We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data-Independent Acquisition.

Exosomes and extracellular vesicles (EV) are increasingly being explored as circulating biomarkers, but their heterogenous composition will likely mandate the development of multiplexed EV technologies. Iteratively multiplexed analyses of near single EVs have been challenging to implement beyond a few colors during spectral sensing. Here we developed a multiplexed analysis of EV technique (MASEV) to interrogate thousands of individual EVs during 5 cycles of multi-channel fluorescence staining for 15 EV biomarkers. Contrary to the common belief, we show that: several markers proposed to be ubiquitous are less prevalent than believed; multiple biomarkers concur in single vesicles but only in small fractions; affinity purification can lead to loss of rare EV subtypes; and deep profiling allows detailed analysis of EV, potentially improving the diagnostic content. These findings establish the potential of MASEV for uncovering fundamental EV biology and heterogeneity and increasing diagnostic specificity. 

Multiplexed analysis of EV reveals specific biomarker composition with diagnostic impact

mRNA vaccines have emerged as a revolutionary tool to generate rapid and precise immune responses against infectious diseases and cancers. Compared with conventional vaccines such as inactivated viruses, viral vectors, protein subunits or DNA-based vaccines, mRNA vaccines stand out owing to multiple advantages, including simplicity of design, fast production, enhanced safety and high efficacy. Nevertheless, efficient and targeted delivery of mRNA molecules remains a significant challenge owing to their inherent instability and susceptibility to degradation. Nanotechnology offers innovative solutions to surmount these obstacles and amplify the potency of mRNA vaccines. This Primer aims to outline a modular approach to developing biomaterials and nanotechnology for mRNA vaccines, with a focus on particle design, formulation evaluation and therapeutic applications. We delve into the underlying mechanisms of nanoparticle-facilitated mRNA protection, cellular uptake, endosomal escape and immune stimulation. We underscore the critical parameters that impact the manufacturing and clinical implementation of nanomaterial-based mRNA vaccines. Finally, we present the current limitations and future perspectives in the advancement of nanotechnology-enhanced mRNA vaccines for broad applications in prophylactic and therapeutic interventions. mRNA vaccines produce rapid and precise immune responses against infectious diseases and cancers. Chen et al. discuss the development of biomaterials and nanotechnology for mRNA vaccines, how these are designed and evaluated and the underlying mechanisms of cellular uptake and immune stimulation. 

Nanotechnology-based mRNA vaccines

Single extracellular vesicle analysis for early cancer detection.

Commonly used methods for extracellular vesicles' enrichment: Implications in downstream analyses and use.

Detecting protein markers in extracellular vesicles (EVs) is becoming a useful tool for basic research and clinical diagnoses. Most EV protein assays, however, require lengthy processes—conjugating affinity ligands onto sensing substrates and affixing EVs with additional labels to maximize signal generation. Here, we present an iPEX (impedance profiling of extracellular vesicles) system, an all-electrical strategy toward fast, multiplexed EV profiling. iPEX adopts one-step electropolymerization to rapidly functionalize sensor electrodes with antibodies; it then detects EV proteins in a label-free manner through impedance spectroscopy. The approach streamlines the entire EV assay, from sensor preparation to signal measurements. We achieved (i) fast immobilization of antibodies (<3 min) per electrode; (ii) high sensitivity (500 EVs/mL) without secondary labeling; and (iii) parallel detection (quadruple) in a single chip. A potential clinical utility was demonstrated by directly analyzing plasma samples from glioblastoma multiforme patients.

https://pubs.acs.org/doi/pdf/10.1021/acscentsci.1c01193

Multielectrode Spectroscopy Enables Rapid and Sensitive Molecular Profiling of Extracellular Vesicles

Electrochemiluminescence (ECL) has an inherently low background and enables precise chemical reactions through electrical control. Here, we report an advanced ECL system, termed ECLipse (ECL in paired signal electrode). We physically separated ECL generation from target detection: These two processes were carried out in isolated chambers and coupled through an electrode. The strategy allowed us to minimize cross-chemical reactions, design electrodes for high ECL signals, and integrate multiple sensors in a chip. As a proof of concept, we implemented an eight-plex ECLipse and applied it to detect host factors in human plasma. ECLipse achieved higher signal-to-noise ratio than conventional ECL assays and was >7000-fold more sensitive than enzyme-linked immunosorbent assay. In a pilot clinical study, we could detect septic conditions by measuring host factors [i.e., interleukin-3 (IL-3), IL-6, and procalcitonin (PCT)]. ECLipse assay further revealed distinct IL-3 and IL-6 patterns in patients with severe acute respiratory syndrome coronavirus 2 infection.

https://www.science.org/doi/pdf/10.1126/sciadv.abq4022?download=true

Electrochemiluminescence in paired signal electrode (ECLipse) enables modular and scalable biosensing

Smartphone-integrated lateral flow assay for robust detection of Δ9-Tetrahydrocannabinol.

Lateral flow assays (LFAs) are widely adopted for fast, on-site molecular diagnostics. Obtaining high-precision assay results, however, remains challenging and often requires a dedicated optical setup to control the imaging environment. Here, we describe quick light normalization exam (qLiNE) that transforms ubiquitous smartphones into a robust LFA reader. qLiNE used a reference card, printed with geometric patterns and color standards, for real-time optical calibration: a photo of an LFA test strip was taken along with the card, and the image was processed using a smartphone app to correct shape distortion, illumination brightness, and color imbalances. This approach yielded consistent optical signal, enabling quantitative molecular analyses under different illumination conditions. We adapted qLiNE to detect cortisol, a known stress hormone, in saliva samples at point-of-use settings. The assay was fast (15 min) and sensitive (detection limit, 0.16 ng/mL). The serial qLiNE assay detected diurnal cycles of cortisol levels as well as stress-induced cortisol increase.

Normalizing the Optical Signal Enables Robust Assays with Lateral Flow Biosensors

Extracellular vesicles (EVs) carry information inherited from parental cells, having significant potential for disease diagnosis. In blood, however, EVs are outnumbered >104-fold by low density lipoproteins (LDLs), yet similar in size and density. These fundamental disadvantages often cause LDL spillover into EV isolates, thus confounding assay results. We hypothesized that EVs can be further separated from LDLs based on electric charge: EVs and LDLs have different lipid composition, which can lead to differential surface charge densities. To test this hypothesis, we modeled and quantified the surface charge of EVs and LDLs, and used the information to optimally separate EVs from LDLs via ion-exchange chromatography. Methods: We built an enhanced dual-mode chromatography (eDMC) device which performed i) size-exclusion to remove particles smaller than EVs and LDLs and ii) cation-exchange in an acidic elution to retain LDLs longer than EVs. The performance of the eDMC, in comparison to size-exclusion only, was evaluated by analyzing the yield and purity of the isolated EVs. Results: By measuring and modeling zeta potentials at different buffer pH, we estimated surface charge densities of EVs (-6.2 mC/m2) and LDLs (-3.6 mC/m2), revealing that EVs are more negatively charged than LDLs. Furthermore, the charge difference between EVs and LDLs was maximal at a weak acidic condition (pH = 6.4). By applying these findings, we optimized eDMC operation to enrich EVs directly from plasma, depleting >99.8% of LPPs within 30 min. Minimizing LDL contamination improved analytical signals in EV molecular assays, including single vesicle imaging, bulk protein measurements, and mRNA detection. Conclusions: These developments will promote the translational value of the dual-mode separation - a fast, equipment-free, and non-biased way for EV isolation from plasma samples.

/pdf/characterization-and-modulation-of-surface-charges-to-gjuh15px.pdf

Characterization and modulation of surface charges to enhance extracellular vesicle isolation in plasma

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