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Extracellular vesicles (EVs) function as potent mediators of intercellular communication for many in vivo processes, contributing to both health and disease related conditions. Given their biological origins and diverse functionality from correspondingly unique “cargo” compositions, both endogenous and modified EVs are garnering attention as promising therapeutic modalities and vehicles for targeted therapeutic delivery applications. Their diversity in composition, however, has revealed a significant need for more comprehensive analytical-based characterization methods, and manufacturing processes that are consistent and scalable. In this review, we explore the dynamic landscape of EV research and development efforts, ranging from novel isolation approaches, to their analytical assessment through novel characterization techniques, and to their production by industrial-scale manufacturing process considerations. Expanding the horizon of these topics to EVs for in-human applications, we underscore the need for stringent development and adherence to Good Manufacturing Practice (GMP) guidelines. Wherein, the intricate interplay of raw materials, production in bioreactors, and isolation practices, along with analytical assessments compliant with the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines, in conjunction with reference standard materials, collectively pave the way for standardized and consistent GMP production processes.
RESUMO
BackgroundCOVID-19 pandemic interrupted routine care for individuals living with HIV, putting them at risk of becoming virologically unsuppressed and ill. Often they are at high risk for exposure to SARS-CoV-2 infection and severe disease once infected. For this population, it is urgent to closely monitor HIV plasma viral load (VL) and screen for SARS-COV-2 infection. MethodWe have developed a non-proprietary method to isolate RNA from plasma, nasal secretions (NS), or both. HIV, SARS-CoV-2, and human RP targets in extracted RNA are then RT-qPCR to estimate the VL and classify HIV/SARS-CoV-2 status (i.e., HIV as VL failure or suppressed; SARS-CoV-2 as positive, presumptive positive, negative, or indeterminate). We evaluated this workflow on 133 clinical specimens: 40 plasma specimens (30 HIV-seropositive), 67 NS specimens (31 SARS-CoV-2-positive), and 26 pooled plasma/NS specimens (26 HIV-positive with 10 SARS-CoV-2-positive), and compared the results obtained using the in-house extraction to those using a commercial extraction kit. ResultsIn-house extraction had a detection limit of 200-copies/mL for HIV and 100-copies/mL for SARS-CoV-2. In-house and commercial methods yielded positively correlated HIV VL (R2: 0.98 for contrived samples; 0.81 for seropositive plasma). SARS-CoV-2 detection had 100% concordant classifications in contrived samples, and in clinical NS extracted by in-house method, excluding indeterminate results, was 95% concordant (25 positives, 6 presumptive positives, and 31 negatives) to those using the commercial method. Analysis of pooled plasma/NS showed R2 of 0.91 (contrived samples) and 0.71 (clinical specimens) for HIV VL correlations obtained by both extraction methods, while SARS-CoV-2 detection showed 100% concordance in contrived and clinical specimens. InterpretationOur low-cost workflow for molecular testing of HIV and SARS-CoV-2 could serve as an alternative to current standard assays for laboratories in low-resource settings.
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Biosensors are analytical devices for biomolecule detection that compromise three essential components: recognition moiety, transducer, and signal processor. The sensor converts biomolecule recognition to detectable signals, which has been applied in diverse fields such as clinical monitoring, in vitro diagnostics, food industry etc. Based on signal transduction mechanisms, biosensors can be categorized into three major types: optical biosensors, electrochemical biosensors, and mass-based biosensors. Recently, the need for faster, more sensitive detection of biomolecules has compeled researchers to develop various sensing techniques. In this review, the basic structure and sensing principles of biosensors are introduced. Additionally, the review discusses multiple recent works about nucleic acid and exosome sensing.
