ImmunoLipoplex Nanoparticle Biochip Containing Molecular Probes for Capture and Characterization of Extracellular Vesicles

A method of fabricating an antibody immunolipoplex nanoparticle (Ab-ILN) biochip, said method comprising:

• (1). Providing a substrate which comprises glass, silicon wafer, polymer and ceramics;
• (2). Providing a silane which comprises trimethoxy [3-(oxiranylmethoxy)propyl] silane, triethoxy [3-(oxiranylmethoxy)propyl] silane, trimethoxy(methyl) silane, trimethoxy(propyl) silane and triethoxy(propyl) silane;
• (3). Forming a self-assembly monolayer with the silane on surfaces of the substrate, wherein the self-assembly monolayer containing lipoplex nanoparticles;
• (6). Binding a protein onto the surfaces to form a active site, wherein the protein comprises Protein A;
• and
• (7). Performing a reaction with antibodies to have an antibody conjugation on the active site, so as to form the antibody immunolipoplex nanoparticle (Ab-ILN) biochip, wherein the antibody conjugation is a ligands-receptors interaction which comprises avidin-biotin, digoxigenin-anti-Dig, fluorescein-anti-FITC and hapten linkages of antibody molecules.

A method of fabricating an antibody tethered lipoplex nanoparticle (Ab-TLN) biochip, said method comprising:

• (1). Providing a substrate which comprises glass, silicon wafer, polymer and ceramics;
• (2). Coating gold onto the substrate to form a gold layer on surfaces of the substrate;
• (3). Providing a composition which comprises a tethering molecule, a linker which comprises a biotin-conjugated thiol molecule with ethylene oxide unites and a lateral spacer;
• (4). Forming a self-assembly monolayer with the composition on the gold layer, wherein the self-assembly monolayer containing tethered liposomal nanoparticles;
• (5). Adding an avidin which comprises neutravidin and streptavidin to the self-assembly monolayer, wherein the avidin reacts with the linker to form an avidin-modified surface;
• and
• (6). Performing a reaction with antibodies to have an antibody conjugation on the avidin-modified surface, so as to form the antibody tethered lipoplex nanoparticle (Ab-TLN) biochip, wherein the antibody conjugation is a ligands-receptors interaction which comprises complementary DNA/RNA, avidin-biotin, digoxigenin-anti-Dig, fluorescein-anti-FITC and hapten linkages of antibody molecules.

A method of detecting the presence of a disease or condition in a subject, said method comprising:

• (1). Providing a body fluid sample containing membrane proteins and intra-vesicular DNA/RNA/proteins of extracellular vesicles;
• (2). Providing an antibody-based biochip which comprises an antibody immunolipoplex nanoparticle (Ab-ILN) biochip and an antibody tethered lipoplex nanoparticle (Ab-TLN) biochip;
• (3). Contacting the body fluid sample with the antibody-based biochip, wherein the membrane proteins and the intra-vesicular DNA/RNA/proteins of extracellular vesicles are captured by molecular probes on the antibody-based biochip, wherein the molecular probes comprise molecular beacons and aptamer beacons;
• and
• (4). Characterizing the membrane proteins and the intra-vesicular DNA/RNA/proteins of extracellular vesicles by using instrument which comprises TIRF microscope, fluorescence microscope, plate reader, microplate reader and portable fluorescence detector, so as to detect the presence of a disease or condition in the subject.