Immunolipoplex nanoparticle biochip containing molecular probes for capture and characterization of extracellular vesicles
The present invention disclosed a method of fabricating an antibody immunolipoplex nanoparticle (Ab-ILN) biochip and antibody tethered lipoplex nanoparticle (Ab-TLN) biochip. The aforementioned antibody-based lipoplex nanoparticle biochip or the related array contains molecular probes and is applied for detecting the presence of a disease or condition in a subject obtaining a body fluid sample by capturing and identifying both membrane protein and intra-vesicular DNA/RNA/proteins of extracellular vesicles (EVs).
1. A method of detecting a presence of leukemia or multiple myeloma in a subject, the method comprising:
(a) providing a body fluid sample comprising extracellular vesicles, wherein the extracellular vesicles comprise:
i. cancer-specific membrane proteins; and
ii. intra-vesicular RNA;
(b) providing an antibody-coated biochip surface;
(c) contacting the body fluid sample with the antibody-coated biochip surface, such that the extracellular vesicles comprising i. cancer-specific membrane proteins; and ii. intra-vesicular RNA are preferentially captured on the antibody-coated biochip surface, thereby obtaining captured extracellular vesicles bound to the antibody-coated biochip surface; and
(d) detecting the intra-vesicular RNA of the captured extracellular vesicles bound to the antibody-coated biochip surface by applying lipoplex nanoparticles comprising molecular beacons to the antibody-coated biochip surface.
2. The method according to claim 1 , wherein the body fluid sample comprises a body fluid selected from the group consisting of: blood and serum.
3. The method according to claim 1 , wherein the body fluid sample is a serum sample.
4. The method according to claim 1 , wherein the body fluid sample is a plasma sample.
5. The method according to claim 1 , further comprising flowing the body fluid sample through a microfluidic device fluidly connected to the antibody-coated biochip surface.
6. The method according to claim 1 , wherein the lipoplex nanoparticles comprising molecular beacons further comprise reagents selected from the group consisting of: quantum dots, magnetic particles, Au nanoparticles, and combinations thereof.
7. The method according to claim 1 , wherein obtaining captured extracellular vesicles bound to the antibody-coated biochip surface further comprises washing away extracellular vesicles not bound to the antibody-coated biochip surface.
8. The method according to claim 1 , further comprising:
(e) characterizing a fluorescence expression of the captured extracellular vesicles bound to the antibody-coated biochip surface and the applied lipoplex nanoparticles comprising molecular beacons.
9. The method according to claim 8 , wherein the fluorescence expression is characterized using an instrument selected from the group consisting of: a total internal reflection fluorescence (TIRF) microscope, a fluorescence microscope, a plate reader, a microplate reader, and a portable fluorescence detector.
10. The method according to claim 9 , wherein the fluorescence expression is characterized using a fluorescence microscope.
11. The method according to claim 1 , wherein the extracellular vesicles comprise exosomes.
12. The method according to claim 1 , wherein the extracellular vesicles are exosomes comprising:
i. CD63 or nucleolin; and
ii. intravesicular mRNA or microRNA.
13. The method according to claim 1 , wherein (d) comprises detecting intravesicular messenger RNA or intravesicular lncRNA.
14. The method according to claim 1 , wherein (d) comprises detecting intravesicular microRNA.
15. The method according to claim 1 , wherein the method is for detecting a presence of leukemia.
16. The method according to claim 1 , wherein the method is for detecting a presence of multiple myeloma.