights the necessity of both gradient wettability and extreme wettability contrast and will have broad prospects in directional transport on open-surfaces, biological research, energy conversion and microfluidic mixers.

Parametric Study on the Evaporation Characteristics of Biodiesel-Diesel-Methanol Microemulsion Droplets

ABSTRACT In order to investigate the evaporation characteristics of biodiesel-diesel-methanol microemulsions, an experimental method of droplet evaporation with post processing techniques is established. The droplet normalized square diameter (NSD), droplet temperature and images during evaporation process are used to analyze the evaporation characteristics at different ambient temperatures. The results indicate that the methanol volume percentage, initial volume of droplet and ambient temperature have evident influence on the evaporation characteristics of droplets. When the initial volume of droplet is 0.5 μL, BD95M5 (biodiesel-diesel blend (95%)+methanol (5%)) and BD85M15 (biodiesel-diesel blend (85%)+methanol (15%)) droplets do not show the puffing and micro-explosion phenomena during evaporation process, but BD75M25 (biodiesel-diesel blend (75%)+methanol (25%)) droplet exhibits micro-explosion evidently at ambient temperature of 573K. Puffing is observed for BD85M15 droplet, and micro-explosion is for BD75M25 droplet at ambient temperature of 773K. The droplet lifetime is decreased and the mean evaporation rate (MER) is increased with the increase of ambient temperature and methanol volume percentage in microemulsion. The evaporation process ends at 5.938 s/mm2, 5.417 s/mm2 and 4.792 s/mm2 for BD95M5, BD85M15 and BD75M25, respectively, and MERs of them are 0.087 mm3/s, 0.095 mm3/s and 0.108 mm3/s at ambient temperature of 573K. The MERs of BD95M5, BD85M15 and BD75M25 are 0.225 mm3/s, 0.244 mm3/s and 0.309 mm3/s, respectively, at ambient temperature of 773K. With the increase of initial volume of droplet, the occurrence of puffing and micro-explosion is strengthened. The MER of droplets with initial volume of 0.6 μL is the largest, which increases by 13.1% and 12.8% compared with 0.4 μL droplets and 0.5 μL droplets, respectively.

Dust mitigation by a water droplet in between movable and modified wetting states surfaces

A novel approach for mitigating environmental dust from hydrophobic surfaces using a water droplet is presented. A sessile droplet is sandwiched between two parallel plates, one of which is moveable and hydrophilic while the other is stationary and hydrophobic. Investigations are conducted into how plate spacing affects the dust mitigation rate and the droplet's level motion. The high-speed camera analyzes the droplet motion for various plate spacing, dusty regions, and droplet sizes. In a controlled laboratory setting, the movement of fluid and dust particles inside a droplet is simulated. The results showed that when a droplet is still, it effectively reduces dust. The droplet meniscus expands by decreasing the gap between the droplet and the surface, increasing the dust removal rate. While the Magdeburg force and surface tension influence the droplet's adhesion to a hydrophobic surface, surface tension remains the primary factor affecting droplet pinning on a hydrophilic plate, more so than pinning on a dusty hydrophobic surface. When compressing, a current is created in the droplet fluid, greatly accelerating the rate at which dust is removed from the hydrophobic surface. We also move a dangling droplet over a dirty surface to evaluate its cleaning effectiveness and find that a 60 µL droplet has a 97% cleaning effectiveness and can remove dust from up to 450 mm2 of surface area. Our study provides insight into the unique method of removing dust from active surfaces and sheds light on droplet pinning forces generated by the Magdeburg effect in nano-cavities during vertical and horizontal movement.

Detection of influenza virus and Streptococcus pneumoniae in air sampled from co-infected ferrets and analysis of their influence on pathogen stability.

Secondary infection with Streptococcus pneumoniae has contributed significantly to morbidity and mortality during multiple influenza virus pandemics and remains a common threat today. During a concurrent infection, both pathogens can influence the transmission of each other, but the mechanisms behind this are unclear. In this study, condensation air sampling and cyclone bioaerosol sampling were performed using ferrets first infected with the 2009 H1N1 pandemic influenza virus (H1N1pdm09) and secondarily infected with S. pneumoniae strain D39 (Spn). We detected viable pathogens and microbial nucleic acid in expelled aerosols from co-infected ferrets, suggesting that these microbes could be present in the same respiratory expulsions. To assess whether microbial communities impact pathogen stability within an expelled droplet, we performed experiments measuring viral and bacterial persistence in 1 µL droplets. We observed that H1N1pdm09 stability was unchanged in the presence of Spn. Further, Spn stability was moderately increased in the presence of H1N1pdm09, although the degree of stabilization differed between airway surface liquid collected from individual patient cultures. These findings are the first to collect both pathogens from the air and in doing so, they provide insight into the interplay between these pathogens and their hosts.IMPORTANCEThe impact of microbial communities on transmission fitness and environmental persistence is under-studied. Environmental stability of microbes is crucial to identifying transmission risks and mitigation strategies, such as removal of contaminated aerosols and decontamination of surfaces. Co-infection with S. pneumoniae is very common during influenza virus infection, but little work has been done to understand whether S. pneumoniae alters stability of influenza virus, or vice versa, in a relevant system. Here, we demonstrate that influenza virus and S. pneumoniae are expelled by co-infected hosts. Our stability assays did not reveal any impact of S. pneumoniae on influenza virus stability, but did show a trend towards increased stability of S. pneumoniae in the presence of influenza viruses. Future work characterizing environmental persistence of viruses and bacteria should include microbially complex solutions to better mimic physiologically relevant conditions.

First Report of Xanthomonas hydrangeae Causing Leaf Spot on Oakleaf Hydrangea (Hydrangea quercifolia) in Tuscany (Italy).

Hydrangeas (Hydrangea L.) are popular ornamental plants used in urban landscapes and gardens worldwide for the beauty of their large flowers. In June 2022, dark brown/purple and irregular water-soaked spots coalescing into large areas of necrosis were observed on the leaves of potted Hydrangea quercifolia Bartr. plants growing in two ornamentals nurseries in Pistoia, Tuscany, Italy. Isolations, using two symptomatic plants/nursery, were performed by excising small portions of leaf tissue from the margin of the lesions, and macerating them in 100 μl of sterile distilled water (SDW). After 15 min of incubation, a loopful of the resulting suspension was streaked on yeast extract-dextrose-CaCO3 agar (YDCA) amended with 60 mg L-1 cycloheximide. Mucoid, convex and yellow colonies appeared on YDCA after incubation at 28°C for 48h. After colony purification on yeast extract-nutrient-agar (YNA), two isolates from each nursery were subject to amplification and sequence analysis of the 16S rRNA using universal primers FD1/RD1, for genus identification (Vauterin et al. 2000; Weisburg et al. 1991). All 16S rRNA sequences (OP441051) were identical and BLASTn searches indicated that the isolates belong to the genus Xanthomonas [99.9% nucleotide identity with X. hydrangeae strain LMG 31885 (LR990741.1) and 99.8% with strain LMG 31884T (NR_181958.1)]. For classification at species level, fragments of the housekeeping genes gyrB, rpoD, dnaK, and fyuA, were amplified according to Young et al. (2008). Both strands were sequenced and the consensus sequences aligned using MUSCLE as implemented in MEGA X (Kumar et al. 2018). Homologous sequences were once again identical between the isolates. A neighbor joining phylogenetic analysis of the concatenated fragments, was carried out, using the Tuscan isolate HyQ-Tu1, the type/pathotype strains of the seven pathovars of X.hortorum, proposed by Morinière et al. (2020), the four X.hydrangeae strains characterized by Dia et al. (2021) and the type strain of X.populi as the outgroup. The analysis indicated that HyQ-Tu1 isolate clusters within the X. hydrangeae branch of the recently described X. hortorum - X. hydrangeae species complex (Morinière et al. 2020; Dia et al. 2021; 2022). In agreement with this result, isolates tested positive to the LAMP assay specific for members of the complex's clade C (X. hydrangeae) (Dia et al. 2022). Based on molecular evidence, the isolates were identified as X. hydrangeae (Dia et al. 2021; Oren and Garrity, 2022). Three healthy H. quercifolia potted plants were inoculated by rubbing a 10 µl droplet of a bacterial suspension of X. hydrangeae HyQ-Tu1 adjusted to an OD600 of 0.3 (approx. 108 CFU/ml) in SDW on the adaxial surface of two leaves per plant. Two control leaves/plant were inoculated with SDW. Each inoculated leaf was enclosed for 24h in a polyethylene bag and all plants were maintained in a greenhouse at 28°C. Nine days post inoculation (DPI), leaf spots similar to those observed on naturally infected plants started to become evident on the bacteria-inoculated leaves while control leaves remained asymptomatic throughout the trial (21 DPI). Koch's postulates were fulfilled by re-isolating the bacterium from the symptomatic tissues, obtaining a positive amplification with the clade C-specific LAMP assay (Dia et al. 2022), and confirming that the gyrB sequence was 100% identical to that of X. hydrangeae HyQ-Tu1. Housekeeping gene sequences were submitted to GenBank (OP456006-9). Members of the X. hortorum - X. hydrangeae species complex have been reported to affect H. quercifolia in the USA (Uddin et al. 1996) and H. quercifolia and H. arborescens L. in Belgium (Cottyn et al. 2021). To the best of our knowledge, this is the first documentation of X. hydrangeae causing disease on H. quercifolia in Italy. Further work is required to verify the presence of the bacterium in other European countries and to assess the economic impact that it causes within and outside nurseries.

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants.

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied. Nanomaterials fabricated on rigid substrate (backing) are particularly difficult to successfully apply to curved plant structures. This study describes the process for microfabricating vertically aligned carbon nanofiber arrays and transferring them from a rigid to a flexible substrate. We detail and demonstrate how these fibers (on either rigid or flexible substrates) can be used for transient transformation or dye (e.g., fluorescein) delivery to plants. We show how VACNFs can be transferred from rigid silicon substrate to a flexible SU-8 epoxy substrate to form flexible VACNF arrays. To overcome the hydrophobic nature of SU-8, fibers in the flexible film were coated with a thin silicon oxide layer (2-3 nm). To use these fibers for delivery to curved plant organs, we deposit a 1 µL droplet of dye or DNA solution on the fiber side of VACNF films, wait 10 min, place the films on the plant organ and employ a swab with a rolling motion to drive fibers into plant cells. With this method, we have achieved dye and DNA delivery in plant organs with curved surfaces.

P-722 Impact of blastocyst quality and post-thaw embryo culture factors in non-invasive PGT-A informativity on vitrified blastocysts.

Abstract Study question What are the most important factor/s involved in the informativity of non-invasive PGT-A (niPGT-A) of vitrified blastocysts? Summary answer The day of vitrification and post-thaw time in culture of vitrified blastocysts are the most important factors for niPGT-A informativity, without correlation with blastocyst quality. What is known already niPGT-A is a technique that allows the non-invasive study of the chromosomal content of blastocysts. The informativity and concordance rates for fresh (Rubio, 2020) and frozen-thawed embryos (Garcia-Pascual, 2022) have been related with the time in culture and the day of media collection. However, there is a need to understand the optimal conditions needed to obtain optimal informativity for frozen-thawed vitrified blastocyst undergoing niPGT-A. Study design, size, duration Prospective study including 135 spent blastocyst media (SBM) and the corresponding blastocyst samples from thawed day-5 and day-6 blastocysts from PGT-A (73) and niPGT-A (62) cycles collected from January 2021 until March 2022. Blastocysts were discarded under IRB approval. The embryos were divided into 3 groups depending on the day of development and the time in culture after thawing: group-1, day-5 blastocysts cultured 8h; group-2, day-5 blastocysts cultured 24h and group-3, day-6 blastocysts cultured 8h. Participants/materials, setting, methods Media and blastocysts were obtained from IVF patients (29-42 years-old). After thawing, each blastocyst was washed to remove remaining cumulus cells and individually transferred to a 10 μL droplet of fresh media or to an EmbryoScope dish with 20 μL per well. SBM and blastocysts were collected, frozen and analysed by Next Generation Sequencing. Correlation between concordance rates (overall agreement between SBM and blastocyst samples), informativity, embryo quality and time in culture were evaluated. Main results and the role of chance All the embryos had a good embryo quality (BB or greater using Gardner Criteria) but different expansion degree. Informativity was significantly lower when day-5 embryos were cultured for only 8 hours (group 1), regardless of the degree of expansion (expansion degree 3, 28.57%; degree 4, 64.29% and degree 5-6, 57.14%, p &amp;lt; 0.0005). The concordance rates between SBM and whole blastocysts were very high and no significant different in relation to the expansion degree when comparing day-5 embryos cultured for 8 hours (group 1, 90.5%) with 24 hours (group 2, 93.6%) or with day-6 embryos cultured for 8 hours (group 3, 92.3%). The data suggest that the important factor impacting the informativity rate is the day of vitrification, and the time in culture after thawing, but not the expansion degree of the embryos. Regarding the concordance rates, there is no impact of the time in culture nor the expansion degree. Limitations, reasons for caution The main limitation of this study is that all the embryos analysed had fair or good quality but different expansion degrees. Wider implications of the findings The high concordance rates observed in frozen blastocysts opens the possibility of expanding the use of niPGT-A to frozen blastocyst without an impact of the expansion degree in the results. Trial registration number Not applicable

P-243 A commonly used temperature probe could result in setting heated surfaces within the IVF lab too high

Abstract Study question Which temperature probe (thermocouple Type T or Pt100) is the most accurate and robust for measuring medium temperatures in dishes on a heated surface? Summary answer A Pt100 probe attached to the bottom of the dish provides a more accurate and repeatable measurement than the commonly used thermocouple Type T probe. What is known already The success of an IVF cycle is dependent on several variables. Ensuring the gametes and embryos are exposed to appropriate temperatures is a critical variable during incubation and manipulation. There are different temperature measurement probes available for this purpose. Their specific properties could make them more, or less suitable for the various temperature measurements required in the laboratory. Study design, size, duration The aim of the study was to determine the best method (thermocouple Type T probe placed in the medium or a Pt100 sensor attached to the bottom of a dish) to measure temperature in medium held in two types of dish placed on a heated surface. Measurements were made in triplicate and standard deviation determined. Participants/materials, setting, methods The temperature of the medium in two types of dish (5 well and 40mm direct surface contact dishes, Vitrolife) were measured with two types of probes. The commonly used thermocouple Type T, which is composed of two wires, a copper and copper nickel alloy, that are welded together, and a resistance sensor composed of a small platinum plate attached to the bottom of the dish (Pt100). Main results and the role of chance With a 100 μl droplet under 5.0 ml of oil in a 40 mm dish (with the lid on) the tip of the Type T probe measured a temperature 3.2 ± 0.26 °C lower than the Pt100 probe. The Type T probe also measured a lower temperature in the 5 well dish. With a well containing 500 μl (lid on) it measured a temperature 2.1 ± 0.15 °C lower than the Pt100 probe. When the entire length of the Type T probe was placed inside of an incubator set at 37.0 °C the measured temperature was 0.1 °C lower than the Pt100 probe. This suggests the length of the Type T probe within the environment to be measured is critical, i.e., the lower temperatures obtained with the Type T probe when measuring medium temperature on heated surfaces depends on the method used rather than the probe itself. The reason for the lower temperature is correlated to the high temperature conductivity of the copper in the Type T probe. This also makes the measurement less repeatable as different factors (e.g., length of probe submerged, ambient temperature, air flow, etc.) will affect the reading. The Pt100 is not influenced by such factors. Limitations, reasons for caution The dishes used in this investigation create a uniform direct contact with the heated surface. This means that the heated surface will have a temperature close to that measured by the Pt100. This will not be the case with dishes with an air gap design. Wider implications of the findings This investigation highlights the importance of using the correct type of thermometer probe for the purpose. It is likely that when using a thermocouple Type T probe, temperature in the medium can be 2-3 °C higher than measured, which could impact gametes/embryos and lead to deleterious results. Trial registration number Not applicable

Comparison of a standard environmental surface sampling method and a composite approach for select healthcare pathogens

Background: Hospital surfaces are known to contribute to the spread of healthcare-associated infections (HAIs). Environmental sampling is often performed to locate a reservoir or to evaluate intervention strategies in healthcare facilities. Composite sampling is commonly practiced in other fields of environmental sampling and involves collection of multiple samples combined entirely or partially to form a new sample. We compared a standard CDC surface whole-tool sampling method with a composite sampling approach. Methods:Acinetobacter baumannii (AB), Klebsiella pneumoniae that produce K. pneumoniae carbapenemase (KPC), vancomycin-resistant Enterococcus faecalis (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and Clostridioides difficile spores were suspended in an artificial soil and deposited as 40 µL droplets (~104 CFU total) onto steel coupons of surface areas 323 cm2, 645 cm2, or 1,290 cm2 and dried for 2 hours. The surfaces were sampled with a single pass of a cellulose sponge— either the larger side of the sponge (face) or the smaller side of the sponge (edge)—and the optimal surface area was determined. Recovery from the optimal surface area with a single pass sampling was compared to the recovery using a standard CDC method in which all sides were used (ie, whole-tool method) to sample a standard area (645 cm2). Recovery was determined by culture and total CFU were determined for each optimal surface area. Theoretical composites were constructed using the mean total CFU of optimal surface area; 2×((face) + (edge)). Significance was set at P ≤ .05. Results: Total CFU recovery using the whole-tool method was significantly greater than the single pass sample recovery for MRSA (18,300 vs 16,600 CFU) and VRE (27,600 vs 26,400 CFU) (P &lt; .05). When comparing the theoretical composite method to the standard whole-tool area (625 cm2), the theoretical composite total CFU was significantly greater than the whole-tool method for all organisms. For example, VRE recovery with the standard CDC whole-tool method was 27,600 CFU from 625 cm2, yet a theoretical composite approach recovered 79,800 CFU from an area of 1,290 cm2. Conclusions: Many factors influence recovery when sampling the environment, and composite sampling is a promising approach when sampling large surface areas. Using a theoretical composite of single-pass samples, the potential for improved detection with composite sampling was demonstrated. A composite sampling approach will reduce time and resources for sampling and sample processing, allowing larger surface areas to be investigated which will improve infection control strategies.Disclosures: None

Superspreading Surfactant on Hydrophobic Porous Substrates

The wetting behavior of droplets of aqueous surfactant solutions over hydrophobic thin PVDF porous membrane and non-porous hydrophobic PVDF film is investigated for small (~10 μL) droplets of aqueous trisiloxane surfactant solutions: superspreader S 240. The time dependencies of contact angle, droplet radius, wetted area and volume were monitored as well as penetration into the porous substrate. It is shown that the fast spreading of droplets of trisiloxane solutions takes place both in the case of porous and non-porous substrates at a concentration above some critical concentration. It was found that the trisiloxane droplets penetrate into the hydrophobic porous substrates and disappear much faster than on a corresponding hydrophobic non-porous substrate, which was not observed before. This phenomenon is referred to as “superpenetration”.

Spontaneous Transport Mechanics of Water Droplets under a Synergistic Action of Designed Pattern and Non-Wetting Gradient.

The controllable spontaneous transport of water droplets on solid surfaces has a broad application background in daily life. Herein, a patterned surface with two different non-wetting characteristics was developed to control the droplet transport behavior. Consequently, the patterned surface exhibited great water-repellant properties in the superhydrophobic region, and the water contact angle reached 160° ± 0.2°. Meanwhile, the water contact angle on the wedge-shaped hydrophilic region dropped to 22° after UV irradiation treatment. On this basis, the maximum transport distance of water droplets could be observed on the sample surface with a small wedge angle of 5° (10.62 mm), and the maximum average transport velocity of droplets was obtained on the sample surface with a large wedge angle of 10° (218.01 mm/s). In terms of spontaneous droplet transport on an inclined surface (4°), both the 8 μL droplet and 50 μL droplet could move upward against gravity, which showed that the sample surface possessed an obvious driving force for droplet transport. Surface non-wetting gradient and the wedge-shaped pattern provided unbalanced surface tension to produce the driving forces in the process of droplet transport, and the Laplace pressure as well is produced inside the water droplet during this process. This work provides a new strategy to develop a patterned superhydrophobic surface for droplet transport.

Ultra-High-Throughput and Low-Volume Analysis of Intact Proteins with LAP-MALDI MS.

High-throughput (HTP) mass spectrometry (MS) is a rapidly growing field, with many techniques evolving to accommodate ever increasing sample analysis rates. Many of these techniques, such as AEMS and IR-MALDESI MS, require volumes of at least 20-50 μL for analysis. Here, liquid atmospheric pressure-matrix-assisted laser desorption/ionization (LAP-MALDI) MS is presented as an alternative for ultra-high-throughput analysis of proteins requiring only femtomole quantities of protein in 0.5 μL droplets. By moving a 384-well microtiter sample plate with a high-speed XY-stage actuator, sample acquisition rates of up to 10 samples per second have been achieved at a data acquisition rate of 200 spectra per scan. It is shown that protein mixture solutions with concentrations of ≤2 μM can be analyzed at this speed, while individual protein solutions can be analyzed at concentrations of ≤0.2 μM. Thus, LAP-MALDI MS provides a promising platform for multiplexed HTP protein analysis.

Boosted energy harvesting in droplet electrochemical cell with non-equilibrium electrical double layer

Droplet energy harvesting technologies, such as droplet triboelectric nanogenerators (D-TENGs) and droplet-based electricity generators (DEGs), which are not restricted by geographical location, have been vigorously developed in recent years. However, achieving electricity generation with high performance is still challenging. Here, we demonstrate a new approach for droplet-based energy harvesting device named droplet electrochemical cell (DECC) which could enhance energy generation performance by utilizing the formation of non-equilibrium electrical double layers. The DECC is designed by drawing inspiration from the zinc-copper primary battery system and demonstrated remarkable results when tested with dynamically impinging water droplets. The peak current and transferred charge generated by a 100 μL droplet are found to be 400 μA and 3.2 μC, respectively. The short circuit current is also found to increase by approximately 30 times when the dynamic impinging frequency of the droplets is increased. This study provides valuable insights into the development of energy harvesting devices based on droplets.

Asymmetric electrodes for droplet directional actuation by a square wave on an open surface

The large-scale electrowetting-on-dielectric (EWOD) digital microfluidic platforms always require complex electrode-addressing schemes and high-cost fabrication methods of dielectric and hydrophobic layers. Hence, the voltage polarity-dependent electrowetting was used to actuate droplets with asymmetric printed circuit board (PCB) electrodes on an open slippery liquid-infused porous surface (SLIPS) chip. The V-shaped, convex-shaped, and curve-shaped coplanar asymmetric electrode arrays were designed to achieve the directional motion of a droplet by a square wave voltage signal. The operation ranges of voltage amplitude and frequency for continuously actuating a 10 µl droplet were obtained on the three asymmetric electrode arrays. Our results found that the curve-shaped electrode array has a broader voltage operation range than the other two for continuous pumping of a 10 µl droplet. It is because the droplet has a long effective width of contact line with the front curve-shaped electrode and does not overlap with the rear electrode during the movement. Meanwhile, a reconfigurable convex-shaped electrode structure was also proposed to achieve bidirectional actuation of a droplet by controlling the square wave voltage. This work provides a low-cost, straightforward electrode-addressing scheme for designing digital microfluidic devices.

High Precision Dynamic Manipulations of Large‐Volume‐Range Water‐Based Droplets by a Soft Gripper with Robust Water‐Repellent Superhydrophobic Surface

AbstractDroplet manipulation has emerged as a key enabling technology in various scientific and engineering fields with great potentials in advanced applications such as bioanalysis and reagent microreactors. The current strategies generally involve external stimuli or chemical/structural surface gradients to provide the driving force for in plane droplet manipulations, which suffer from low efficiency, poor controllability, and small volume ranges (generally Vmax/Vmin &lt; 10) due to their inherent limitations. Herein, a dynamic gripping based droplet manipulation method is proposed by employing a flexible gripper with a notch covered by superhydrophobic silicon rubber membrane (SRM) between the gripper fingers to dynamically pick up, constrain and release droplets, capable of consecutively manipulating large‐volume‐range (Vmax/Vmin &gt;130) droplets with minimal liquid loss. The robust Cassie‐to‐Wenzel transition (CWT) resistance and mechanical stability of the produced superhydrophobic surface well support the dynamic operations of various droplets, where the superhydrophobic surface presents great water‐repellent performance during stretching and impinging experiments as well as remains superhydrophobicity even after mechanical abrasion against 600 grit SiC sandpaper for 15 m at an applied pressure of 3.2 kPa. Lossless manipulations of 3–180 µL droplets have been experimentally validated, where an application of the proposed method in droplet‐based microreactors for chemical analysis and bioassay is demonstrated.