Paper-based loop-mediated isothermal amplification and CRISPR integrated platform for on-site nucleic acid testing of pathogens.

We report the development and initial validation of a paper-based nucleic acid testing platform that integrates Loop-mediated isothermal amplification (LAMP) with clustered regularly interspaced short palindromic repeats (CRISPR) technology, referred to as PLACID (Paper-based LAMP-CRISPR Integrated Diagnostics). LAMP eliminates the need for thermal cycling, resulting in simplified instrumentation, and the CRISPR-associated protein (Cas 12a) system eliminates false positive signals from LAMP products, resulting in highly selective and sensitive assays. We optimized the assay to perform both amplification and detection entirely on paper, eliminating the need for complex fluid handling steps and lateral flow assay transfers. Additionally, we engineered a smartphone-operated system that includes a low-powered, non-contact IR heating chamber to actuate paper-based LAMP and CRISPR reactions and enable the detection of fluorescent signals from the paper. The platform demonstrates high specificity and sensitivity in detecting nucleic acid targets with a limit of detection of 50 copies/µL. We integrate an equipment-free sample preparation separation technology designed to streamline the preparation of crude samples prior to nucleic acid testing. The practical utility of our platform is demonstrated by the successful detection of spiked SARS-CoV-2 RNA fragments in saliva, E. Coli in soil, and pathogenic E. Coli in clinically fecal samples of infected patients. Furthermore, we demonstrate that the paper-based LAMP CRISPR chips employed in our assays possess a shelf life of several weeks, establishing them as viable candidates for on-site diagnostics.

High-Resolution Single Particle Zeta Potential Characterisation of Biological Nanoparticles using Tunable Resistive Pulse Sensing.

Physicochemical properties of nanoparticles, such as size, shape, surface charge, density, and porosity play a central role in biological interactions and hence accurate determination of these characteristics is of utmost importance. Here we propose tunable resistive pulse sensing for simultaneous size and surface charge measurements on a particle-by-particle basis, enabling the analysis of a wide spectrum of nanoparticles and their mixtures. Existing methodologies for measuring zeta potential of nanoparticles using resistive pulse sensing are significantly improved by including convection into the theoretical model. The efficacy of this methodology is demonstrated for a range of biological case studies, including measurements of mixed anionic, cationic liposomes, extracellular vesicles in plasma, and in situ time study of DNA immobilisation on the surface of magnetic nanoparticles. The high-resolution single particle size and zeta potential characterisation will provide a better understanding of nano-bio interactions, positively impacting nanomedicine development and their regulatory approval.

A standardized method to determine the concentration of extracellular vesicles using tunable resistive pulse sensing.

BACKGROUND: Understanding the pathogenic role of extracellular vesicles (EVs) in disease and their potential diagnostic and therapeutic utility is extremely reliant on in-depth quantification, measurement and identification of EV sub-populations. Quantification of EVs has presented several challenges, predominantly due to the small size of vesicles such as exosomes and the availability of various technologies to measure nanosized particles, each technology having its own limitations. MATERIALS AND METHODS: A standardized methodology to measure the concentration of extracellular vesicles (EVs) has been developed and tested. The method is based on measuring the EV concentration as a function of a defined size range. Blood plasma EVs are isolated and purified using size exclusion columns (qEV) and consecutively measured with tunable resistive pulse sensing (TRPS). Six independent research groups measured liposome and EV samples with the aim to evaluate the developed methodology. Each group measured identical samples using up to 5 nanopores with 3 repeat measurements per pore. Descriptive statistics and unsupervised multivariate data analysis with principal component analysis (PCA) were used to evaluate reproducibility across the groups and to explore and visualise possible patterns and outliers in EV and liposome data sets. RESULTS: PCA revealed good reproducibility within and between laboratories, with few minor outlying samples. Measured mean liposome (not filtered with qEV) and EV (filtered with qEV) concentrations had coefficients of variance of 23.9% and 52.5%, respectively. The increased variance of the EV concentration measurements could be attributed to the use of qEVs and the polydisperse nature of EVs. CONCLUSION: The results of this study demonstrate the feasibility of this standardized methodology to facilitate comparable and reproducible EV concentration measurements.

High resolution particle characterization to expedite development and regulatory acceptance of nanomedicines.

The pharmaceutical industry as well as European and US governing agencies have indicated the need for more accurate, high resolution, characterization of complex drug materials, nanomedicines, to facilitate their development and eventual approval. In particular, accurately measuring the size, zeta-potential, and concentration of nanomedicines is desired. Herein we demonstrate the comprehensive and high resolution analysis capabilities of tunable resistive pulse sensing (TRPS) on the most widely approved nanomedicines to-date, liposomal particles. The number-based size distribution, concentration and volume fraction of liposomes formed by extrusion through a 100 nm or 200 nm Nucleopore filter membrane are shown as well as how freeze-thaw aggregation changes individual liposomes and the overall size distribution. In addition, the simultaneous size and zeta-potential analysis capabilities of TRPS is used to characterize the homogeneity and difference between liposomes made with and without the addition of PEGylated phospholipids.

Monitoring aptamer-protein interactions using tunable resistive pulse sensing.

Aptamers are short single-stranded pieces of DNA or RNA capable of binding to analytes with specificity and high affinity. Due to their comparable selectivity, stability, and cost, over the last two decades, aptamers have started to challenge antibodies in their use on many technology platforms. The binding event often leads to changes in the aptamer's secondary and tertiary structure; monitoring such changes has led to the creation of many new analytical sensors. Here, we demonstrate the use of a tunable resistive pulse sensing (TRPS) technology to monitor the interaction between several DNA aptamers and their target, thrombin. We immobilized the aptamers onto the surface of superparamagnetic beads, prior to their incubation with the thrombin protein. The protein binding to the aptamer caused a conformational change resulting in the shielding of the polyanion backbone; this was monitored by a change in the translocation time and pulse frequency of the particles traversing the pore. This signal was sensitive enough to allow the tagless detection of thrombin down to nanomolar levels. We further demonstrate the power of TRPS by performing real time detection and characterization of the aptamer-target interaction and measuring the association rates of the thrombin protein to the aptamer sequences.

Characterization of a nanoparticulate drug delivery system using scanning ion occlusion sensing.

PURPOSE: To explore the application of scanning ion occlusion sensing (SIOS) as a novel technology for characterization of nanoparticles. METHODS: Liposomes were employed as model nanoparticles. The size distribution of the liposomes was measured by both SIOS and dynamic light scattering (DLS). Particle number concentration was determined based on particle translocation rate. The ability of SIOS and DLS to resolve bimodal samples was evaluated by measuring a mixture of 217 and 355 nm standard nanoparticles. Opsonization of liposomes by plasma was also studied using SIOS. RESULTS: SIOS was shown to measure the size of different liposomes with higher sensitivity than DLS and it requires a smaller sample volume than DLS. With appropriate calibration, SIOS could be used to determine particle number concentrations. In comparison, SIOS analysis of the mixture showed accurate resolution of the population as a bimodal distribution over a wide range of number ratios of the particles. SIOS could detect plasma opsonization of liposomes by demonstrating a increase in particle size and also changes in the particle translocation rate. CONCLUSION: SIOS is a useful technology for nanoparticle characterization. It shows some advantages over DLS and is clearly a useful tool for the study of nanoparticle drug delivery systems.