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

Abstract

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).

What is claimed is: 
     
         1 . 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). Coating gold onto the substrate to form a gold layer on surfaces of the substrate;   (3). Providing a composition which comprises a lipidic anchor 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 lipoplex 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;   (6). Binding a protein onto the avidin-modified surface to form an active site, wherein the protein comprises biotin-conjugated 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.   
     
     
         2 . The method according to  claim 1 , wherein the lipidic anchor molecule comprises 20-tetradecyloxy-3,6,9,12,15,18,22-heptaoxahexatricontane-1-thiol. 
     
     
         3 . The method according to  claim 1 , wherein the lateral spacer comprises 2-mercaptoethanol, 6-mercaptohexanol and 16-mercaptohexadecanoic acid. 
     
     
         4 . The method according to  claim 1 , wherein the antibodies being receptors which comprise moieties for binding to target extracellular vesicles, microvesicles, exosomes and circulating cell-free particles which is selected from one of the groups consisting of viruses, bacteria and antigens. 
     
     
         5 . The method according to  claim 1 , wherein the lipoplex nanoparticles are lyophilized lipoplex nanoparticles. 
     
     
         6 . The method according to  claim 1 , wherein the lipoplex nanoparticles further comprising reagents which comprise molecular beacons, quantum dots, messenger RNA, microRNA, lncRNA, genomic DNA, circulating tumor DNA, drug, DNA/RNA, magnetic particles, Au nanoparticles and proteins. 
     
     
         7 . The method according to  claim 1 , wherein the antibody immunolipoplex nanoparticle (Ab-ILN) biochip is applied for finger prick capillary blood assay. 
     
     
         8 . 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.   
     
     
         9 . The method according to  claim 8 , wherein the antibodies being receptors which comprise moieties for binding to target extracellular vesicles, microvesicles, exosomes and circulating cell-free particles which is selected from one of the groups consisting of viruses, bacteria and antigens. 
     
     
         10 . The method according to  claim 8 , wherein the lipoplex nanoparticles are lyophilized lipoplex nanoparticles. 
     
     
         11 . The method according to  claim 8 , wherein the lipoplex nanoparticles further comprise molecular beacons, quantum dots, messenger RNA, microRNA, lncRNA, genomic DNA, circulating tumor DNA, drug, DNA/RNA, magnetic particles, Au nanoparticles and proteins. 
     
     
         12 . The method according to  claim 8 , wherein the antibody immunolipoplex nanoparticle (Ab-ILN) biochip is applied for finger prick capillary blood assay. 
     
     
         13 . 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.   
     
     
         14 . The method according to  claim 13 , wherein the tethering molecules comprise 20-tetradecyloxy-3,6,9,12,15,18,22-heptaoxahexatricontane-1-thiol,29-hexadecyloxy-3,6,9,12,15,18,21,24,27,31-decaoxaheptatetracontan-1-thiol, 20-(Z-octadec-9-enyloxy)-3,6,9,12,15,18,22-heptaoxatetracont-31-ene-1-thiol, and thiolipids with ethylene oxide units. 
     
     
         15 . The method according to  claim 13 , wherein the lateral spacer comprises 2-mercaptoethanol, 6-mercaptohexanol and 16-mercaptohexadecanoic acid. 
     
     
         16 . The method according to  claim 13 , wherein the tethered liposomal nanoparticles being formed from a lipid mixture which comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3β-[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol), 1,2-di-O-octadecenyl-3-dimethylammonium propane (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), L-α-phosphatidylcholine (EggPC, SoyPC), Cholesterol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), saturated fatty acid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG), and PEG phospholipids. 
     
     
         17 . The method according to  claim 13 , wherein the antibodies being receptors which comprise moieties for binding to target extracellular vesicles, microvesicles, exosomes and circulating cell-free particles which are selected from one of the groups consisting of viruses, bacteria and antigens. 
     
     
         18 . The method according to  claim 13 , wherein the tethered liposomal nanoparticles are lyophilized liposomal nanoparticles. 
     
     
         19 . The method according to  claim 13 , wherein the tethered liposomal nanoparticles further comprise molecular beacons, quantum dots, messenger RNA, microRNA, lncRNA, genomic DNA, circulating tumor DNA, drug, DNA/RNA, magnetic particles, Au nanoparticles and proteins. 
     
     
         20 . The method according to  claim 13 , wherein the antibody tethered lipoplex nanoparticle (Ab-TLN) biochip being applied for finger prick capillary blood assay. 
     
     
         21 . 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.   
     
     
         22 . The method according to  claim 21 , wherein the body fluid sample comprises blood, serum, urine, sputum and saliva from the subject. 
     
     
         23 . The method according to  claim 21 , wherein the antibody-based biochip is connected to a microfluidic setup, so a cell culture medium or the body fluid sample is able to be brought onto the antibody-based biochip. 
     
     
         24 . The method according to  claim 21 , wherein surfaces of the antibody-based biochip having ligands which comprises antibody molecules, peptides, carbohydrates, DNA and RNA, and wherein said ligands being for detecting specific extracellular vesicles surface biomarkers. 
     
     
         25 . The method according to  claim 21 , wherein the antibody immunolipoplex nanoparticle (Ab-ILN) biochip having lipoplex nanoparticles which comprise reagents being selected from one of the groups or the combinations consisting of molecular beacons, quantum dots, messenger RNA, microRNA, lncRNA, genomic DNA, circulating tumor DNA, drug, DNA/RNA, magnetic particles, Au nanoparticles and proteins. 
     
     
         26 . The method according to  claim 21 , wherein the tethered lipoplex nanoparticle (Ab-TLN) biochip having tethered liposomal nanoparticles which comprise reagents being selected from one of the groups or the combinations consisting of molecular beacons, quantum dots, messenger RNA, microRNA, lncRNA, genomic DNA, circulating tumor DNA, drug, DNA/RNA, magnetic particles, Au nanoparticles and proteins. 
     
     
         27 . The method according to  claim 21 , wherein the disease or condition comprises a cancer, an antigen and extracellular vesicles derived from a cancer lymphomas (Hodgkins and non-Hodgkins), B cell lymphoma, T cell lymphoma, myeloid leukemia, leukemias, mycosis fungoides, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas, AIDS related lymphomas or sarcomas, metastatic cancers, bladder cancer, brain cancer, nervous system cancer, squamous cell carcinoma of head and neck, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, hematopoietic cancers, testicular cancer, colon-rectal cancers, prostatic cancer, pancreatic cancer, and cancer cachexia. 
     
     
         28 . The method according to  claim 27 , wherein the antigen comprises extracellular vesicles, exosome, proteins, peptides, and nucleic acids from the cancer.