Multi-level and patient-adaptive biomarker detection methods for high sensitivity and specificity disease detection and assays

A method of detecting disease correlates using extracellular vesicles, whereby detection of the disease correlates occurs over a surface of the extracellular vesicles without proximity constraints, comprising:

• obtaining a sample of biofluid;
• using one or more binders to attach to extracellular vesicles correlative to a disease or target organ tissue from the biofluid;
• measuring biomarkers on the attached extracellular vesicles;
• upon the measurement not meeting a predetermined detection threshold, disconnecting the binders from the extracellular vesicles;
• using one or more different binders to attach to the extracellular vesicles;
• and
• repeating the disconnecting and the using one or more different binders until the measurement meets the predetermined detection threshold.

A method for detecting lung disease, whereby disease on interior lung regions exposed to sputum and disease on exterior lung regions exposed to blood are both detected for increased sensitivity, comprising:

• obtaining a sample of sputum from a subject;
• obtaining a sample of blood from the subject;
• performing extracellular vesicle analysis on each of the samples;
• and
• using results from both said samples to detect lung disease.

A method of detecting a cancer-correlative biomarker in a biofluid sample from a subject, whereby segregation ensures approximate single-EV analysis to prevent cross-contamination, comprising:

• accessing a biological sample obtained from the subject comprising extracellular vesicles;
• segregating the extracellular vesicles such that approximately one of the extracellular vesicles enters each of a plurality of chambers;
• lysing the segregated extracellular vesicles within the chambers to release intravesicular biomolecules, wherein the lysing renders a cancer-correlative biomarker detectable that is otherwise undetectable or less detectable in a non-lysed sample;
• and
• detecting the released cancer-correlative biomarker.

A method of detecting a cancer-correlative biomarker in a biological sample from a subject, comprising:

• providing a biological sample comprising extracellular vesicles obtained from said subject;
• segregating the extracellular vesicles such that approximately one of the extracellular vesicles enters each of a plurality of chambers;
• lysing the segregated extracellular vesicles within the chambers to release proteins;
• partially denaturing the released proteins to expose secondary structural features while maintaining detectable primary sequence information;
• and
• analyzing the exposed secondary structural features to identify cancer-correlative motifs.

A system for patient-adaptive analysis of extracellular vesicles for disease detection, comprising:

• supports configured to attach binders for serial capture and release of the extracellular vesicles;
• a detacher configured to disconnect the binders from the supports;
• a quantifier configured for mid-cycle quantification of the extracellular vesicles to determine progression based on a predetermined range;
• a plurality of chambers configured to segregate the extracellular vesicles such that approximately one of the extracellular vesicles enters each of the chambers;
• lysing reagents configured to lyse the segregated extracellular vesicles within the chambers to release intravesicular contents;
• and denaturation reagents configured for gradient-based denaturation of proteins within the released intravesicular contents.

A system for performing extracellular vesicle-based disease detection, comprising:

• binders on supports for serial capture and release of extracellular vesicles;
• a detacher for disconnecting the binders from the supports;
• a quantifier for mid-cycle extracellular vesicle counting;
• a plurality of chambers for segregating the extracellular vesicles such that approximately one of the extracellular vesicles enters each of said chambers;
• lysing reagents for lysing the segregated extracellular vesicles within the chambers to release intravesicular contents;
• and
• denaturation reagents for denaturing proteins within said released intravesicular contents.