Bidirectional linkage of DNA barcodes for the multiplexed mapping of higher-order protein interactions in cells.

Capturing the full complexity of the diverse hierarchical interactions in the protein interactome is challenging. Here we report a DNA-barcoding method for the multiplexed mapping of pairwise and higher-order protein interactions and their dynamics within cells. The method leverages antibodies conjugated with barcoded DNA strands that can bidirectionally hybridize and covalently link to linearize closely spaced interactions within individual 3D protein complexes, encoding and decoding the protein constituents and the interactions among them. By mapping protein interactions in cancer cells and normal cells, we found that tumour cells exhibit a larger diversity and abundance of protein complexes with higher-order interactions. In biopsies of human breast-cancer tissue, the method accurately identified the cancer subtype and revealed that higher-order protein interactions are associated with cancer aggressiveness.

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.

Analytical device miniaturization for the detection of circulating biomarkers.

Diverse (sub)cellular materials are secreted by cells into the systemic circulation at different stages of disease progression. These circulating biomarkers include whole cells, such as circulating tumour cells, subcellular extracellular vesicles and cell-free factors such as DNA, RNA and proteins. The biophysical and biomolecular state of circulating biomarkers carry a rich repertoire of molecular information that can be captured in the form of liquid biopsies for disease detection and monitoring. In this Review, we discuss miniaturized platforms that allow the minimally invasive and rapid detection and analysis of circulating biomarkers, accounting for their differences in size, concentration and molecular composition. We examine differently scaled materials and devices that can enrich, measure and analyse specific circulating biomarkers, outlining their distinct detection challenges. Finally, we highlight emerging opportunities in biomarker and device integration and provide key future milestones for their clinical translation.

A hydrogel-based mechanical metamaterial for the interferometric profiling of extracellular vesicles in patient samples.

The utility of mechanical metamaterials for biomedical applications has seldom been explored. Here we show that a metamaterial that is mechanically responsive to antibody-mediated biorecognition can serve as an optical interferometric mask to molecularly profile extracellular vesicles in ascites fluid from patients with cancer. The metamaterial consists of a hydrogel responsive to temperature and redox activity functionalized with antibodies to surface biomarkers on extracellular vesicles, and is patterned into micrometric squares on a gold-coated glass substrate. Through plasmonic heating, the metamaterial is maintained in a transition state between a relaxed form and a buckled state. Binding of extracellular vesicles from the patient samples to the antibodies on the hydrogel causes it to undergo crosslinking, induced by free radicals generated via the activity of horseradish peroxidase conjugated to the antibodies. Hydrogel crosslinking causes the metamaterial to undergo fast chiral re-organization, inducing amplified changes in its mechanical deformation and diffraction patterns, which are detectable by a smartphone camera. The mechanical metamaterial may find broad utility in the sensitive optical immunodetection of biomolecules.

A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.

PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.

Accessible detection of SARS-CoV-2 through molecular nanostructures and automated microfluidics.

Accurate and accessible nucleic acid diagnostics is critical to reducing the spread of COVID-19 and resuming socioeconomic activities. Here, we present an integrated platform for the direct detection of SARS-CoV-2 RNA targets near patients. Termed electrochemical system integrating reconfigurable enzyme-DNA nanostructures (eSIREN), the technology leverages responsive molecular nanostructures and automated microfluidics to seamlessly transduce target-induced molecular activation into an enhanced electrochemical signal. Through responsive enzyme-DNA nanostructures, the technology establishes a molecular circuitry that directly recognizes specific RNA targets and catalytically enhances signaling; only upon target hybridization, the molecular nanostructures activate to liberate strong enzymatic activity and initiate cascading reactions. Through automated microfluidics, the system coordinates and interfaces the molecular circuitry with embedded electronics; its pressure actuation and liquid-guiding structures improve not only analytical performance but also automated implementation. The developed platform establishes a detection limit of 7 copies of RNA target per µl, operates against the complex biological background of native patient samples, and is completed in <20 min at room temperature. When clinically evaluated, the technology demonstrates accurate detection in extracted RNA samples and direct swab lysates to diagnose COVID-19 patients.

Surfactant-guided spatial assembly of nano-architectures for molecular profiling of extracellular vesicles.

The controlled assembly of nanomaterials into desired architectures presents many opportunities; however, current preparations lack spatial precision and versatility in developing complex nano-architectures. Inspired by the amphiphilic nature of surfactants, we develop a facile approach to guide nanomaterial integration - spatial organization and distribution - in metal-organic frameworks (MOFs). Named surfactant tunable spatial architecture (STAR), the technology leverages the varied interactions of surfactants with nanoparticles and MOF constituents, respectively, to direct nanoparticle arrangement while molding the growing framework. By surfactant matching, the approach achieves not only tunable and precise integration of diverse nanomaterials in different MOF structures, but also fast and aqueous synthesis, in solution and on solid substrates. Employing the approach, we develop a dual-probe STAR that comprises peripheral working probes and central reference probes to achieve differential responsiveness to biomarkers. When applied for the direct profiling of clinical ascites, STAR reveals glycosylation signatures of extracellular vesicles and differentiates cancer patient prognosis.

Collaborative Equilibrium Coupling of Catalytic DNA Nanostructures Enables Programmable Detection of SARS-CoV-2.

Accessible and adaptable nucleic acid diagnostics remains a critical challenge in managing the evolving COVID-19 pandemic. Here, an integrated molecular nanotechnology that enables direct and programmable detection of SARS-CoV-2 RNA targets in native patient specimens is reported. Termed synergistic coupling of responsive equilibrium in enzymatic network (SCREEN), the technology leverages tunable, catalytic molecular nanostructures to establish an interconnected, collaborative architecture. SCREEN mimics the extraordinary organization and functionality of cellular signaling cascades. Through programmable enzyme-DNA nanostructures, SCREEN activates upon interaction with different RNA targets to initiate multi-enzyme catalysis; through system-wide favorable equilibrium shifting, SCREEN directly transduces a single target binding into an amplified electrical signal. To establish collaborative equilibrium coupling in the architecture, a computational model that simulates all reactions to predict overall performance and optimize assay configuration is developed. The developed platform achieves direct and sensitive RNA detection (approaching single-copy detection), fast response (assay reaction is completed within 30 min at room temperature), and robust programmability (across different genetic loci of SARS-CoV-2). When clinically evaluated, the technology demonstrates robust and direct detection in clinical swab lysates to accurately diagnose COVID-19 patients.

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.

Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2.

Despite the importance of nucleic acid testing in managing the COVID-19 pandemic, current detection approaches remain limited due to their high complexity and extensive processing. Here, we describe a molecular nanotechnology that enables direct and sensitive detection of viral RNA targets in native clinical samples. The technology, termed catalytic amplification by transition-state molecular switch (CATCH), leverages DNA-enzyme hybrid complexes to form a molecular switch. By ratiometric tuning of its constituents, the multicomponent molecular switch is prepared in a hyperresponsive state-the transition state-that can be readily activated upon the binding of sparse RNA targets to turn on substantial enzymatic activity. CATCH thus achieves superior performance (~8 RNA copies/µl), direct fluorescence detection that bypasses all steps of PCR (<1 hour at room temperature), and versatile implementation (high-throughput 96-well format and portable microfluidic assay). When applied for clinical COVID-19 diagnostics, CATCH demonstrated direct and accurate detection in minimally processed patient swab samples.

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.

Barcoded DNA nanostructures for the multiplexed profiling of subcellular protein distribution.

Massively parallel DNA sequencing is established, yet high-throughput protein profiling remains challenging. Here, we report a barcoding approach that leverages the combinatorial sequence content and the configurational programmability of DNA nanostructures for high-throughput multiplexed profiling of the subcellular expression and distribution of proteins in whole cells. The barcodes are formed by in situ hybridization of tetrahedral DNA nanostructures and short DNA sequences conjugated with protein-targeting antibodies, and by nanostructure-assisted ligation (either enzymatic or chemical) of the nanostructures and exogenous DNA sequences bound to nanoparticles of different sizes (which cause these localization sequences to differentially distribute across subcellular compartments). Compared with linear DNA barcoding, the nanostructured barcodes enhance the signal by more than 100-fold. By implementing the barcoding approach on a microfluidic device for the analysis of rare patient samples, we show that molecular subtypes of breast cancer can be accurately classified and that subcellular spatial markers of disease aggressiveness can be identified.

Design and synthesis of magnetic nanoparticles for biomedical diagnostics.

Sensitive and quantitative characterization of clinically relevant biomarkers can facilitate disease diagnosis and treatment evaluation. Magnetic nanomaterials and their biosensing strategies have recently received considerable attention. Magnetic signals experience little interference from native biological background as most biological molecules have negligible magnetic susceptibilities and thus appear transparent to external magnetic fields. Because of this unique property, magnetic sensing can be applied to both in vivo deep tissue imaging as well as ex vivo point-of-care diagnostics. To exploit this mode of magnetic detection, new advancements in both magnetic material syntheses and sensing technologies have been made. This review focuses on recent developments of magnetic nanomaterials as image contrast agents and diagnostic sensors. These developments have not only enabled precise control of magnetic nanomaterial properties but also expanded the reach of magnetic detection for biomedical diagnostics.