Integrated Antigenic and Nucleic Acid Detection in Single Virions and Extracellular Vesicles with Viral Content.

Virion-mediated outbreaks are imminent and despite rapid responses, continue to cause adverse symptoms and death. Therefore, tunable, sensitive, high-throughput assays are needed to help diagnose future virion-mediated outbreaks. Herein, we developed a tunable in situ assay to selectively enrich virions and extracellular vesicles (EVs) and simultaneously detect antigens and nucleic acids at a single-particle resolution. The Biochip Antigen and RNA Assay (BARA) enhanced sensitivities compared to quantitative reverse-transcription polymerase chain reaction (qRT-PCR), enabling the detection of virions in asymptomatic patients, genetic mutations in single virions, and enabling the continued long-term expression of viral RNA in the EV-enriched subpopulation in the plasma of patients with post-acute sequelae of COVID-19. BARA revealed highly accurate diagnoses of COVID-19 by simultaneously detecting the spike glycoprotein and nucleocapsid-encoding RNA in saliva and nasopharyngeal swab samples. Altogether, the single-particle detection of antigens and viral RNA provides a tunable framework for the diagnosis, monitoring, and mutation screening of current and future outbreaks. This article is protected by copyright. All rights reserved.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

An immunogold single extracellular vesicular RNA and protein (Au SERP) biochip to predict responses to immunotherapy in non-small cell lung cancer patients.

Conventional PD-L1 immunohistochemical tissue biopsies only predict 20%-40% of non-small cell lung cancer (NSCLC) patients that will respond positively to anti-PD-1/PD-L1 immunotherapy. Herein, we present an immunogold biochip to quantify single extracellular vesicular RNA and protein (Au SERP) as a non-invasive alternative. With only 20 µl of purified serum, PD-1/PD-L1 proteins on the surface of extracellular vesicles (EVs) and EV PD-1/PD-L1 messenger RNA (mRNA) cargo were detected at a single-vesicle resolution and exceeded the sensitivities of their bulk-analysis conventional counterparts, ELISA and qRT-PCR, by 1000 times. By testing a cohort of 27 non-responding and 27 responding NSCLC patients, Au SERP indicated that the single-EV mRNA biomarkers surpass the single-EV protein biomarkers in predicting patient responses to immunotherapy. Dual single-EV PD-1/PD-L1 mRNA detection differentiated responders from non-responders with an accuracy of 72.2% and achieved an NSCLC diagnosis accuracy of 93.2%, suggesting the potential for Au SERP to provide enhanced immunotherapy predictions and cancer diagnoses within the clinical setting.

Microfluidic harvesting of breast cancer tumor spheroid-derived extracellular vesicles from immobilized microgels for single-vesicle analysis.

Investigating cellular and vesicular heterogeneity in breast cancer remains a challenge, which encourages the development of controllable in vitro systems that mimic the tumor microenvironment. Although three-dimensional cell culture better recapitulates the heterogeneity observed in tumor growth and extracellular vesicle (EV) biogenesis, the physiological relevance is often contrasted with the control offered by two-dimensional cell culture. Therefore, to challenge this misconception we developed a novel microfluidic system harboring highly tunable three-dimensional EV microbioreactors (EVµBRs) to model micrometastatic EV release in breast cancer while capitalizing on the convenient, low-volume, and sterile interface provided by microfluidics. The diameter and cellular occupancy of the EVµBRs could be precisely tailored to various configurations, supporting the formation of breast cancer tumor spheroids. To immobilize the EVµBRs within a microchannel and facilitate EV extraction, oxygen inhibition in free-radical polymerization was repurposed to rapidly generate two-layer hydrodynamic traps in situ using a digital-micromirror device (DMD)-based ultraviolet (UV) projection system. Breast cancer tumor spheroid-derived EVs were harvested with as little as 20 µL from the microfluidic system and quantified by single-EV immunofluorescence for CD63 and CD81. Despite the low-volume extraction, differences in biomarker expression and coexpression of the tetraspanins on single EVs were observed. Furthermore, the EVµBRs were capable of recapitulating heterogeneity at a cellular and vesicular degree, indicating the utility and robustness of the microfluidic system to investigate physiologically relevant EVs in breast cancer and other disease models.

Chemical Transformation of Astaxanthin from Haematococcus pluvialis Improves Its Antioxidative and Anti-inflammatory Activities.

Astaxanthin is a strong antioxidant, but the effect of esterification on its biological activities remains unclear. Here, we chemically synthesized three forms of astaxanthin (nonesterified (Ast-N), monoesterified (Ast-mE), and diesterified (Ast-dE) forms) using esterified astaxanthin (Ast-E) in natural extract from Haematococcus pluvialis and characterized them by spectrophotometry and high-performance liquid chromatography (HPLC). Additionally, the antioxidant and anti-inflammatory activities of the samples containing three forms of astaxanthin at different ratios were evaluated. The sample containing the maximum level of Ast-mE compared to those of Ast-N and Ast-dE showed the highest antioxidant and anti-inflammatory activities. We also observed the greatest increase in expression of genes related to antioxidant and anti-inflammatory effects in samples containing the highest Ast-mE. These results provide a foundation for in-depth investigation of astaxanthin and other antioxidant molecules, allowing for the development of a practical and cost-effective strategy to improve antioxidant or anti-inflammatory activities of natural extracts that can be used as dietary supplements.