Thomas F. Scherr

A Planar Microfluidic Mixer Based on Logarithmic Spirals

A passive, planar micromixer design based on logarithmic spirals is presented. The device was fabricated using polydimethylsiloxane soft photolithography techniques, and mixing performance was characterized via numerical simulation and fluorescent microscopy. Mixing efficiency initially declined as Reynolds number increased, and this trend continued until a Reynolds number of 15 where a minimum was reached at 53%. Mixing efficiency then began to increase reaching a maximum mixing efficiency of 86% at Re = 67. Three-dimensional simulations of fluid mixing in this design were compared to other planar geometries such as the Archimedes spiral and Meandering-S mixers. The implementation of logarithmic curvature offers several unique advantages that enhance mixing, namely a variable cross-sectional area and a logarithmically varying radius of curvature that creates 3-D Dean vortices. These flow phenomena were observed in simulations with multilayered fluid folding and validated with confocal microscopy. This design provides improved mixing performance over a broader range of Reynolds numbers than other reported planar mixers, all while avoiding external force fields, more complicated fabrication processes, and the introduction of flow obstructions or cavities that may unintentionally affect sensitive or particulate-containing samples. Due to the planar design requiring only single-step lithographic features, this compact geometry could be easily implemented into existing micro-total analysis systems requiring effective rapid mixing.

A numerical study on distributions during cryoprotectant loading caused by laminar flow in a microchannel

In this work, we conduct a computational study on the loading of cryoprotective agents into cells in preparation for cryopreservation. The advantages of microfluidics in cryopreserving cells include control of fluid flow parameters for reliable cryoprotectant loading and reproducible streamlined processing of samples. A 0.25 m long, three inlet T-junction microchannel serves as an idealized environment for this process. The flow field and concentration distribution are determined from a computational fluid dynamics study and cells are tracked as inert particles in a Lagrangian frame. These particles are not confined to streamlines but can migrate laterally due to the Segre-Sildeberg effect for particles in a shear flow. During this tracking, the local concentration field surrounding the cell is monitored. This data are used as input into the Kedem-Katchalsky equations to numerically study passive solute transport across the cell membrane. As a result of the laminar flow, each cell has a unique pathline in the flow field resulting in different residence times and a unique external concentration field along its path. However, in most previous studies, the effect of a spatially varying concentration field on the transport across the cell membrane is ignored. The dynamics of this process are investigated for a population of cells released from the inlet. Using dimensional analysis, we find a governing parameter α, which is the ratio of the time scale for membrane transport to the average residence time in the channel. For [Formula: see text], cryoprotectant loading is completed to within 5% of the target concentration for all of the cells. However, for [Formula: see text], we find the population of cells does not achieve complete loading and there is a distribution of intracellular cryoprotective agent concentration amongst the population. Further increasing α beyond a value of 2 leads to negligible cryoprotectant loading. These simulations on populations of cells may lead to improved microfluidic cryopreservation protocols where more consistent cryoprotective agent loading and freezing can be achieved, thus increasing cell survival.

Microfluidics and numerical simulation as methods for standardization of zebrafish sperm cell activation

Sperm cell activation plays a critical role in a range of biological and engineering processes, from fertilization to cryopreservation protocol evaluation. Across a range of species, ionic and osmotic effects have been discovered that lead to activation. Sperm cells of zebrafish (Danio rerio) initiate motility in a hypoosmotic environment. In this study, we employ a microfluidic mixer for the purpose of rapidly diluting the extracellular medium to initiate the onset of cell motility. The use of a microchannel offers a rapid and reproducible mixing profile throughout the device. This greatly reduces variability from trial to trial relative to the current methods of analysis. Coupling these experiments with numerical simulations, we were able to investigate the dynamics of intracellular osmolality as each cell moves along its path through the micromixer. Our results suggest that intracellular osmolality, and hence intracellular ion concentration, only slightly decreases, contrary to the common thought that larger changes in these parameters are required for activation. Utilizing this framework, microfluidics for controlled extracellular environments and associated numerical modeling, has practical applicability in standardizing high-throughput aquatic sperm activation, and more fundamentally, investigations of the intracellular environment leading to motility.

Mobile phone imaging and cloud-based analysis for standardized malaria detection and reporting

Rapid diagnostic tests (RDTs) have been widely deployed in low-resource settings. These tests are typically read by visual inspection, and accurate record keeping and data aggregation remains a substantial challenge. A successful malaria elimination campaign will require new strategies that maximize the sensitivity of RDTs, reduce user error, and integrate results reporting tools. In this report, an unmodified mobile phone was used to photograph RDTs, which were subsequently uploaded into a globally accessible database, REDCap, and then analyzed three ways: with an automated image processing program, visual inspection, and a commercial lateral flow reader. The mobile phone image processing detected 20.6 malaria parasites/microliter of blood, compared to the commercial lateral flow reader which detected 64.4 parasites/microliter. Experienced observers visually identified positive malaria cases at 12.5 parasites/microliter, but encountered reporting errors and false negatives. Visual interpretation by inexperienced users resulted in only an 80.2% true negative rate, with substantial disagreement in the lower parasitemia range. We have demonstrated that combining a globally accessible database, such as REDCap, with mobile phone based imaging of RDTs provides objective, secure, automated, data collection and result reporting. This simple combination of existing technologies would appear to be an attractive tool for malaria elimination campaigns.

A two-magnet strategy for improved mixing and capture from biofluids

Magnetic beads are a popular method for concentrating biomolecules from solution and have been more recently used in multistep pre-arrayed microfluidic cartridges. Typical processing strategies rely on a single magnet, resulting in a tight cluster of beads and requiring long incubation times to achieve high capture efficiencies, especially in highly viscous patient samples. This report describes a two-magnet strategy to improve the interaction of the bead surface with the surrounding fluid inside of a pre-arrayed, self-contained assay-in-a-tube. In the two-magnet system, target biomarker capture occurs at a rate three times faster than the single-magnet system. In clinically relevant biomatrices, we find a 2.5-fold improvement in biomarker capture at lower sample viscosities with the two-magnet system. In addition, we observe a 20% increase in the amount of protein captured at high viscosity for the two-magnet configuration relative to the single magnet approach. The two-magnet approach offers a means to achieve higher biomolecule extraction yields and shorter assay times in magnetic capture assays and in self-contained processor designs.

Ceragenin mediated selectivity of antimicrobial silver nanoparticles.

The understanding that common broad-spectrum antimicrobials disrupt natural microbial flora important in acquiring nutrients and preventing infection has resulted in a paradigm shift favoring more selective antimicrobials. This work explores silver nanoparticles conjugated with ceragenin, or cationic antimicrobials (CSA-SNPs), as a potential Gram-positive selective antimicrobial. Herein, CSA-SNPs are characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, and high-performance liquid chromatography-electrospray time-of-flight mass spectrometry (HPLC-ESI-TOF-MS). The antimicrobial properties are determined through minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) and time-kill studies. Spatial selectivity of the conjugate nanoparticle was evaluated using confocal imaging, MATLAB statistical analysis, and video monitored interactions between bacteria and CSA-SNPs via laser trapping techniques. Cytotoxicity was also determined by live/dead staining and flow cytometry. Average particle size, as determined through TEM analysis, and hydrodynamic diameter, as determined via DLS, are 63.5 ± 38.8 and 102.23 ± 2.3 nm, respectively. The zeta potential of the SNP before and after CSA attachment is -18.23 and -8.34 mV, respectively. MIC/MBC data suggest that CSA-SNPs are 8 times more effective against Staphylococcus aureus than SNPs alone. Furthermore, MATLAB analysis of confocal imaging found that 70% of CSA-SNPs are within 2 μm of S. aureus, whereas this percentage falls to below 40% with respect to Escherichia coli. These results are bolstered further by laser trapping experiments demonstrating selective adherence of CSA-SNPs conjugates with bacterial strains. Cytotoxicity studies of CSA-SNPs against 3T3 fibroblasts indicate 50% cell viability at 50 ppm.

A handheld orbital mixer for processing viscous samples in low resource settings

Clinical diagnostics play an important role in infectious disease monitoring. Often these diseases are endemic in settings that lack laboratory resources, which limits the types of tools that are available. Mixing is a common procedure in clinical diagnostic assays and is not immune to these challenges. In this report, we characterize the efficacy of a handheld therapeutic massager that has been repurposed into a mixer. Using metal affinity functionalized magnetic beads and a histidine-tagged fluorescent label, we find that this inexpensive orbital mixer is effective over a range of clinically relevant sample volumes and viscosities. Even with larger sample volumes (>200 μL) and more viscous sample matrices (3× ηH2O), the mixer binds more than 90% of the biomarkers in solution in less than 90 seconds. In a clinical assay for the detection of Plasmodium falciparum histidine-rich protein 2, we find there is no statistical difference in performance between our inexpensive and portable orbital mixer and a standard laboratory benchtop vortexer.

Application of mass transfer theory to biomarker capture by surface functionalized magnetic beads in microcentrifuge tubes

In many diagnostic assays, specific biomarker extraction and purification from a patient sample is performed in microcentrifuge tubes using surface-functionalized magnetic beads. Although assay binding times are known to be highly dependent on sample viscosity, sample volume, capture reagent, and fluid mixing, the theoretical mass transport framework that has been developed and validated in engineering has yet to be applied in this context. In this work, we adapt this existing framework for simultaneous mass transfer and surface reaction and apply it to the binding of biomarkers in clinical samples to surface-functionalized magnetic beads. We discuss the fundamental fluid dynamics of vortex mixing within microcentrifuge tubes as well as describe how particles and biomolecules interact with the fluid. The model is solved over a wide range of parameters, and we present scenarios when a simplified analytical expression would be most accurate. Next, we review of some relevant techniques for model parameter estimation. Finally, we apply the mass transfer theory to practical use-case scenarios of immediate use to clinicians and assay developers. Throughout, we highlight where further characterization is necessary to bridge the gap between theory and practical application.

In vitro evaluation of thermal frontally polymerized thiol‐ene composites as bone augments

Because of the large number of total knee replacement (TKR) surgeries conducted per year, and with projections of increased demand to almost a million primary TKR surgeries per year by 2030 in the United States alone, there is a need to discover more efficient working materials as alternatives to current bone cements. There is a need for surgeons and hospitals to become more efficient and better control over the operative environment. One area of inefficiency is the cement steps during TKR. Currently the surgeon has very little control over cement polymerization. This leads to an increase in time, waste, and procedural inefficiencies. There is a clear need to create an extended working time, moldable, osteoconductive, and osteoinductive bone augment as a substitution for the current clinically used bone cement where the surgeon has better control over the polymerization process. This study explored several compositions of pentaerythritol-co-trimethylolpropane tris-(3-mercaptopropionate) hydroxyapatite composite materials prepared via benzoyl peroxide-initiated thermal frontal polymerization. The 4:1 acrylate to thiol ratio containing augment material shows promise with a maximal propagation temperature of 160°C ± 10°C, with mechanical strength of 3.65 MPa, and 111% cytocompatibility, relative to the positive control. This frontally polymerized material may have application as an augment with controlled polymerization supporting cemented implants.

In vitro characterization of polyesters of aconitic acid, glycerol, and cinnamic acid for bone tissue engineering

In this study, a novel class of polyesters of glycerol, aconitic acid, and cinnamic acid were synthesized along with their hydroxyapatite (HA) composites, and studied for their potential application in bone defect repair. An osteogenic study was conducted with human adipose derived mesenchymal stem cells (hASCs) to determine the osteoinductive ability of aconitic acid–glycerol (AG) polyesters, AG:HA (80:20), aconitic acid–glycerol–cinnamic acid (AGC) polyesters, and AGC:HA (80:20) to serve as bone scaffolds. The results indicate that AGC scaffolds have the highest mechanical strength in comparison to AG, AG:HA (80:20), and AGC:HA (80:20) scaffolds due to its low porosity. It was determined by cytotoxicity and osteogenesis experiments that hASCs cultured for 21 days on AG:HA (80:20) scaffolds in stromal medium exhibited a greater number of live cells than control PCL:HA composites. Moreover, hASCs cultured on foamed AG:HA (80:20) scaffolds resulted in the highest levels of mineralization, increased alkaline phosphatase (ALP) expression, and the greatest osteocalcin (OCN) expression after 21 days. Overall, AG:HA (100:0 and 80:20) scaffolds had higher mechanical strength and cytocompatibility than the PCL:HA control. In vitro osteogenic study demonstrated that AG:HA (100:0 and 80:20) synthesized using sugarcane industry by-products hold potential as scaffolds for bone tissue engineering applications.