Analysis of exosomes and methods of diagnosing cancer

An exosome and cancer technology, applied in the field of exosome analysis and cancer diagnosis

Inactive Publication Date: 2019-03-19
THE HONG KONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods remain challenging for clinical application in terms of throughput and cost

Method used

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  • Analysis of exosomes and methods of diagnosing cancer
  • Analysis of exosomes and methods of diagnosing cancer
  • Analysis of exosomes and methods of diagnosing cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1 - Construction of exosome immune complexes on beads

[0113] Digital ELISAs have been demonstrated in various microfluidic platforms. Exosome solutions are obtained from biological fluids and prepared by ultracentrifugation, ultrafiltration, density gradient separation, and immunoaffinity capture methods. Since antigens are present on the surface of exosomes, they can be recognized by specific antibodies. A pair of antibodies that identify exosomes is built on the beads as an immune complex. Construction of immune complexes on beads as figure 1 shown. Antibodies that recognize biomarkers (such as CD63) on the surface of exosomes and beads (such as Dynabeads TM or agarose beads) binding. The beads were then incubated with the exosome solution. After incubation, collect the beads magnetically or by centrifugation. After thorough washing, the target exosomes bound to the beads were purified from the sample solution. Next, exosomes are detected using a sec...

Embodiment 2

[0114] Example 2-digital quantification of target exosomes

[0115] Digital quantification of immune complex beads bound to target exosomes by specific protein biomarkers. The immune complex-constructed bead solution is flowed into the channel to mix the solution with the flow of another channel's matrix (eg, FDG) and form droplets of the mixture. Instead of using droplets as compartments, microwells fabricated on flat chips can also be used to divide the sample solution. A sample with beads can first be dropped onto the chip and scraped into the wells. Substrate (eg FDG) solution is then added to each compartment. The microwell chip is then sealed on top to isolate each individual space for reactions. Microfluidic workflows such as figure 2 shown. After incubation, the droplets / wells of beads with constructed immune complexes emit a color or a fluorescent or electrochemical signal for detection. Signals can be detected by fluorescence microscopy or electrochemical sens...

Embodiment 3

[0118] Example 3 - Exosome Isolation

[0119] By constructing immune complexes on beads and encapsulating them into droplets, signals from labeled fluoresceins or chemiluminescence can be used as triggers for droplet sorting. Droplets containing target exosomes can be isolated by droplet sorting techniques including electrical sorting, mechanical sorting, or acoustic sorting. image 3 Schematic illustration of the isolation of fluorescent exosomes with desired information.

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Abstract

The invention relates to quantification, isolation, and characterization of exosomes. In some embodiments, exosomes can be quantified by contacting a sample with a capture bead comprising a bead and afirst binding agent, and a second binding agent. The first binding agent binds to a first biomolecule in the exosomes to produce a first complex and the second binding agent binds to a second biomolecule in the exosomes of the first complex to produce a second complex. The first complexes and the second complexes are quantified based on a detectable signal conjugated to the second binding agent.A microwell or a droplet generation is utilized to quantify the first complexes and the second complexes. In optional embodiments, quantification of the exosomes is used to diagnose a cancer in a subject. In such methods, the first and the second binding agents bind to cancer biomarkers present in the exosomes.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 606687, filed October 5, 2017, the disclosure of which is incorporated by reference in its entirety, including any figures, tables or drawings. Background technique [0003] Exosomes have been positioned as effective biomarkers for cancer diagnosis. Exosomes are heterogeneous membranous particles with a diameter of 30–150 nm secreted from cells by plasma membrane fusion of multivesicular bodies (MVBs). Exosomes shed from tumor tissues carry many biomarkers, such as transmembrane and cytoplasmic proteins (CD9, CD63, CD81, etc.), lipids, DNA and microRNA. Special proteins such as Glypican-1 (GPC1), fibronectin (FN), prostate-specific membrane antigen (PSMA) and functional nucleic acids such as microRNA-145 have clinical significance in early cancer diagnosis. Furthermore, exosomes are widely present in human biological fluids, such a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/543G01N33/574
CPCG01N33/535G01N33/54326G01N33/5436G01N33/574G01N33/57488G01N2333/4722G16H50/20
Inventor 姚舒懷许潇楠刘春辰胡宇郑磊
Owner THE HONG KONG UNIV OF SCI & TECH
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