Molecular nanotags
Inventors
Jones, Jennifer C. • Morales-Kastresana, Aizea • Berzofsky, Jay A. • Welsh, Joshua • Rosner, Ari
Assignees
US Department of Health and Human Services
Publication Number
US-11536719-B2
Publication Date
2022-12-27
Expiration Date
2037-10-23
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Abstract
A molecular nanotag is disclosed that includes a core nanoparticle with a diameter of less than about 100 nm, with an optional shell surrounding the core, and an armor bound to the surface of the core nanoparticle, or if present, to the surface of the shell. The molecular nanotag also includes a functionalized end with a fixed number of binding sites that can selectively bind to a molecular targeting ligand. Any one of, or any combination of, the core, the shell and the armor contribute to fluorescence, light scattering and/or ligand binding properties of the molecular tag that are detectable by microscopy or in a devices that measures intensity or power of fluorescence and light scattering. The light scattering intensity or power of the assembled structure is detectable above the specific level of the reference noise of a device detecting the light scattering intensity or power, its fluorescence intensity or power has sufficient brightness for detection above the limit of detection for the instrument, and ligand specificity is conferred by the ligand binding component. Methods of biomarker and biosignature detection using the molecular tags are also disclosed.
Core Innovation
The invention discloses molecular nanotags that comprise a core nanoparticle less than about 100 nm in diameter, optionally surrounded by a shell, and an armor bound to the core or shell surface. The armor has a first portion attached to the nanoparticle surface and a second portion with a fixed number of functional binding sites that selectively bind to a molecular targeting ligand. Any combination of the core, shell, and armor contribute to fluorescence, light scattering, and/or ligand binding properties that are detectable by microscopy or devices measuring fluorescence and light scattering intensity or power, such as high resolution flow cytometers configured for nanoscale particle detection and sorting.
The problem being solved is the prior inability to detect, analyze, and sort nanoscale particles, particularly extracellular vesicles (EVs) smaller than about 200 nm, with single epitope sensitivity. Existing flow cytometers cannot resolve signals from nanoparticles under approximately 500 nm due to signal loss caused by sample debris and electronic noise. There is a need for reagents and methods enabling functional sorting and enumeration of single molecules on such small particles, overcoming the limitations imposed by current detection tools and instruments.
The disclosed molecular nanotags address this gap by enabling multiparametric detection and sorting of nanoscale particles or vesicles bearing a single epitope. The nanotags provide controlled valency (e.g., monovalence) allowing one-to-one binding to target molecules, enabling counting and characterization of individual molecular components on nanoscale particles, such as surface receptors on EVs. By using cores with high refractive index materials (e.g., quantum nanocrystals or noble metal nanoparticles) and an armor that restricts binding sites, the molecular nanotags generate light scattering and fluorescence signals above instrument noise thresholds, ensuring detectability at the single molecule level.
Claims Coverage
The patent includes one independent claim addressing a method for detecting single target molecules using nanoscale molecular tags in a flow cytometer, supported by multiple dependent claims further specifying structural and functional aspects.
Molecular tag composition and target-specific detection
A nanoscale molecular tag comprising a core nanoparticle with a diameter of 30-80 nm made of a noble metal, an optional shell selected from gold, silver, both, nucleic acids or PEG, and an armor bound to the core or shell surface. The armor reduces nanoparticle valency to one functional binding site with a functionalized end that specifically binds a target molecule via a first binding partner capable of binding to a second binding partner or ligand selected from benzylguanine/SNAP-tag, biotin/streptavidin, oligonucleotide complements, aptamers, receptors and ligands, antibodies and antigens, etc. Any one or combination of core, shell, and armor contribute fluorescence, light scattering, and/or ligand binding properties detectable above instrument noise.
Flow cytometric detection method with multiparametric scattering and fluorescence
Methods analyze samples contacted with molecular nanotags using a flow cytometer configured for resolving small particles, detecting individual nanotags bound to the target by side scatter or forward scatter, fluorescence, or combinations thereof, including use of at least two side scatter channels with trigger and detection roles. Detection includes identifying parallel subthreshold events indicative of particles below the trigger threshold.
Nanoparticle core and armor properties enhancing detection sensitivity
The core nanoparticle comprises nanomaterials with high refractive indices, such as gold or silver, and may include quantum nanocrystals. The core and optional shell possess cumulative optical properties resulting in collected power exceeding device detection limits for one or more scattering wavelengths. The armor includes fluorescent or scattering-contributing polymers such as phosphorothioate DNA and enables fixed monovalent binding sites for ligand-specific targeting.
Target specificity and ligand options
The functional ligand on the molecular nanotag can include tumor-associated antigens, peptide tags, or fluorophores, with examples including prostate specific membrane antigen (PSMA), EGFR, HER-2/neu, EpCAM, CD24, CD133, CD47, PD-L1, GPC-1, Muc-1, and others.
The claims collectively cover a molecular nanotag structure with nanoscale noble metal cores, optional shells, and polymer-based armors providing monovalent target binding, suitable for detection by flow cytometry through fluorescence and light scattering above noise levels. The method claims include detecting single molecules in samples using such nanotags with multiparametric flow cytometric detection, supporting label specificity, detection sensitivity, and multiplexing potential.
Stated Advantages
Enables detection and quantification of single molecules and sub-200 nm nanoscale particles with single epitope sensitivity.
Allows functional sorting and downstream analysis of nanoscale particles preserving biological function.
Facilitates multiplexed detection of a plurality of different targets simultaneously or contemporaneously.
Compatible with high resolution flow cytometers capable of fluorescence and light scattering measurements.
Provides controlled valency in nanotags, permitting accurate enumeration of ligands per target particle.
Supports broader applications including biomedical diagnostics, therapeutics, environmental and biodefense uses.
Documented Applications
Detection, sorting, and analysis of extracellular vesicles (EVs) and submicron biological particles for clinical and research purposes.
Identification and quantification of tumor-associated antigens on EVs, including prostate specific membrane antigen (PSMA) to aid prostate cancer diagnosis and prognosis.
Detection of viral pathogen antigens on submicron particles using specific molecular nanotags.
Profiling biomarkers and biosignatures related to diseases such as cancer, autoimmune diseases, inflammatory diseases, and cardiovascular disorders using labeled EVs.
Monitoring radiation exposure biomarkers and immune system cell antigens in biological samples.
Use in multiplexed flow cytometry to detect multiple distinct ligand targets simultaneously on nanoscale particles.
Application in environmental samples and broad nanoscale biosignature detection for industrial, clinical, and biodefense contexts.
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