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.

Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability.

Despite recent advances in nucleic acid delivery systems with the success of LNP vehicles, adeno-associated virus (AAV) remains the leading platform for targeted gene delivery due to its low immunogenicity to humans, high transduction efficiency, and range of serotypes with varying tropisms. Depending on the therapeutic goals and serotype used, different production conditions may be more amenable, generating an ever-growing need for rapid yet robust analytical techniques to support the high-quality manufacturing of AAV. A critical bottleneck exists for assessing full capsids where rapid, high-throughput techniques capable of analyzing a range of serotypes are needed. Here, we present a rapid, high-throughput analytical technique, microfluidic electrophoresis, for the assessment of full capsids compatible with AAV1, AAV2, AAV6, AAV8, and AAV9 without the need for assay modifications or optimizations, and AAV5 with some constraints. The method presented in this study uses a mathematical formulation we developed previously with a reference standard to combine the independently obtained capsid protein and single-stranded DNA (ssDNA) profiles to estimate the percentage of full capsids in a sample of unknown concentration. We assessed the ability to use a single serotype (AAV8) as the reference standard regardless of the serotype of the sample being analyzed so long as the melting temperature (Tm) of the capsids is within 12 °C from the Tm of AAV8. Using this method, we are able to characterize samples ±6.1% with an average analytical turnaround time of <5 min/sample, using only 10 µL/sample at a concentration of 2.5 × 1012 VG/mL.

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.

An Optimized CoBRA Method for the Microfluidic Electrophoresis Detection of Breast Cancer Associated RASSF1 Methylation.

Although breast cancer screening assays exist, many are inaccessible and have high turnaround times, leaving a significant need for better alternatives. Hypermethylation of tumor suppressor genes is a common epigenetic marker of breast cancer. Methylation tends to occur most frequently in the promoter and first exon regions of genes. Preliminary screening tests are crucial for informing patients whether they should pursue more involved testing. We selected RASSF1, previously demonstrated to be aberrantly methylated in liquid biopsies from breast cancer patients, as our gene of interest. Using CoBRA as our method for methylation quantification, we designed unique primer sets that amplify a portion of the CpG island spanning the 5' end of the RASSF1 first exon. We integrated the CoBRA approach with a microfluidics-based electrophoresis quantification system (LabChip) and optimized the assay such that insightful results could be obtained without post-PCR purification or concentration, two steps traditionally included in CoBRA assays. Circumventing these steps resulted in a decreased turnaround time and mitigated the laboratory machinery and reagent requirements. Our streamlined technique has an estimated limit of detection of 9.1 ng/µL of input DNA and was able to quantify methylation with an average error of 4.3%.

Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids.

Adeno-associated virus (AAV) has shown great potential in gene therapy due to its low immunogenicity, lack of pathogenicity to humans, and ability to provide long-term gene expression in vivo. However, there is currently a need for fast, high-throughput characterization systems that require low volumes for the determination of its sample composition in terms of full and empty capsids since empty capsids are a natural byproduct of AAV synthesis. To address this need, the following study proposes a high-throughput electrophoresis-mediated microfluidics approach that is independent of sample input concentration to estimate the composition of a given sample by combining its protein and ssDNA information relative to a standard. Using this novel approach, we were able to estimate the percentage of full capsids of six AAV8 samples with an average deviation from the actual percentage of 4%. The experiments used for these estimations were conducted with samples of varying percentages of full capsids (21-75%) and varying concentrations (5 × 1011-1 × 1012 VP/mL) with a total volume requirement of 3-10 µL for triplicate analysis of the sample. This method offers a rapid way to evaluate the quality and purity of AAV products. We believe that our method addresses the critical need as recognized by the gene and molecular therapy community.

Electrokinetic characterization of synthetic protein nanoparticles.

The application of nanoparticle in medicine is promising for the treatment of a wide variety of diseases. However, the slow progress in the field has resulted in relatively few therapies being translated into the clinic. Anisotropic synthetic protein nanoparticles (ASPNPs) show potential as a next-generation drug-delivery technology, due to their biocompatibility, biodegradability, and functionality. Even though ASPNPs have the potential to be used in a variety of applications, such as in the treatment of glioblastoma, there is currently no high-throughput technology for the processing of these particles. Insulator-based electrokinetics employ microfluidics devices that rely on electrokinetic principles to manipulate micro- and nanoparticles. These miniaturized devices can selectively trap and enrich nanoparticles based on their material characteristics, and subsequently release them, which allows for particle sorting and processing. In this study, we use insulator-based electrokinetic (EK) microdevices to characterize ASPNPs. We found that anisotropy strongly influences electrokinetic particle behavior by comparing compositionally identical anisotropic and non-anisotropic SPNPs. Additionally, we were able to estimate the empirical electrokinetic equilibrium parameter (eE EEC) for all SPNPs. This particle-dependent parameter can allow for the design of various separation and purification processes. These results show how promising the insulator-based EK microdevices are for the analysis and purification of clinically relevant SPNPs.

Determination of the Empirical Electrokinetic Equilibrium Condition of Microorganisms in Microfluidic Devices.

The increased concern regarding emerging pathogens and antibiotic resistance has drawn interest in the development of rapid and robust microfluidic techniques to analyze microorganisms. The novel parameter known as the electrokinetic equilibrium condition (EEEC) was presented in recent studies, providing an approach to analyze microparticles in microchannels employing unique electrokinetic (EK) signatures. While the EEEC shows great promise, current estimation approaches can be time-consuming or heavily user-dependent for accurate values. The present contribution aims to analyze existing approaches for estimating this parameter and modify the process into an accurate yet simple technique for estimating the EK behavior of microorganisms in insulator-based microfluidic devices. The technique presented here yields the parameter called the empirical electrokinetic equilibrium condition (eEEEC) which works well as a value for initial approximations of trapping conditions in insulator-based EK (iEK) microfluidic systems. A total of six types of microorganisms were analyzed in this study (three bacteria and three bacteriophages). The proposed approach estimated eEEEC values employing images of trapped microorganisms, yielding high reproducibility (SD 5.0-8.8%). Furthermore, stable trapping voltages (sTVs) were estimated from eEEEC values for distinct channel designs to test that this parameter is system-independent and good agreement was obtained when comparing estimated sTVs vs. experimental values (SD 0.3-19.6%). The encouraging results from this work were used to generate an EK library of data, available on our laboratory website. The data in this library can be used to design tailored iEK microfluidic devices for the analysis of microorganisms.

Creation of an electrokinetic characterization library for the detection and identification of biological cells.

The rising concern over drug-resistant microorganisms has increased the need for rapid and portable detection systems. However, the traditional methods for the analysis of microorganisms can be both resource and time intensive. This contribution presents an alternative approach for the characterization of microorganisms using a microscale electrokinetic technique. The present study aims to develop and validate a library with a novel parameter referred to as the electrokinetic equilibrium condition for each strain, which will allow for fast identification of the studied bacterial and yeast cells in electrokinetic (EK) microfluidic devices. To create the library, experiments with six organisms of interest were conducted using insulator-based EK devices with circle-shaped posts. The organisms included one yeast strain, Saccharomyces cerevisiae; one salmonella strain, Salmonella enterica; two species from the same genus, Bacillus cereus and Bacillus subtilis; and two Escherichia coli strains. The results from these experiments were then analyzed with a mathematical model in COMSOL Multiphysics®, which yielded the electrokinetic equilibrium condition for each distinct strain. Lastly, to validate the applicability EK library, the COMSOL model was used to estimate the trapping conditions needed in a device with oval-shaped posts for each organism, and these values were then compared with experimentally obtained values. The results suggest the library can be used to estimate trapping voltages with a maximum relative error of 12%. While the proposed electrokinetic technique is still a novel approach and the analysis of additional microorganisms would be needed to expand the library, this contribution further supports the potential of microscale electrokinetics as a technique for the rapid and robust characterization of microbes. Graphical abstract.

Analysis of Bacteriophages with Insulator-Based Dielectrophoresis.

Bacterial viruses or phages have great potential in the medical and agricultural fields as alternatives to antibiotics to control nuisance populations of pathogenic bacteria. However, current analysis and purification protocols for phages tend to be resource intensive and have numbers of limitations, such as impacting phage viability. The present study explores the potential of employing the electrokinetic technique of insulator-based dielectrophoresis (iDEP) for virus assessment, separation and enrichment. In particular, the application of the parameter "trapping value" (Tv) is explored as a standardized iDEP signature for each phage species. The present study includes mathematical modeling with COMSOL Multiphysics and extensive experimentation. Three related, but genetically and structurally distinct, phages were studied: Salmonella enterica phage SPN3US, Pseudomonas aeruginosa phage ϕKZ and P. chlororaphis phage 201ϕ2-1. This is the first iDEP study on bacteriophages with large and complex virions and the results illustrate their virions can be successfully enriched with iDEP systems and still retain infectivity. In addition, our results indicate that characterization of the negative dielectrophoretic response of a phage in terms of Tv could be used for predicting individual virus behavior in iDEP systems. The findings reported here can contribute to the establishment of protocols to analyze, purify and/or enrich samples of known and unknown phages.

Identification of Essential Genes in the Salmonella Phage SPN3US Reveals Novel Insights into Giant Phage Head Structure and Assembly.

Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ϕKZ, have long genomes packaged into large, atypical virions. Many aspects of ϕKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ϕKZ and related phages, ∼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase ß subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a "missing" ß' subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution. IMPORTANCE: In recent years, there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage ϕKZ are of great interest due to their virions being among the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited, as a large proportion of their proteins have not been characterized and/or have been deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics, and proteomics and in doing so revealed new information regarding giant phage head structure and assembly and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as a model phage for the growing group of phages related to ϕKZ.