Assessment of pDNA isoforms using microfluidic electrophoresis.

The recent rise in nucleic acid-based vaccines and therapies has resulted in an increased demand for plasmid DNA (pDNA). As a result, there is added pressure to streamline the manufacturing of these vectors, particularly their design and construction, which is currently considered a bottleneck. A significant challenge in optimizing pDNA production is the lack of high-throughput and rapid analytical methods to support the numerous samples produced during the iterative plasmid construction step and for batch-to-batch purity monitoring. pDNA is generally present as one of three isoforms: supercoiled, linear, or open circular. Depending on the ultimate use, the desired isoform may be supercoiled in the initial stages for cell transfection or linear in the case of mRNA synthesis. Here, we present a high-throughput microfluidic electrophoresis method capable of detecting the three pDNA isoforms and determining the size and concentration of the predominant supercoiled and linear isoforms from 2 to 7 kb. The limit of detection of the method is 0.1 ng/µL for the supercoiled and linear isoforms and 0.5 ng/µL for the open circular isoform, with a maximum loading capacity of 10-15 ng/µL. The turnaround time is 1 min/sample, and the volume requirement is 10 µL, making the method suitable for process optimization and batch-to-batch analysis. The results presented in this study will enhance the understanding of electrophoretic transport in microscale systems dependent on molecular conformations and potentially aid technological advances in diverse areas relevant to microfluidic devices.

Enzymatic isolation and microfluidic electrophoresis analysis of residual dsRNA impurities in mRNA vaccines and therapeutics.

The versatility, rapid development, and ease of production scalability of mRNA therapeutics have placed them at the forefront of biopharmaceutical research. However, despite their vast potential to treat diseases, their novelty comes with unsolved analytical challenges. A key challenge in ensuring sample purity has been monitoring residual, immunostimulatory dsRNA impurities generated during the in vitro transcription of mRNA. Here, we present a method that combines an enzyme, S1 nuclease, to identify and isolate dsRNA from an mRNA sample with a microfluidic electrophoresis analytical platform to characterize the impurity. After the method was developed and optimized, it was tested with clinically relevant, pseudouridine-modified 700 and 1800 bp dsRNA and 818-4451 nt mRNA samples. While the treatment impacted the magnitude of the fluorescent signal used to analyze the samples due to the interference of the buffer with the labeling of the sample, this signal loss was mitigated by 8.8× via treatment optimization. In addition, despite the mRNA concentration being up to 400× greater than that of the dsRNA, under every condition, there was a complete disappearance of the main mRNA peak. While the mRNA peak was digested, the dsRNA fragments remained physically unaffected by the treatment, with no change to their migration time. Using these samples, we detected 0.25% dsRNA impurities in mRNA samples using 15 µL with an analytical runtime of 1 min per sample after digestion and were able to predict their size within 8% of the expected length. The short runtime, sample consumption, and high throughput compatibility make it suitable to support the purity assessment of mRNA during purification and downstream.

A microfluidic electrophoretic dual dynamic staining method for the identification and relative quantitation of dsRNA contaminants in mRNA vaccines.

mRNA vaccines (i.e., COVID-19 vaccine) offer various advantages over traditional vaccines in preventing and reducing disease and shortening the time between pathogen discovery and vaccine creation. Production of mRNA vaccines results in several nucleic acid and enzymatic by-products, most of which can be detected and removed; however, double-stranded RNA (dsRNA) contaminants pose a particular challenge. Current purification and detection platforms for dsRNA vary in effectiveness, with problems in scalability for mass mRNA vaccine production. Effectively detecting dsRNA is crucial in ensuring the safety and efficacy of the vaccines, as these strands can cause autoimmune reactions with length-symptom dependency and enhance mRNA degradation. We present a new microfluidics method to rapidly identify and quantify dsRNA fragments in mRNA samples. Our innovation exploits the differences in the dynamic staining behavior between mRNA and dsRNA molecules to detect dsRNA contaminants in a high throughput approach. The limit of detection of the system for dsRNA was estimated to be between 17.7-76.6 pg µL-1 with a maximum loading capacity of mRNA of 12.99 ng µL-1. Based on these estimated values, our method allows for the detection of dsRNA contaminants present in percentages as low as 0.14-0.59% compared to the total mRNA concentration. Here, we discuss the molecular mechanism of the dynamic staining behavior of dsRNA and mRNA for two different stains. We believe our method will accelerate the mRNA vaccine development from initial development to quality control workflows.

Electro-DBS: A Simple Method to Rapidly Extract Genomic DNA from Dried Blood Spots.

Dried blood spots (DBSs) have been used for more than 50 years and are used as a method of sample collection for pharmacological testing, genetic analyses, monitoring of viral infections, and more. Protocols for nucleic acid extraction from DBSs involve several steps that require specialized equipment and can be lengthy. Thus, we sought to explore ways to reduce the analytical burden of DBSs. We developed a DBS extraction method that uses the synergistic action of electrophoretic and diffusive transport mechanisms to extract genomic DNA (gDNA) through the DBS matrix. This method (which we termed "Electro-DBS") reduces the time of extraction from 40 min to 5 min and removes the need for heat, shaking, and DNA purification steps while maintaining gDNA quality and yield. We found that the electrophoretic transport speeds up gDNA elution, allowing for the diffusive transport of polymerase chain reaction (PCR) inhibitors to be minimized due to a reduced elution time. Overall, this work added mechanistic insight into the extraction of DNA from DBSs and developed a method that was ideal for point of care and automation.

Sequence to size-based separation using microfluidic electrophoresis for targeted cell-free DNA analysis.

The aim of this research is to present a new method to identify and separate target DNA of the same size, in base pairs (bp), into different sizes based on the targeted sequences. This sequence-specific analysis can then be used to evaluate the presence of multiple targeted analytes in a sample without the need for fluorescence detection. This work displays the feasibility of this method using multiple different 150 bp target sequences separated via microfluidic electrophoresis into 230 bp to 330 bp peaks. Using a combination of denaturation, hybridization, ligation, purification, and universal amplification, this represents a simple, robust method for targeted analysis of short DNA sequences. This work shows a limit of detection of 3 pg (∼1.825 x 107 copies) of input DNA using 20 PCR cycles and the ability for the method to be used for short DNA sequences extracted from a plasma sample, most importantly cell-free DNA. Overall, this method has the potential to be used for mutation detection and multiplexed analysis without the need for multiple fluorophores or significant optimization due to varying melting temperatures between PCR primers and can qualitatively evaluate the presence of specific target sequences in a variety of molecular diagnostic applications.

A microfluidic platform for high-purity cell free DNA extraction from plasma for non-invasive prenatal testing.

OBJECTIVES: Increase the yield and purity of cell-free DNA (cfDNA) extracted from plasma for non-invasive prenatal testing (NIPT) as inefficiencies in this extraction and purification can dramatically affect the sensitivity and specificity of the test. METHODS: This work integrates cfDNA extraction from plasma with a microfluidic chip platform by combining magnetic bead-based extraction and electroosmotic flow on the microfluidic chip. Various wash buffers and voltage conditions were simulated using COMSOL Multiphysics Modeling and tested experimentally. RESULTS: When performing the first wash step of this assay on the microfluidic chip with 300 V applied across the channel there was a six-fold increase in the A260 /A230 ratio showing a significant improvement (p value 0.0005) in the purity of the extracted sample all while maintaining a yield of 68.19%. These values are critical as a high yield results in more sample to analyze and an increase in A260 /A230 ratio corresponds to a decrease in salt contaminants such as guanidinium thiocyanate which can interfere with downstream processes during DNA library preparation and potentially hinder the NIPT screening results. CONCLUSIONS: This technique has the potential to improve NIPT outcomes and other clinically relevant workflows that use cfDNA as an analyte such as cancer detection.

Interaction of Cyanobacteria with Nanometer and Micron Sized Polystyrene Particles in Marine and Fresh Water.

Microplastics and nanoplastics are emerging pollutants, widespread both in marine and in freshwater environments. Cyanobacteria are also ubiquitous in water and play a vital role in natural ecosystems, using photosynthesis to produce oxygen. Using photography, fluorescence microscopy and cryogenic and scanning electron microscopy (cryo-SEM, SEM) we investigated the physicochemical response of one of the most predominant seawater cyanobacteria (Synechococcus elongatus, PCC 7002) and freshwater cyanobacteria (S. elongatus Nageli PCC 7942) when exposed to 10 µm diameter polystyrene (microPS) and 100 nm diameter polystyrene (nanoPS) particles. Marine and freshwater cyanobacteria formed aggregates with the nanoPS, bound together by extracellular polymeric substances (EPS), and these aggregates sedimented. The aggregates were larger, and the sedimentation was more rapid for the marine system. Aggregate morphologies were qualitatively different for the microPS samples, with the bacteria linking up a small number of particles, all held together by EPS. There was no sedimentation in these samples. The cyanobacteria remained alive after exposure to the particles. The particle size- and salt concentration-dependent response of cyanobacteria to these anthropogenic stressors is an important factor to consider for a proper understanding of the fate of the particles as well as the bacteria.

Synergistic use of electroosmotic flow and magnetic forces for nucleic acid extraction.

Nucleic acid sample preparation is essential for biological sample-based diagnostics. It is crucial that diagnostic tests be both specific and sensitive as to provide the most accurate diagnosis possible. Inefficient sample preparation can hinder the specificity and sensitivity of these tests since carryover contaminants can inhibit downstream processes, such as amplification. Microfluidic devices have been used previously to extract nucleic acids from a biological sample due to lower reagent volumes and ease of use. A novel microfluidic chip has been designed for nucleic acid sample preparation which combines electroosmotic flow and magnetic bead-based extraction to isolate DNA from a plasma sample. A steady electric field was incorporated into the microfluidic chip design, which when combined with a glass clover slip and a voltage differential, creates electroosmotic flow. With the goal of isolating nucleic acids into a clean, inhibitor free solution, the electroosmotic flow is the driving force and separation mechanism purifying the DNA sample captured on magnetic beads in the microfluidic chip system. Carryover volume, or the volume of unwanted sample contaminants that accompany the nucleic acids into the final elution buffer, was minimized to 0.22 ± 0.03%. In combination with magnetic bead based nucleic acid extraction techniques, a 15% increase in DNA extraction yield is reported for the microfluidic chip with the voltage applied versus without. Although the literature on nucleic acid separation in microfluidic chips is abundant, this is the first to combine microfluidic chip design, magnetic bead-based isolation and electroosmotic flow.

Microfluidic Immiscible Phase Filtration System for the Isolation of Small Numbers of Cells from Whole Blood.

Isolation of circulating tumor cells (CTCs) has generated clinical and academic interest due to the important role that CTCs play in cancer metastasis and diagnosis. Here, we present a PDMS and glass prototype of a microfluidic device for the immunomagnetic, immiscible phase filtration based capture, and isolation of MCF-7 breast cancer cells, from various sample matrices including PBS-based buffer, blood plasma, and unprocessed whole blood. Following optimization of surface energy of an oil-water interface, microfluidic geometry, and bead-binding kinematics, our microfluidic device achieved 95 ± 4% recovery of target cells from PBS-based buffer with 95% purity, 90 ± 3% recovery of target cells from blood plasma and recovery of ~70 ± 5% from unprocessed whole blood with purity >99% with 1 ml blood samples with 1,000 spiked target cells. From quantitative studies to assess the nonspecific carryover of contaminants from whole blood, we found that our system accomplishes a >175 fold depletion in platelets, >900 fold depletion in erythrocytes, and >1,700 fold depletion in leukocytes with respect to unprocessed whole blood, enabling us to avoid sample pre-processing. In addition, we found that ~95% of the isolated target cells were viable, making them suitable for subsequent molecular and cellular studies. We quantify and propose mechanisms for the carryover of platelet, erythrocyte, and leukocyte contamination in purified samples, rather than relying on sample pre-processing. These results validate the continued study of our platform for extraction of CTCs from patient samples and other rare cell isolation applications. © 2019 International Society for Advancement of Cytometry.

Mathematical model to reduce loop mediated isothermal amplification (LAMP) false-positive diagnosis.

Loop mediated isothermal amplification (LAMP) is a nucleic acid amplification technique performed under isothermal conditions. The output of this amplification technique includes multiple different sizes of deoxyribonucleic acid (DNA) structures which are identified by a banding pattern on gel electrophoresis plots. Although this is a specific amplification technique, the complexity of the primer design and amplification still lead to the issue of obtaining false-positive results, especially when a positive reading is determined solely by whether there is any banding pattern in the gel electrophoresis plot. Here, we first performed extensive LAMP experiments and evaluated the DNA structures using microchip electrophoresis. We then developed a mathematical model derived from the various components that make up an entire LAMP structure to predict the full LAMP structure size in base pairs. This model can be implemented by users to make predictions for specific, DNA size dependent, banding patterns on their gel electrophoresis plots. Each prediction is specific to the target sequence and primers used and therefore reduces incorrect diagnosis errors through identifying true-positive and false-positive results. This model was accurately tested with multiple primer sets in house and was also translatable to different DNA and RNA types in previously published literature. The mathematical model can ultimately be used to reduce false-positive LAMP diagnosis errors for applications ranging from tuberculosis diagnostics to E. coli to numerous other infectious diseases.

Rapid electrophoretic recovery of DNA from dried blood spots.

Large-scale genetic screening of neonatal dried blood spots for episomal DNA has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of dried blood spots remains one of the most time consuming, costly, and labor-intensive parts of DNA analysis. In the present study, we developed and optimized a rapid methodology using only 50 V and heat to extract episomal DNA from dried blood spots prepared from diagnostic cord blood samples. This electric field DNA extraction is the first methodology to use an electric field to extract episomal DNA from a dried blood spot. This 25-minute procedure has one of the lowest times for the extraction of episomal DNA found within the literature and this novel procedure not only negates the need for costly treatment and wash steps, but reduces the time of manual procedures by more than 30 min while retaining the 75-80% of the yield. Combined with real-time PCR, this novel method of electric field extraction not only provides an effective tool for the large scale genetic analysis of neonates, but a key step forward in the simplification and standardization of diagnostic testing.

Behavior of Marine Bacteria in Clean Environment and Oil Spill Conditions.

Alcanivorax borkumensis is a bacterial community that dominates hydrocarbon-degrading communities around many oil spills. The physicochemical conditions that prompt bacterial binding to oil/water interfaces are not well understood. To provide key insights into this process, A. borkumensis cells were cultured either in a clean environment condition (dissolved organic carbon) or in an oil spill condition (hexadecane as the sole energy source). The ability of these bacteria to bind to the oil/water interface was monitored through interfacial tension measurements, bacterial cell hydrophobicity, and fluorescence microscopy. Our experiments show that A. borkumensis cells cultured in clean environment conditions remain hydrophilic and do not show significant transport or binding to the oil/water interface. In sharp contrast, bacteria cultured in oil spill conditions become partially hydrophobic and their amphiphilicity drives them to oil/water interfaces, where they reduce interfacial tension and form the early stages of a biofilm. We show that it is A. borkumensis cells that attach to the oil/water interface and not a synthesized biosurfactant that is released into solution that reduces interfacial tension. This study provides key insights into the physicochemical properties that allow A. borkumensis to adhere to oil/water interfaces.

Current Status of Point-of-Care Testing for Human Immunodeficiency Virus Drug Resistance.

Healthcare delivery has advanced due to the implementation of point-of-care testing, which is often performed within minutes to hours in minimally equipped laboratories or at home. Technologic advances are leading to point-of-care kits that incorporate nucleic acid-based assays, including polymerase chain reaction, isothermal amplification, ligation, and hybridization reactions. As a limited number of single-nucleotide polymorphisms are associated with clinically significant human immunodeficiency virus (HIV) drug resistance, assays to detect these mutations have been developed. Early versions of these assays have been used in research. This review summarizes the principles underlying each assay and discusses strategic needs for their incorporation into the management of HIV infection.

Effects of Flow and Bulk Vesicle Concentration on Supported Lipid Bilayer Formation.

Supported lipid bilayers (SLBs) have been used extensively in a variety of biotechnology applications and fundamental studies exploring lipid behavior. Despite their widespread use, various physicochemical parameters have yet to be thoroughly investigated for their impact on SLB formation. In this work, we have studied the importance of flow in inducing the rupture of surface adsorbed chicken egg-derived l-α-phosphatidylcholine (egg PC) vesicles on silica and gold surfaces via quartz crystal microbalance with dissipation monitoring (QCM-D). On silica at 25 °C, egg PC vesicles were found to adsorb in a flattened configuration (∼13 nm thick, compared to bulk vesicle diameters of ∼165 nm) but only undergo a transition to a stable SLB under flow conditions. In the absence of flow, an increase in system temperature to 37 °C was able to promote vesicle rupture and SLB formation on silica with a 10 times lower rupture time, compared to rupture under continuous flow (175 µL/min flow rate). Gold surfaces, with their increased hydrophobicity, led to less vesicle flattening once adsorbed (structures ∼60 nm thick), and did not support vesicle rupture or SLB formation, even at flow rates of up to 650 µL/min. We also showed that, under continuous flow conditions, vesicle adsorption rates on silica surfaces follow Langmuir kinetics, with an inverse dependence on bulk vesicle concentration, while an empirical power law dependence of vesicle rupture time on bulk vesicle concentration was observed. Ultimately, this work elicits fundamental insight into the importance of flow and bulk vesicle concentration in the adsorbed vesicle rupture process during SLB formation using QCM-D.

Archaeal RNA ligase from thermoccocus kodakarensis for template dependent ligation.

Nicking-sealing RNA ligases play a significant biological role in host defense and cellular repair, and have become an important molecular tool in biomedical engineering. Due to the propensity for RNA to form secondary structures, RNA modifying enzymes with elevated optimum temperatures are highly desired. Current characterized double stranded RNA ligases, such as the bacteriophage T4 RNA ligase 2, while possessing good template dependency, are not active at elevated temperatures. The few characterized RNA ligases from thermophiles exhibit high template independency. We synthesize and characterize here, KOD RNA ligase (KOD1Rnl), a thermostable and template dependent RNA ligase from the archaeon, Thermoccocus Kodakarensis. We disclose that a 13 time reduction in template independent ligation can be achieved with the addition of a single stranded DNase, such as RecJ. We also elucidate the effects of the presence of blood proteins on the activity of KOD1Rnl. Template dependent and thermostable RNA ligases, such as KOD RNA ligase, can be utilized in RNA detection, modification and sequencing.

Microfluidic Sample Preparation for Medical Diagnostics.

Fast and reliable diagnoses are invaluable in clinical care. Samples (e.g., blood, urine, and saliva) are collected and analyzed for various biomarkers to quickly and sensitively assess disease progression, monitor response to treatment, and determine a patient's prognosis. Processing conventional samples entails many manual time-consuming steps. Consequently, clinical specimens must be processed by skilled technicians before antigens or nucleic acids are detected, and these are often present at dilute concentrations. Recently, several automated microchip technologies have been developed that potentially offer many advantages over traditional bench-top extraction methods. The smaller length scales and more refined transport mechanisms that characterize these microfluidic devices enable faster and more efficient biomarker enrichment and extraction. Additionally, they can be designed to perform multiple tests or experimental steps on one integrated, automated platform. This review explores the current research on microfluidic methods of sample preparation that are designed to aid diagnosis, and covers a broad spectrum of extraction techniques and designs for various types of samples and analytes.

Interaction of Alcanivorax borkumensis with a Surfactant Decorated Oil-Water Interface.

Alcanivorax borkumensis is a hydrocarbon degrading bacterium linked to oil degradation around oil spill sites. It is known to be a surface bacterium leading to substantial interaction with the oil-water interface. Because of its abundance in oil spill regions, it has great potential to be used actively in oil spill remediation. Dispersants are thought to be important in the creation of oil-in-water emulsions that are meant to aid in the biodegradation process by bacteria. Although it is likely that some sort of dispersant will be used again in the case of another oil spill, to date, no studies have shown the impact of dispersants on the bacteria population. Corexit 9500 was the main dispersant used during the Deepwater Horizon oil spill, but little is known about its effect on the bacteria community. We built an experimental platform to quantitatively measure the transient growth of Alcanivorax borkumensis at the interface of oil and water. To our knowledge, this is the first study of how A. borkumensis interacts with a surfactant decorated oil-water interface. We use COREXIT EC9500A, cetylytrimethylamonium bromide, dioctyl sulfosuccinate sodium salt, l-α-phosphatidylcholine, sodium dodecyl sulfate, and Tween 20 to investigate the impact of dispersants on Alcanivorax borkumensis. We assess the impact of these dispersants on the growth rate, lag time, and maximum concentration of Alcanivorax borkumensis. We show that the charge, structure, and surface activity of these surfactants greatly impact the growth of A. borkumensis. Our results indicated that out of the surfactants tested only Tween 20 assists Acanivorax borkumensis growth. The results of this study will be important in the decision of dispersant use in the future.

Multilayer spheroids to quantify drug uptake and diffusion in 3D.

There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil's inhibition of Pgp and GJIC were similar (IC50: 8 µM). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.

Dynamics of a capillary invasion in a closed-end capillary.

The position of fluid invasion in an open capillary increases as the square root of time and ceases when the capillary and hydrostatic forces are balanced, when viscous and inertia terms are negligible. Although this fluid invasion into open-end capillaries has been well described, detailed studies of fluid invasion in closed-end capillaries have not been explored thoroughly. Thus, we demonstrated, both theoretically and experimentally, a fluid invasion in closed-end capillaries, where the movement of the meniscus and the invasion velocity are accompanied by adiabatic gas compression inside the capillary. Theoretically, we found the fluid oscillations during invasion at short time scales by solving the one-dimensional momentum balance. This oscillatory motion is evaluated to determine which physical forces dominate the different conditions, and is further described by a damped driven harmonic oscillator model. However, this oscillating motion is not observed in the experiments. This inconsistency is due to the following: first, a continuous decrease in the radius of the curvature caused by decreasing the invasion velocity and increasing pressure inside the closed-end capillary, and second, the shear stress increase in the short time scale by the plug like velocity profile within the entrance length. The viscous term of modified momentum equation can be written as K(8µl/rc(2))(dl/dt) by using the multiplying factor K, which represents the increase of shear stress. The K is 7.3, 5.1, and 4.8 while capillary aspect ratio χc is 740, 1008, and 1244, respectively.

Phase and steady shear behavior of dilute carbon black suspensions and carbon black stabilized emulsions.

We use para-amino benzoic acid terminated carbon black (CB) as a model particulate material to study the effect of salt-modulated attractive interactions on phase behavior and steady shear stresses in suspensions and particle-stabilized emulsions. Surprisingly, the suspension displayed a yield stress at a CB volume fraction of ϕCB = 0.008. The yield stress scaled with CB concentration with power law behavior; the power law exponent changed abruptly at a critical CB concentration, suggesting a substantial change in network structure. Cryogenic scanning electron microscopy revealed structural differences between the networks found in each scaling regime. Randomly oriented pores with thick CB boundaries were observed in the scaling region above the critical particle concentration, suggesting a strong gel network, and long, oriented pores were found in the scaling region below the critical particle concentration, suggesting a weak network influenced by an induced shear stress. These findings correlate with the existence of gels and transient networks. Transient networks break down under gravitational forces over time periods of 12-24 hours. The yield stresses of CB-gels containing oil emulsion droplets were found to scale with carbon black concentration similar to the CB-gels without oil. These results offer insight into salt-induced attractive colloidal networks and the difference in structure and yield-stress behavior between transient networks and gels. Furthermore, CB offers the ability to stabilize an oil phase in discrete droplets and contain them within a rigid network structure.