Method for multiplex detection of exosomes using magnetic beads and molecular beacons

Cancer diagnostic technology for simultaneous detection of exosomal proteins and miRNA using magnetic beads and molecular beacons

This technology provides diagnostic information and a kit for the multiplex detection of protein and miRNA biomarkers in a single sample. It utilizes antibody-conjugated magnetic beads that bind to exosome surface proteins, fluorescently labeled detection antibodies, and molecular beacons that recognize internal exosomal genes. Diagnostic information for various cancers is provided by comparing measured fluorescence intensity against a control group.

Biological samples such as blood, saliva, and urine contain a mixture of proteins and genetic materials from various tissues at varying concentrations, making it difficult to identify the tissue of origin even when biomarkers are detected. Furthermore, varying expression levels across tissues make accurate diagnosis based on single markers difficult, and the presence of serum RNases poses challenges for the stable measurement of free genetic material.

By capturing exosomes with CD9, CD63, and CD81 antibodies and simultaneously reading surface proteins like EpCAM and EGFR alongside internal miRNAs like miR-21 via fluorescence, this technology enables more specific identification of exosomes secreted by diseased tissue. It can be applied to non-invasive liquid biopsy diagnostic kits for various cancers, including prostate cancer, allowing for rapid and accurate multiplex biomarker analysis from small volumes of bodily fluid.

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Key Features:

• A step of preparing a mixture by adding antibody-conjugated magnetic beads, fluorescently labeled surface protein detection antibodies, and molecular beacons to a subject's tissue or biological sample
• A step of obtaining cancer diagnostic information by measuring the fluorescence intensity of the mixture and comparing the subject's fluorescence intensity with that of a control
• Magnetic beads composed of one or more of Fe(III), Mg, Mn, Ni, Zn, and Co, with antibodies against one of CD9, CD63, or CD81 attached to specifically bind to exosome surface proteins
• A multiplex detection kit comprising molecular beacons that detect miRNAs such as miR-21 and miR-574 by labeling both ends of a nucleic acid, which binds complementarily to target genes inside exosomes, with a fluorophore and a quencher

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This invention was developed with support from the Nano-Biofluidic Research Center of the Ministry of Science, ICT and Future Planning.

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