Multiplexed RNA profiling by regenerative catalysis enables blood-based subtyping of brain tumors.

Current technologies to subtype glioblastoma (GBM), the most lethal brain tumor, require highly invasive brain biopsies. Here, we develop a dedicated analytical platform to achieve direct and multiplexed profiling of circulating RNAs in extracellular vesicles for blood-based GBM characterization. The technology, termed 'enzyme ZIF-8 complexes for regenerative and catalytic digital detection of RNA' (EZ-READ), leverages an RNA-responsive transducer to regeneratively convert and catalytically enhance signals from rare RNA targets. Each transducer comprises hybrid complexes - protein enzymes encapsulated within metal organic frameworks - to configure strong catalytic activity and robust protection. Upon target RNA hybridization, the transducer activates directly to liberate catalytic complexes, in a target-recyclable manner; when partitioned within a microfluidic device, these complexes can individually catalyze strong chemifluorescence reactions for digital RNA quantification. The EZ-READ platform thus enables programmable and reliable RNA detection, across different-sized RNA subtypes (miRNA and mRNA), directly in sample lysates. When clinically evaluated, the EZ-READ platform established composite signatures for accurate blood-based GBM diagnosis and subtyping.

Extracellular vesicle drug occupancy enables real-time monitoring of targeted cancer therapy.

Current technologies to measure drug-target interactions require complex processing and invasive tissue biopsies, limiting their clinical utility for cancer treatment monitoring. Here we develop an analytical platform that leverages circulating extracellular vesicles (EVs) for activity-based assessment of tumour-specific drug-target interactions in patient blood samples. The technology, termed extracellular vesicle monitoring of small-molecule chemical occupancy and protein expression (ExoSCOPE), utilizes bio-orthogonal probe amplification and spatial patterning of molecular reactions within matched plasmonic nanoring resonators to achieve in situ analysis of EV drug dynamics. It measures changes in drug occupancy and protein composition in molecular subpopulations of EVs. When used to monitor various targeted therapies, the ExoSCOPE revealed EV signatures that closely reflected cellular treatment efficacy. We further applied the technology for clinical cancer diagnostics and treatment monitoring. Using a small volume of blood, the ExoSCOPE accurately classified disease status and rapidly distinguished between targeted treatment outcomes, within 24 h after treatment initiation.

Head-to-head comparison of amplified plasmonic exosome Aß42 platform and single-molecule array immunoassay in a memory clinic cohort.

BACKGROUND AND PURPOSE: Various blood biomarkers reflecting brain amyloid-ß (Aß) load have recently been proposed with promising results. However, to date, no comparative study amongst blood biomarkers has been reported. Our objective was to examine the diagnostic performance and cost effectiveness of three blood biomarkers on the same cohort. METHODS: Using the same cohort (n = 68), the performances of the single-molecule array (Simoa) Aß40, Aß42, Aß42/Aß40 and the amplified plasmonic exosome (APEX) Aß42 blood biomarkers were compared using amyloid positron emission tomography (PET) as the reference standard. The extent to which these blood tests can reduce the recruitment cost of clinical trials was also determined by identifying amyloid positive (Aß+) participants. RESULTS: Compared to Simoa biomarkers, APEX-Aß42 showed significantly higher correlations with amyloid PET retention values and excellent diagnostic performance (sensitivity 100%, specificity 93.3%, area under the curve 0.995). When utilized for clinical trial recruitment, our simulation showed that pre-screening with blood biomarkers followed by a confirmatory amyloid PET imaging would roughly half the cost (56.8% reduction for APEX-Aß42 and 48.6% for Simoa-Aß42/Aß40) compared to the situation where only PET imaging is used. Moreover, with 100% sensitivity, APEX-Aß42 pre-screening does not increase the required number of initial participants. CONCLUSIONS: With its high diagnostic performance, APEX is an ideal candidate for Aß+ subject identification, monitoring and primary care screening, and could efficiently enrich clinical trials with Aß+ participants whilst halving recruitment costs.

Exosome-templated nanoplasmonics for multiparametric molecular profiling.

Exosomes are nanoscale vesicles distinguished by characteristic biophysical and biomolecular features; current analytical approaches, however, remain univariate. Here, we develop a dedicated platform for multiparametric exosome analysis-through simultaneous biophysical and biomolecular evaluation of the same vesicles-directly in clinical biofluids. Termed templated plasmonics for exosomes, the technology leverages in situ growth of gold nanoshells on vesicles to achieve multiselectivity. For biophysical selectivity, the nanoshell formation is templated by and tuned to distinguish exosome dimensions. For biomolecular selectivity, the nanoshell plasmonics locally quenches fluorescent probes only if they are target-bound on the same vesicle. The technology thus achieves multiplexed analysis of diverse exosomal biomarkers (e.g., proteins and microRNAs) but remains unresponsive to nonvesicle biomarkers. When implemented on a microfluidic, smartphone-based sensor, the platform is rapid, sensitive, and wash-free. It not only distinguished biomarker organizational states in native clinical samples but also showed that the exosomal subpopulation could more accurately differentiate patient prognosis.

Biomarker Organization in Circulating Extracellular Vesicles: New Applications in Detecting Neurodegenerative Diseases.

Neurodegenerative diseases are heterogeneous disorders characterized by a progressive loss of function and/or death of nerve cells, leading to severe cognitive and functional decline. Due to the complex pathology, early detection and intervention are critical to the development of successful treatments; however, current diagnostic approaches are limited to subjective, late-stage clinical findings. Extracellular vesicles (EVs) have recently emerged as a promising circulating biomarker for neurodegenerative diseases. Actively released by diverse cells, EVs are nanoscale membrane vesicles. They abound in blood, readily cross the blood-brain barrier, and carry diverse molecular cargoes in different organizational states: these molecular cargoes are inherited from the parent cells or bound to the EV membrane through surface associations. Specifically, EVs have been found to be associated with several important pathogenic proteins of neurodegenerative diseases, and their involvement could alter disease progression. This article provides an overview of EVs as circulating biomarkers of neurodegenerative diseases and introduces new technological advances to characterize the biophysical properties of EV-associated biomarkers for accurate, blood-based detection of neurodegenerative diseases.

New Sensors for Extracellular Vesicles: Insights on Constituent and Associated Biomarkers.

Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles released by cells into the circulation. As an emerging class of circulating biomarkers, EVs contain a trove of molecular information and play important roles in mediating intercellular communication. These EV molecular cargoes are differentially organized in the vesicles; they could be inherited from the parent cells or bound to the EV membrane through surface interactions. While the inherited constituents could serve as cell surrogate biomarkers, extravesicular association could reflect structural states of the bound molecules, revealing distinct subpopulations with different biophysical and/or biochemical properties. Despite the clinical potential of EVs and their diverse contents, conventional sensing technologies have limited compatibility to reveal nanoscale EV features. Complementary analytical platforms are being developed to address these technical challenges and expand the biomedical applications of EVs, to establish novel correlations and empower new diagnostics. This article provides a perspective on recent developments in sensor technologies to profile the diverse contents-different molecular types, quantities, and organizational states-in extracellular vesicles.

Subtyping of circulating exosome-bound amyloid ß reflects brain plaque deposition.

Despite intense interests in developing blood measurements of Alzheimer's disease (AD), the progress has been confounded by limited sensitivity and poor correlation to brain pathology. Here, we present a dedicated analytical platform for measuring different populations of circulating amyloid ß (Aß) proteins - exosome-bound vs. unbound - directly from blood. The technology, termed amplified plasmonic exosome (APEX), leverages in situ enzymatic conversion of localized optical deposits and double-layered plasmonic nanostructures to enable sensitive, multiplexed population analysis. It demonstrates superior sensitivity (~200 exosomes), and enables diverse target co-localization in exosomes. Employing the platform, we find that prefibrillar Aß aggregates preferentially bind with exosomes. We thus define a population of Aß as exosome-bound (Aß42+ CD63+) and measure its abundance directly from AD and control blood samples. As compared to the unbound or total circulating Aß, the exosome-bound Aß measurement could better reflect PET imaging of brain amyloid plaques and differentiate various clinical groups.