Identifying Viral Vector Characteristics by Nanopore Sensing.

Using viral vectors as gene delivery vehicles for gene therapy necessitates their quality control. Here, we report on nanopore sensing for nondestructively inspecting genomes inside the nanoscale cargoes at the single-molecule level. Using ionic current measurements, we motion-tracked the adeno-associated virus (AAV) vectors as they translocated through a solid-state nanopore. Considering the varying contributions of the electrophoretic forces from the negatively charged internal polynucleotides of different lengths, the nanocargoes carrying longer DNA moved more slowly in the nanochannel. Moreover, ion blockage characteristics revealed their larger volume by up to approximately 3600 nm3 in proportion to the length of single-stranded DNA packaged inside, thereby allowing electrical discriminations of AAV vectors by the gene-derived physical features. The present findings can be a promising tool for the enhanced quality control of AAV products by enabling the screening of empty and intermediate vectors at the single-particle level.

Challenges in the analysis of pharmaceutical lentiviral vector products by orthogonal and complementary physical (nano)particle characterization techniques.

Lentiviral vectors (LVVs) are used as a starting material to generate chimeric antigen receptor (CAR) T cells. Therefore, LVVs need to be carefully analyzed to ensure safety, quality, and potency of the final product. We evaluated orthogonal and complementary analytical techniques for their suitability to characterize particulate matter (impurities and LVVs) in pharmaceutical LVV materials at development stage derived from suspension and adherent manufacturing processes. Microfluidic resistive pulse sensing (MRPS) with additional manual data fitting enabled the assessment of mode diameters for particles in the expected LVV size range in material from adherent production. LVV material from a suspension process, however, contained substantial amounts of particulate impurities which blocked MRPS cartridges. Sedimentation-velocity analytical ultracentrifugation (SV-AUC) resolved the LVV peak in material from adherent production well, whereas in more polydisperse samples from suspension production, presence of particulate impurities masked a potential signal assignable to LVVs. In interferometric light microscopy (ILM) and nanoparticle tracking analysis (NTA), lower size detection limits close to âˆ¼ 70 nm resulted in an apparent peak in particle size distributions at the expected size for LVVs emphasizing the need to interpret these data with care. Interpretation of data from dynamic light scattering (DLS) was limited by insufficient size resolution and sample polydispersity. In conclusion, the analysis of LVV products manufactured at pharmaceutical scale with current state-of-the-art physical (nano)particle characterization techniques was challenging due to the presence of particulate impurities of heterogeneous size. Among the evaluated techniques, MRPS and SV-AUC were most promising yielding acceptable results at least for material from adherent production.

Critical parameters to standardize the size and concentration determination of nanomaterials by nanoparticle tracking analysis.

The size and concentration are critical for the diagnostic and therapeutic applications of nanomaterials but the accurate measurement remains challenging. Nanoparticle tracking analysis (NTA) is widely used for size and concentration determination. However, highly repeatable standard operating procedures (SOPs) are absent. We adopted the "search-evaluate-test" strategy to standardize the measurement by searching the critical parameters. The particles per frame are linearly proportional to the sample concentration and the measured results are more accurate and repeatable when the concentration is 108-109 particles/ml. The optimal detection threshold is around 5. The optimal camera level is such that it allows clear observation of particles without diffractive rings and overexposure. The optimal speed is ≤ 50 in AU and âˆ¼ 10 µl/min in flow rate. We then evaluated the protocol using polydisperse polystyrene particles and we found that NTA could discriminate particles in bimodal mixtures with high size resolution but the performance on multimodal mixtures is not as good as that of resistive pulse sensing (RPS). We further analyzed the polystyrene particles, SiO2 particles, and biological samples by NTA following the SOPs. The size and concentration measured by NTA differentially varies to those determined by RPS and transmission electron microscopy.

Assessment of Tunable Resistive Pulse Sensing (TRPS) Technology for Particle Size Distribution in Vaccine Formulations - A Comparative Study with Dynamic Light Scattering.

PURPOSE: A comparative assessment was performed to evaluate the potential of particle sizing by an ensemble based conventional dynamic light scattering (DLS) technique and an emerging technology based on tunable resistive pulse sensing (TRPS) using particle by particle approach by evaluating three different types of vaccine formulations representing three case studies and showing the limitation of each technique, instrument variability, sensitivity, and the resolution in mixed population. METHODS: Three types of in-house vaccine formulations- a protein antigen, an outer membrane vesicle and viral particles were simultaneously evaluated by TRPS based Exoid and two DLS instruments-Zetatrac and Zetasizer for particle size distribution, aggregates, and resolution of polydisperse species. RESULTS: The data from first case study show the risk of possible size overestimation and size averaging in polydisperse samples in DLS measurements which can be addressed by the TRPS analysis. It also shows how TRPS may be utilized only to large size antigens due to its limited size range. The second case study highlights the difference in the sensitivities of two DLS instruments working on the same principle. The third case study show that how TRPS can better resolve the large aggregate species compare to DLS in polydisperse samples. CONCLUSION: This analysis shows that TRPS can be used as an orthogonal technique in addition to conventional DLS based methods for more precise and in-depth characterization. Both techniques are efficient in size characterization and produce comparable results, however the choice will depend on the type of formulation and size range to be evaluated.

Integrated In-Plane Nanofluidic Devices for Resistive-Pulse Sensing.

Single-particle (or digital) measurements enhance sensitivity (10- to 100-fold improvement) and uncover heterogeneity within a population (one event in 100 to 10,000). Many biological systems are significantly influenced by rare or infrequent events, and determining what species is present, in what quantity, and the role of that species is critically important to unraveling many questions. To develop these measurement systems, resistive-pulse sensing is used as a label-free, single-particle detection technique and can be combined with a range of functional elements, e.g., mixers, reactors, filters, separators, and pores. Virtually, any two-dimensional layout of the micro- and nanofluidic conduits can be envisioned, designed, and fabricated in the plane of the device. Multiple nanopores in series lead to higher-precision measurements of particle size, shape, and charge, and reactions coupled directly with the particle-size measurements improve temporal response. Moreover, other detection techniques, e.g., fluorescence, are highly compatible with the in-plane format. These integrated in-plane nanofluidic devices expand the toolbox of what is possible with single-particle measurements.

Particle Size and Concentration Measurement Using the Spectradyne nCS1 Instrument.

This protocol describes the use of the Spectradyne nCS1 instrument to measure the particles per mL concentration and size of nanoparticles. The Spectradyne nCS1 is a particle-analyzing instrument that uses microfluidic resistive pulse sensing, rather than optical measurements, to determine the size and concentration of samples. The size and concentration of a sample are determined by measuring the changes in voltage as particles travel through a nano-constriction in the microfluidic cartridge. This method also has the advantage over optical techniques in that measurements are not dependent on the type of material being measured (e.g., refractive index of the sample itself is not needed for accurate analysis), and only microliters (typically 5 µL) of a sample are needed for analysis.

Simultaneous Multipass Resistive-Pulse Sensing and Fluorescence Imaging of Liposomes.

Simultaneous multipass resistive-pulse sensing and fluorescence imaging have been used to correlate the size and fluorescence intensity of individual E. coli lipid liposomes composed of E. coli polar lipid extracts labeled with membrane-bound 3,3-dioctadecyloxacarbocyanine (DiO) fluorescent molecules. Here, a nanopipet serves as a waveguide to direct excitation light to the resistive-pulse sensing zone at the end of the nanopipet tip. Individual DiO-labeled liposomes (>50 nm radius) were multipassed back and forth through the orifices of glass nanopipets' 110-150 nm radius via potential switching to obtain subnanometer sizing precision, while recording the fluorescence intensity of the membrane-bound DiO molecules. Fluorescence was measured as a function of liposome radius and found to be approximately proportional to the total membrane surface area. The observed relationship between liposome size and fluorescence intensity suggests that multivesicle liposomes emit greater fluorescence compared to unilamellar liposomes, consistent with all lipid membranes of the multivesicle liposomes containing DiO. Fluorescent and nonfluorescent liposomes are readily distinguished from each other in the same solution using simultaneous multipass resistive-pulse sensing and fluorescence imaging. A fluorescence "dead zone" of ∼1 µm thickness just outside of the nanopipet orifice was observed during resistive-pulse sensing, resulting in "on/off" fluorescent behavior during liposome multipassing.

Three-Dimensional Homodyne Light Detection (3D-HLD) for High-Throughput Submicron Particle Analysis in (Highly Concentrated) Protein Biopharmaceuticals, Viral Vectors, and LNPs.

During biopharmaceutical development, particle monitoring and characterization are crucial. Notably, particles can be impurities considered as critical quality attribute, or active pharmaceutical ingredient (e.g., viral vectors) or drug delivery system (e.g., lipid nanoparticles) itself. Three-dimensional homodyne light detection (3D-HLD) is a novel technique that can characterize particles in the ∼0.2 µm to 2.0 µm size range. We evaluated 3D-HLD for the analysis of high concentration protein formulations (up to 200 mg/mL), where formulation refractive index and background noise became limiting factors with increasing protein concentration. Sample viscosity however did not impact 3D-HLD results, in contrast to comparative analyses with NTA and MRPS. We also applied 3D-HLD in high-throughput screenings at high protein concentration or of lipid nanoparticle and viral vector formulations, where impurities were analyzed in the presence of a small (<0.2 µm) particulate active pharmaceutical ingredient. 3D-HLD turned out to be in good agreement with or a good complement to other state-of-the-art particle characterization techniques, including BMI, MRPS, and DLS. The main application of 3D-HLD is high-throughput particle analysis at low sample volume. Follow-up investigation of the optimized particle sizing approach and of detection settings could further improve the understanding of the method and potentially increase ease of operation.

θ-Nanopipette for Single-Cell Resistive-Pulse Profiling of DNA Repair Proteins Accompanied by Drug Evaluation.

Single-cell analysis of the DNA repair protein is important but remains unachieved. Exploration of nanopipettte technologies in single-cell electroanalysis has recently seen rapid growth, while the θ-nanopipette represents an emerging technological frontier with its potential largely veiled. Here a θ-nanopipette is first applied for single-cell resistive-pulse sensing (RPS) of the important DNA repair protein O6-alkylguanine DNA alkyltransferase (hAGT). The removal of alkyl mutations by hAGT could restore the damaged aptamer linking with a structural DNA carrier, allowing the selective binding of the aptamer to thrombin with precisely matched size to produce distinct RPS signals when passing through the orifice. Kinetic analysis of hAGT repair was studied. Meanwhile, the device shows the simultaneous on-demand infusion of inhibitors to inactivate the hAGT activity, indicative of its potential in drug screening for enhanced chemotherapy. This work provides a new paradigm for θ-nanopipette-based single-cell RPS of a DNA repair protein accompanied by drug evaluation.

Rapid Detection of Microparticles Using a Microfluidic Resistive Pulse Sensor Based on Bipolar Pulse-Width Multiplexing.

Rapid and accurate analysis of micro/nano bio-objects (e.g., cells, biomolecules) is crucial in clinical diagnostics and drug discovery. While a traditional resistive pulse sensor can provide multiple kinds of information (size, count, surface charge, etc.) about analytes, it has low throughput. We present a unique bipolar pulse-width, multiplexing-based resistive pulse sensor for high-throughput analysis of microparticles. Signal multiplexing is enabled by exposing the central electrode at different locations inside the parallel sensing channels. Together with two common electrodes, the central electrode encodes the electrical signal from each sensing channel, generating specific bipolar template waveforms with different pulse widths. Only one DC source is needed as input, and only one combined electrical output is collected. The combined signal can be demodulated using correlation analysis and a unique iterative cancellation scheme. The accuracy of particle counting and sizing was validated using mixtures of various sized microparticles. Results showed errors of 2.6% and 6.1% in sizing and counting, respectively. We further demonstrated its accuracy for cell analysis using HeLa cells.

Simultaneous Determination of the Size and Shape of Single α-Synuclein Oligomers in Solution.

Soluble oligomers of amyloid-forming proteins are implicated as toxic species in the context of several neurodegenerative diseases. Since the size and shape of these oligomers influence their toxicity, their biophysical characterization is essential for a better understanding of the structure-toxicity relationship. Amyloid oligomers are difficult to characterize by conventional approaches due to their heterogeneity in size and shape, their dynamic aggregation process, and their low abundance. This work demonstrates that resistive pulse measurements using polymer-coated solid-state nanopores enable single-particle-level characterization of the size and shape of individual αSyn oligomers in solution within minutes. A comparison of the resulting size distribution with single-particle analysis by transmission electron microscopy and mass photometry reveals good agreement with superior resolution by nanopore-based characterization. Moreover, nanopore-based analysis has the capability to combine rapid size analysis with an approximation of the oligomer shape. Applying this shape approximation to putatively toxic oligomeric species that range in size from 18 ± 7 aggregated monomers (10S) to 29 ± 10 aggregated monomers (15S) and in concentration from picomolar to nanomolar revealed oligomer shapes that agree well with previous estimates by cryo-EM with the added advantage that nanopore-based analysis occurs rapidly, in solution, and has the potential to become a widely accessible technique.

Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product.

Characterization of particulate impurities such as aggregates is necessary to develop safe and efficacious adeno-associated virus (AAV) drug products. Although aggregation of AAVs can reduce the bioavailability of the virus, only a limited number of studies focus on the analysis of aggregates. We explored three technologies for their capability to characterize AAV monomers and aggregates in the submicron (<1 µm) size range: (i) mass photometry (MP), (ii) asymmetric flow field flow fractionation coupled to a UV-detector (AF4-UV/Vis) and (iii) microfluidic resistive pulse sensing (MRPS). Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids, consistent with sedimentation velocity analytical ultracentrifugation results. MRPS and AF4-UV/Vis enabled the detection and quantification of aggregate content. The developed AF4-UV/Vis method separated AAV monomers from smaller aggregates, thereby enabling a quantification of aggregates <200 nm. MRPS was experienced as a straightforward method to determine the particle concentration and size distribution between 250-2000 nm, provided that the samples do not block the microfluidic cartridge. Overall, within this study we explored the benefits and limitations of the complementary technologies for assessing aggregate content in AAV samples.

Recent Advances in Biomolecular Detection Based on Aptamers and Nanoparticles.

The fast, accurate detection of biomolecules, ranging from nucleic acids and small molecules to proteins and cellular secretions, plays an essential role in various biomedical applications. These include disease diagnostics and prognostics, environmental monitoring, public health, and food safety. Aptamer recognition (DNA or RNA) has gained extensive attention for biomolecular detection due to its high selectivity, affinity, reproducibility, and robustness. Concurrently, biosensing with nanoparticles has been widely used for its high carrier capacity, stability and feasibility of incorporating optical and catalytic activity, and enhanced diffusivity. Biosensors based on aptamers and nanoparticles utilize the combination of their advantages and have become a promising technology for detecting of a wide variety of biomolecules with high sensitivity, reliability, specificity, and detection speed. Via various sensing mechanisms, target biomolecules have been quantified in terms of optical (e.g., colorimetric and fluorometric), magnetic, and electrical signals. In this review, we summarize the recent advances in and compare different aptamer-nanoparticle-based biosensors by nanoparticle types and detection mechanisms. We also share our views on the highlights and challenges of the different nanoparticle-aptamer-based biosensors.

Tunable nanochannel resistive pulse sensing device using a novel multi-module self-assembly.

Nanochannel-based resistive pulse sensing (nano-RPS) system is widely used for the high-sensitive measurement and characterization of nanoscale biological particles and biomolecules due to its high surface to volume ratio. However, the geometric dimensions and surface properties of nanochannel are usually fixed, which limit the detections within particular ranges or types of nanoparticles. In order to improve the flexibility of nano-RPS system, it is of great significance to develop nanochannels with tunable dimensions and surface properties. In this work, we proposed a novel multi-module self-assembly (MS) strategy which allows to shrink the geometric dimensions and tune surface properties of the nanochannels simultaneously. The MS-tuned nano-RPS device exhibits an enhanced signal-to-noise ratio (SNR) for nanoparticle detections after shrunk the geometric dimensions by MS strategy. Meanwhile, by tuning the surface charge, an enhanced resolution for viral particles detection was achieved with the MS-tuned nano-RPS devices by analyzing the variation of pulse width due the tuned surface charge. The proposed MS strategy is versatile for various types of surface materials and can be potentially applied for nanoscale surface reconfiguration in various nanofluidic devices.

Glass Capillary-Based Nanopores for Single Molecule/Single Cell Detection.

A glass capillary-based nanopore (G-nanopore), due to its tapered tip, easy tunability in orifice size, and especially its flexible surface modifications that can be tailored to effectively capture and enhance the ionic current signal of single entities (single molecules, single cells, and single particles), offers a powerful and nanoconfined sensing platform for diverse biological measurements of single cells and single molecules. Compared with other artificial two-dimensional solid-state nanopores, its conical tip and high spatial and temporal resolution characteristics facilitate noninvasive single molecule and selected area (subcellular) single cell detections (e.g., DNA mutations, highly expressed proteins, and small molecule markers that reflect the change characteristics of the tumor), as a small G-nanopore (≤100 nm) does negligible damage to cell functions and cell membrane integrity when inserted through the cell membrane. In this brief review, we summarize the preparation of G-nanopores and discuss the advantages of them as solid-state sensing platforms for single molecule and single cell detection applications as well as for cancer diagnosis and treatment applications. We also describe the current bottlenecks that limit the widespread use of G-nanopores in clinical applications and provide an outlook on future developments. The brief review will provide the reader with a quick survey of this field and facilitate the rapid development of a G-nanopore sensing platform for future tumor diagnosis and personalized medicine based on single-molecule/single-cell bioassay.

Characterization Challenges of Self-Assembled Polymer-SPIONs Nanoparticles: Benefits of Orthogonal Methods.

Size and zeta potential are critical physicochemical properties of nanoparticles (NPs), influencing their biological activity and safety profile. These are essential for further industrial upscale and clinical success. However, the characterization of polydisperse, non-spherical NPs is a challenge for traditional characterization techniques (ex., dynamic light scattering (DLS)). In this paper, superparamagnetic iron oxide nanoparticles (SPIONs) were coated with polyvinyl alcohol (PVAL) exhibiting different terminal groups at their surface, either hydroxyl (OH), carboxyl (COOH) or amino (NH2) end groups. Size, zeta potential and concentration were characterized by orthogonal methods, namely, batch DLS, nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), asymmetric flow field flow fractionation (AF4) coupled to multi-angle light scattering (MALS), UV-Visible and online DLS. Finally, coated SPIONs were incubated with albumin, and size changes were monitored by AF4-MALS-UV-DLS. NTA showed the biggest mean sizes, even though DLS PVAL-COOH SPION graphs presented aggregates in the micrometer range. TRPS detected more NPs in suspension than NTA. Finally, AF4-MALS-UV-DLS could successfully resolve the different sizes of the coated SPION suspensions. The results highlight the importance of combining techniques with different principles for NPs characterization. The advantages and limitations of each method are discussed here.

Nanopore Sensing of DNA-Histone Complexes on Nucleosome Arrays.

The location of nucleosomes in DNA and their structural stability are critical in regulating DNA compaction, site accessibility, and epigenetic gene regulation. Here, we combine the nanopore platform-based fast and label-free single-molecule detection technique with a voltage-dependent force rupture assay to detect distinct structures on nucleosomal arrays and then to induce breakdown of individual nucleosome complexes. Specifically, we demonstrate direct measurement of distinct nucleosome structures present on individual 12-mer arrays. A detailed event analysis showed that nucleosomes are present as a combination of complete and partial structures, during translocation through the pore. By comparing with the voltage-dependent translocation of the mononucleosomes, we find that the partial nucleosomes result from voltage-dependent structural disintegration of nucleosomes. High signal-to-noise detection of heterogeneous levels in translocation of 12-mer array molecules quantifies the heterogeneity and nucleosomal substructure sizes on the arrays. These results facilitate the understanding of electrostatic interactions responsible for the integrity of the nucleosome structure and possible mechanisms of its unraveling by chromatin remodeling enzymes. This study also has potential applications in chromatin profiling.

Translocation of Specific DNA Nanocarrier through an Ultrasmall Nanopipette: Toward Single-Protein-Molecule Detection with Superior Signal-to-Noise Ratio.

The use of functional DNA nanostructures as carriers to ship proteins through solid-state nanopores has recently seen substantial growth in single-protein-molecule detection (SPMD), driven by the potential of this methodology and implementations that it may enable. Ultrasmall nanopores have exhibited obvious advantages in spatiotemporal biological detection due to the appropriate nanoconfined spaces and unique properties. Herein, a 6.8 nm DNA tetrahedron (TDN) with a target-specific DNA aptamer (TDN-apt) was engineered to carry the representative target of acetylcholinesterase (AChE) through an ultrasmall nanopipet with a 30 nm orifice, underpinning the advanced SPMD of AChE with good performance in terms of high selectivity, low detection limit (0.1 fM), and especially superior signal-to-noise ratio (SNR). The kinetic interaction between TDN-apt and AChE was studied and the practical applicability of the as-developed SPMD toward real samples was validated using serum samples from patients with Alzheimer's disease. This work not only presented a feasible SPMD solution toward low-abundance proteins in complex samples and but also was envisioned to inspire more interest in the design and implementation of synergized DNA nanostructure-ultrasmall nanopore systems for future SPMD development.

Single-particle detection of native SARS-CoV-2 virions by microfluidic resistive pulse sensing.

Microfluidic resistive pulse sensing (MRPS) was used to determine the size -distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) based on detecting nearly 30,000 single virions. However, the ultrastructure of SARS-CoV-2 is thoroughly described, but ensemble properties of SARS-CoV-2, e.g., its particle size distribution, are sparsely reported. According to the MRPS results, the size distribution of SARS-CoV-2 follows a log-normal function with a mean value of 85.1 nm, which corresponds to an approximate diameter of the viral envelope. This result also confirms the low number (< 50) of spike proteins on the surface of the virions.

In-Plane, In-Series Nanopores with Circular Cross Sections for Resistive-Pulse Sensing.

Resistive-pulse sensing with solid-state nanopores is a sensitive, label-free technique for analyzing single molecules in solution. To add functionality to resistive-pulse measurements, direct coupling of the nanopores to other pores and nanoscale fluidic elements, e.g., reactors, separators, and filters, in the same device is an important next step. One approach is monolithic fabrication of the fluidic elements in the plane of the substrate, but methods to generate pores with circular cross sections are needed to improve sensing performance with in-plane devices. Here, we report a fabrication method that directly patterns nanopores with circular cross sections in series and in plane with the substrate. A focused ion beam instrument is used to mill a lamella in a nanochannel and, subsequently, bore a nanopore through the lamella. The diameter and geometry of the nanopore are controlled by the current and dose of the ion beam and by the tilt angle and thickness of the lamella. We fabricated devices with vertical and tilted lamellae and nanopores with diameters from 40 to 90 nm in cylindrical and conical geometries. To test device performance, we conducted resistive-pulse measurements of hepatitis B virus capsids. Current pulses from T = 3 capsids (∼31 nm diameter) and T = 4 capsids (∼35 nm diameter) were well resolved and exhibited relative pulse amplitudes (Δi/i) up to 5 times higher than data obtained on nanopores with rectangular cross sections. For smaller pore diameters (<45 nm), which approach the diameters of the capsids, a dramatic increase in the pulse amplitude was observed for both T = 3 and T = 4 capsids. Two and three pores fabricated in series further improved the resolution between the relative pulse amplitude distributions for the T = 3 and T = 4 capsids by up to 2-fold.