Miniaturization of an enclosed electrospinning process to enhance reproducibility in the fabrication of rapidly dissolving cell-based biosensors.

There is broad interest in producing electrospun films embedded with biological materials. It is well known that electrospinning requires careful control of the process conditions, especially the environmental conditions such as relative humidity (RH). Given that commercial electrospinning systems are expensive (> $10,000) and are typically too large to be used in standard biological safety cabinets (BSC), we designed and built a miniaturized electrospinning box (E-Box) that will fit inside a BSC, and the RH can be easily controlled using simple instrumentation (gas cylinder, regulator, needle valve, rotameter). It uses an inexpensive computerized numerical control machine to control the spinneret positioning and collector rotational speed-all the parts for the device (except the syringe pump and voltage supply) can be purchased for approximately $1000. We demonstrate the usefulness of our design in optimizing the production of Escherichia coli-embedded pullulan-trehalose films to be used as rapidly dissolving biosensors for environmental monitoring. At a fixed electrospinning recipe, we showed that decreasing the RH from approximately 48% to 22% resulted in the average fiber diameter increasing from 240 (± 11) nm to 314 (± 8) nm. We also demonstrate the usefulness of our design in performing sequential electrospinning experiments to evaluate process performance reproducibility. For example, from just 1 mL of a polymer solution, we produced 16 electrospun films (approximately 3 cm by 8 cm each)-from those films we hole-punched approximately 80 biosensor discs which were then used in subsequent experiments to determine the amount of two different biocides (Grotan BK and triclosan) in aqueous samples. The technique developed in this study is ideal for creating electrospun materials in high quantities that are highly reproducible through the precise control of RH.

High-throughput screening to evaluate optimum coagulation conditions via colloidal stability analysis.

Current methods of optimizing the coagulant dosage in wastewater treatment processes typically rely on the use of labor- and material-intensive jar testers, which are inadequate when coagulation processes require frequent adjustments due to variations in properties of the incoming feed. Analytical centrifuges (ACs) employ an integrated optics system that simultaneously monitors the position of the boundary between two separating phases in multiple samples of fairly low volumes (∼2 mL) - thus it was expected that ACs would be ideally suited to study the stability and settling kinetics of coagulation treatment processes. In this study, wastewater samples from a biogas generation facility (known as centrate) were collected in February 2022 (Batch A) and July 2022 (Batch B). A comprehensive screening of the treatment performance for Batch B was conducted at three pHs (5, 6, and 7) and nine concentrations of ferric chloride (0-500 mg-Fe3+/L) - it was found that the front-tracking profiles measured by the integrated optics system could be used to identify the minimal coagulation conditions needed to transition from slow to rapid settling. While the settling velocity was found to be well correlated with the instability index, a dimensionless number between 0 and 1 (where values closer to 1 indicate better separation), it was determined that the percentage of COD removal from the centrate samples increased up to an instability index of approximately 0.5 and then plateaued. Finally, it was found that the front-tracking profiles could be used to estimate the volume of sludge produced at various coagulation conditions. Thus, the results from this study establish ACs as an important screening tool for rapid evaluation of treatment performance while consuming minimal material and time - in this study, a total of 132 screening experiments were conducted using approximately ∼11 L of centrate and ∼6 hours of operator time.

Fouling of virus filtration membranes by monoclonal antibody feeds with low aggregate content.

Membrane fouling by monoclonal antibodies (mAbs) is one of the main challenges in virus-filtration processes. Previous publications attributed membrane fouling to the presence of mAb aggregates in the solution, which block the membrane pores. This fouling mechanism can be solved by a prefilter; however, it was shown that there are mAbs that severely foul the membranes (reduce permeability by 90% and more) even after prefiltering the aggregates, while other mAbs foul the membrane weakly (reduce permeability by ~10% and less). Unfortunately, the differences between the fouling- and the nonfouling mAbs have never been convincingly explained. To get a deeper insight on these differences, we measured the fouling of chemically modified Isoprene-Styrene-4-vinylpyridine (ISV) membranes (TeraPore Technologies) by 8 mAbs exhibiting different hydrophobicity and charge. The results show that mAb solutions with low concentration of aggregates foul ISV membranes via an adsorptive mechanism, and the adsorption is driven mainly by hydrophobic forces between the mAb and the membrane. The charge of the mAbs plays a secondary role in fouling. We want to emphasize that the conclusions pertain to ISV membranes; the insights presented in this paper can potentially be used to engineer new surface chemistries to mitigate fouling of other virus-filtration and/or ultrafiltration membranes.

Tuning the sensitivity of cell-based biosensors for the detection of biocides.

The presence of biocides in wastewater can negatively impact the efficiency of wastewater treatment processes, particularly the process of nitrification. In this paper, we describe the development of cell-based biosensors (CBBs) with tunable levels of sensitivity for rapidly detecting the presence and predicting the type and concentration of biocides. The CBB assay developed is performed by first exposing a panel of bacterial strains (E. coli, B. subtilis, B. cereus) to the sample being tested and to the control sample without biocide, and then adding a fluorescent dye (LIVE/DEAD BacLight). We then compare the fluorescence signals generated by the two samples, and the differences in the signals indicate the presence of a biocide, as previously reported in the literature. We found that the sensitivity of the CBB assay can be improved by 'tuning' the type/salinity of the buffer used to suspend the cells, and by changing the number of cells used in the assay. These changes improved the level of detection (LOD) of the biocide Cetyltrimethylammonium bromide (CTAB) from 10 ppm to 0.625 ppm and the biocide Grotan® BK from 500 ppm to 7.8 ppm. With the optimized conditions for each strain, we also establish that the combined response from the panel of bacterial strains can be used to predict the type and concentration of biocide sample tested. Additionally, we provide evidence that the CBB assay can be performed using a compact, commercially available fluorometer. Overall, the significance of this work will improve point-of-use testing and enable the discrimination between biocide-containing samples of similar toxicity and detection of lower toxicity samples, thereby improving the accuracy of the CBB assay.

Evaluation of Host Cell Impurity Effects on the Performance of Sterile Filtration Processes for Therapeutic Viruses.

Efficient downstream processing represents a significant challenge in the rapidly developing field of therapeutic viruses. While it is known that the terminal sterile filtration step can be a major cause of product loss, there is little known about the effect of host cell impurities (DNA and protein) on filtration performance. In this study, fractions of relatively pure Vero host cell protein and DNA were spiked into a highly pure preparation of vesicular stomatitis virus (VSV). Then, the resulting solutions were sterile filtered using two commercially available 0.22 µm rated microfiltration membranes. A combination of transmembrane pressure measurements, virus recovery measurements, and post-filtration microscopy images of the microfiltration membranes was used to evaluate the sterile filtration performance. It was found that increasing the amount of host cell protein from approximately 1 µg/mL (in the un-spiked VSV preparation) to 25 µg/mL resulted in a greater extent of membrane fouling, causing the VSV recovery to decrease from 89% to 65% in experiments conducted with the highly asymmetric Express PLUS PES membrane and to go as low as 48% in experiments conducted with the symmetric Durapore PVDF membrane. Similar effects were not seen when bovine serum albumin, a common model protein used in filtration studies, was spiked into the VSV preparation, which indicates that the sterile filtration performance is critically dependent on the complex composition of the mixture of host cell proteins rather than the presence of any protein. The results presented in this work provide important insights into the role of host cell impurities on the performance of sterile filtration processes for therapeutic viruses.

Navigating Performance Standards for Face Mask Materials: A Custom-Built Apparatus for Measuring Particle Filtration Efficiency.

Public health agencies have recommended the community use of face masks to reduce the transmission of airborne diseases like COVID-19. Virus transmission is reduced when masks act as efficient filters, thus evaluating mask particle filtration efficiency (PFE) is essential. However, the high cost and long lead times associated with purchasing turn-key PFE systems or hiring certified laboratories hampers the testing of filter materials. There is a clear need for "custom" PFE test systems; however, the variety of standards that prescribe (medical) face mask PFE testing (e.g., ASTM International, NIOSH) vary widely in their protocols and clarity of guidelines. Herein, the development is described of an "in-house" PFE system and method for testing face masks in the context of current standards for medical masks. Pursuant to the ASTM International standards, the system uses an aerosol of latex spheres (0.1 µm nominal size) with particle concentrations upstream and downstream of the mask material measured using a laser particle analyzer. PFE measurements are obtained for a variety of common fabrics and medical masks. The approach described in this work conforms to the current standards for PFE testing while providing the flexibility to adapt to changing needs and filtration conditions.

Drug-impregnated, pressurized gas expanded liquid-processed alginate hydrogel scaffolds for accelerated burn wound healing.

While the benefits of both hydrogels and drug delivery to enhance wound healing have been demonstrated, the highly hydrophilic nature of hydrogels creates challenges with respect to the effective loading and delivery of hydrophobic drugs beneficial to wound healing. Herein, we utilize pressurized gas expanded liquid (PGX) technology to produce very high surface area (~200 m2/g) alginate scaffolds and describe a method for loading the scaffolds with ibuprofen (via adsorptive precipitation) and crosslinking them (via calcium chelation) to create a hydrogel suitable for wound treatment and hydrophobic drug delivery. The high surface area of the PGX-processed alginate scaffold facilitates >8 wt% loading of ibuprofen into the scaffold and controlled in vitro ibuprofen release over 12-24 h. In vivo burn wound healing assays demonstrate significantly accelerated healing with ibuprofen-loaded PGX-alginate/calcium scaffolds relative to both hydrogel-only and untreated controls, demonstrating the combined benefits of ibuprofen delivery to suppress inflammation as well as the capacity of the PGX-alginate/calcium hydrogel to maintain wound hydration and facilitate continuous calcium release to the wound. The use of PGX technology to produce highly porous scaffolds with increased surface areas, followed by adsorptive precipitation of a hydrophobic drug onto the scaffolds, offers a highly scalable method of creating medicated wound dressings with high drug loadings. STATEMENT OF SIGNIFICANCE: While medicated hydrogel-based wound dressings offer clear advantages in accelerating wound healing, the inherent incompatibility between conventional hydrogels and many poorly water-soluble drugs of relevance in wound healing remains a challenge. Herein, we leveraged supercritical fluids-based strategies to both process and subsequently impregnate alginate, followed by post-crosslinking to form a hydrogel, to create a very high surface area alginate hydrogel scaffold loaded with high hydrophobic drug contents (here, >8 wt% ibuprofen) without the need for any pore-forming additives. The impregnated scaffolds significantly accelerated burn wound healing while also promoting regeneration of the native skin morphology. We anticipate this approach can be leveraged to load clinically-relevant and highly bioavailable dosages of hydrophobic drugs in hydrogels for a broad range of potential applications.

Development of dewatering textile materials incorporating slit-pore geometries.

The use of engineering textile materials has emerged as a viable alternative to conventional methods of sludge dewatering in numerous application areas including municipal wastewater, mining, and pulp and paper. Previous studies have focused on the development of empirical ratios between dewatering performance and the porous properties of the textile material, the challenge is that the latter is difficult to characterize using currently available techniques. In this study, a series of dewatering filters were produced using advanced microfabrication techniques to create well-defined slit-pore geometries; a full-factorial design-of-experiments was employed to evaluate the effects of slit-pore dimensions and slit-pore spacing on the cake layer development and overall dewatering performance in constant-rate dewatering tests with municipal digestate that had been pre-treated with a commercial polymer flocculant. The results from this study provide new insights into the importance of the cake layer in textile dewatering and the impact of textile porosity and flocculation conditions on dewatering performance. It was found that an inverse relationship exists between the porosity of a dewatering fabric and both medium and cake resistances between 0.1% and 1.0% filter porosity, while these properties are comparatively independent of pore structure beyond 1.0%. In addition, the efficacy of the polymer pre-treatment conditions employed was determined to have a substantial impact on solids retention.

Development of isoporous microslit silicon nitride membranes for sterile filtration applications.

The widely used 0.2/0.22 µm polymer sterile filters were developed for small molecule and protein sterile filtration but are not well-suited for the production of large nonprotein biological therapeutics, resulting in significant yield loss and production cost increases. Here, we report on the development of membranes with isoporous sub-0.2 µm rectangular prism pores using silicon micromachining to produce microslit silicon nitride (MSN) membranes. The very high porosity (~33%) and ultrathin (200 nm) nature of the 0.2 µm MSN membranes results in a dramatically different structure than the traditional 0.2/0.22 µm polymer sterile filter, which yielded comparable performance properties (including gas and hydraulic permeance, maximum differential pressure tolerance, nanoparticle sieving/fouling behavior). The results from bacteria retention tests, conducted according to the guidance of regulatory agencies, demonstrated that the 0.2 µm MSN membranes can be effectively used as sterile filters. It is anticipated that the results and technologies presented in this study will find future utility in the production of non-protein biological therapeutics and in other biological and biomedical applications.

A Rapid Assay to Assess Nitrification Inhibition Using a Panel of Bacterial Strains and Partial Least Squares Modeling.

As a proof of concept, a rapid assay consisting of a cell-based biosensor (CBB) panel of pure bacterial strains, a fluorescent dye, and partial least squares (PLS) modeling was developed to assess the nitrification inhibition potential of industrial wastewater (WW) samples. The current standard method used to assess the nitrification inhibition potential is the specific nitrification rate (SNR) batch test, which requires approximately 4 h to complete under the watch of an experienced operator. In this study, we exposed the CBB panel of seven bacterial strains (nitrifying and non-nitrifying) to 28 different industrial WW samples and then probed both the membrane integrity and cellular activity using a commercially available "live/dead" fluorescent dye. The CBB panel response acts as a surrogate measurement for the performance of nitrification. Of the seven strains, four (Nitrospira, Escherichia coli, Bacillus subtilis, Bacillus cereus) were identified via the modeling technique to be the most significant contributors for predicting the nitrification inhibition potential. The key outcome from this work is that the CBB panel fluorescence data (collected in approximately 10 min) can accurately predict the outcome of an SNR batch test (that takes 4 h) when performed with the same WW samples and has a strong potential to approximate the chemical composition of these WW samples using PLS modeling. Overall, this is a powerful technique that can be used for point-of-use detection of nitrification inhibition.

Optimization of microorganism preservation conditions for the development of an acute toxicity bioassay for biocides.

Biocides, also referred to as 'microbicides' or 'inhibitors', are widely used in industrial processes (e.g. utility water in cooling towers) to control and/or eliminate the growth of microorganisms. Because of their inherent toxicity, their presence in various sources (e.g. river sediments, potable water) can negatively affect ecosystems. Currently available biocide detection techniques are not suitable for 'point-of-use' applications since they are tedious, complicated, and often require experienced personnel to operate. To address this concern, we sought to develop a simple-to-use toxicity bioassay based on a model microorganism (E. coli) after short (<30 min) exposure to known biocides that can be stored at room temperature (preferably) or in the fridge. Based on recent work and our expertise in polymer-based preservation of biomolecules, we leveraged this knowledge to improve E. coli preservation for biocide detection purposes. A design-of-experiments strategy was used to evaluate 16 different preservation conditions from 5 process parameters (i.e. 25-1 fractional factorial). It was found that pullulan, a sugar-based polymer, improved E. coli culturability by an order of magnitude after three months of storage. Also, it was found that storing E. coli in the fridge in Milli-Q water was favorable for maintaining a high level of culturability. Finally, the toxicity of three common biocides (Cetyltrimethylammonium bromide (CTAB), ProClin™ 300, and Grotan® BK) was evaluated using a fluorescence-based assay across all 16 preservation conditions. The response of the preserved E. coli was biocide specific and at certain conditions did not vary during the entire three-month storage period.

Investigation of the role of flocculation conditions in recuperative thickening on dewatering performance and biogas production.

There is considerable interest in recuperative thickening (RT), the recycling of partially digested solids in an anaerobic digester outlet stream back into the incoming feed, as a 'high-performance' process to increase biogas production, increase system capacity, and improve biosolids stabilization. While polymer flocculation is commonly used in full-scale RT operations, no studies have investigated the effect of flocculation conditions on RT process performance. Our goal was to investigate the effect of polymer type and dosage conditions on dewatering performance and biogas production in a lab-scale RT system. The type of polymer flocculant significantly affected dewatering performance. For example, the 440 LH polymer (low molecular weight (MW) polyacrylamide) demonstrated lower capillary suction time (CST) and filtrate total suspended solids (TSS) values than the C-6267 polymer (high MW polyacrylamide). An examination of the dewatering performance of RT digesters with different polymers found a strong correlation between CST and filtrate TSS. The type of polymer flocculant had no significant effect on biogas productivity or composition; the methane content was greater than 60% in good agreement with typical results. The optimization of the polymer flocculation conditions is a critical task for which the lab-scale RT system used in this work is ideally suited.

Optimization of biomolecule separation by combining microscale filtration and design-of-experiment methods.

There is considerable interest in developing microscale (i.e., high-throughput) methods that enable multiple filtration experiments to be run in parallel with smaller sample amounts and thus reduce the overall required time and associated cost to run the filtration tests. Previous studies to date have focused on simply evaluating the filtration capacity, not the separation performance. In this work, the stirred-well filtration (SWF) method was used in combination with design-of-experiment (DOE) methods to optimize the separation performance for three binary mixtures of bio-molecules: protein-protein, protein-polysaccharide, and protein-DNA. Using the parallel based format of the SWF method, eight constant-flux ultrafiltration experiments were conducted at once to study the effects of stirring conditions, permeate flux, and/or solution conditions (pH, ionic strength). Four separate filtration tests were conducted for each combination of process variables; in total, over 100 separate tests were conducted. The sieving coefficient and selectivity results are presented to match the DOE design format and enable a greater understanding of the effects of the different process variables that were studied. The method described herein can be used to rapidly determine the optimal combination of process factors that give the best separation performance for a range of membrane-based separations applications and thus obviate the need to run a large number of traditional lab-scale tests. Biotechnol. Bioeng. 2016;113: 2131-2139. © 2016 Wiley Periodicals, Inc.

Lab-scale demonstration of recuperative thickening technology for enhanced biogas production and dewaterability in anaerobic digestion processes.

There is growing interest in the use of high performance anaerobic digestion (AD) processes for the production of biogas at wastewater treatment facilities to offset the energy demands associated with wastewater treatment. Recuperative thickening (RT) is a promising technique which involves recycling a portion of the digested solids back to the incoming feed. In general there exists a significant number of knowledge gaps in the field of RT because the studies that have been conducted to date have almost exclusively occurred in pilot plant or full scale trials; this approach greatly limits the amount of process optimization that can be done in a given trial. In this work, a detailed and comprehensive study of RT was conducted at the lab scale; two custom designed digesters (capacity = 1.5 L) were operated in parallel with one acting as a 'control' digester and the other operating under a semi-batch RT mode. There was no significant change in biogas methane composition for the two digesters, however the RT digester had an average biogas productivity over two times higher than the control one. It was found that the recycling of the polymer flocculant back into the RT digester resulted in a significant improvement in dewatering performance. At the highest polymer concentration tested, the capillary suction time (CST) values for flocculated samples for the RT digester were over 6 times lower than the corresponding values for the control digester. Thus, there exists an opportunity to decrease the overall consumption of polymer flocculants through judicious selection of the dose of polymer flocculant that is used both for the thickening and end-stage dewatering steps in RT processes.

Demonstration of FBRM as process analytical technology tool for dewatering processes via CST correlation.

The current challenges associated with the design and operation of net-energy positive wastewater treatment plants demand sophisticated approaches for the monitoring of polymer-induced flocculation. In anaerobic digestion (AD) processes, the dewaterability of the sludge is typically assessed from off-line lab-bench tests - the capillary suction time (CST) test is one of the most common. Focused beam reflectance measurement (FBRM) is a promising technique for real-time monitoring of critical performance attributes in large scale processes and is ideally suited for dewatering applications. The flocculation performance of twenty-four cationic polymers, that spanned a range of polymer size and charge properties, was measured using both the FBRM and CST tests. Analysis of the data revealed a decreasing monotonic trend; the samples that had the highest percent removal of particles less than 50 microns in size as determined by FBRM had the lowest CST values. A subset of the best performing polymers was used to evaluate the effects of dosage amount and digestate sources on dewatering performance. The results from this work show that FBRM is a powerful tool that can be used for optimization and on-line monitoring of dewatering processes.

RAPID-SELEX for RNA aptamers.

Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5 × 10(15) sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance.

Multiplexed microcolumn-based process for efficient selection of RNA aptamers.

We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin-a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 10(8) with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells.

We describe a microfluidic device for the extraction, purification and stretching of human chromosomal DNA from single cells. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane channel was designed to capture a single human cell. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Intact chromosomal DNA is entangled in the array, while other cellular components are washed from the channel. To demonstrate the entrapment principle, a single chromosome was hybridized to whole chromosome paints, and imaged by fluorescence microscopy. DNA extracted from a single cell and small cell populations (less than 100) was released from the device by restriction endonuclease digestion under continuous flow and collected for off-chip analysis. Quantification of the extracted material reveals that the microdevice efficiently extracts essentially all chromosomal DNA. The device described represents a novel platform to perform a variety of analyses on chromosomal DNA at the single cell level.

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel.

Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.

Separation of plasmid DNA isoforms by highly converging flow through small membrane pores.

Plasmid DNA isoforms can be separated by both agarose gel electrophoresis and a variety of chromatographic methods, but both of these approaches have significant shortcomings in terms of scalability, throughput, and/or resolution. This study provides the first demonstration that the supercoiled, linear, and open-circular isoforms of plasmid DNA can be effectively separated based on differences in their elongational flexibility in the highly converging flow field that is established during membrane ultrafiltration. Data were obtained with plasmids from 3 to 17 kbp in size using commercially available cellulose ultrafiltration membranes with pores an order of magnitude smaller than the DNA root-mean-square radius of gyration. High-resolution separations were achieved by controlling the filtrate flux between the critical flux values required for transmission of the individual isoforms. The separation behavior in ultrafiltration was very different than that observed in size exclusion chromatography or agarose gel electrophoresis due to differences in the underlying separation mechanisms. The simplicity of the ultrafiltration process makes this approach attractive for a wide range of applications, including large-scale purification of plasmid DNA for gene therapy.