Method for exosomal biomarker detection by electric field-induced release and measurement

Abstract

The molecules harbored in exosomes play important roles in biological science. A highly desirable goal for exosome research is the rapid, simple, simultaneous tracking and quantification of exosome harbored molecules. Disclosed herein are methods and devices for inducing the release and measurement of biomolecules harbored in exosomes. The disclosed method, Electric Field Induced Release and Measurement (EFIRM) technique, uses an electrical field to simultaneously disrupt exosomes to release the contents and measure the harbored exosomal RNA/proteins. The exosome vesicle contents can be released within minutes. This provides a potential on-site method for the detection of exosome-harbored biomolecules.

1 .- 25 . (canceled) 
     
     
         26 . A method for detecting a biomarker present in an exosome, the method comprising:
 (a) providing a biological fluid comprising exosomes;   (b) contacting the biological fluid with an exosome extraction reagent to form a complex between the exosome and the exosome extraction reagent, the exosome extraction reagent comprising a first affinity moiety bound to a magnetic bead, wherein the first affinity binding moiety specifically binds to a surface-exposed exosome marker;   (c) separating the complex formed between the exosome and the exosome extraction reagent from the biological fluid;   (d) contacting a solid phase with the complex formed between the exosome and the exosome extraction reagent, the solid phase comprising a first contact region under the influence of a magnetic field, a second contact region, and a detection reagent immobilized at the second contact region, the detection reagent comprising a second affinity moiety that specifically binds to the biomarker, wherein the biomarker is selected from a protein, a nucleic acid and an mRNA;   (e) immobilizing the complex formed between the exosome and the exosome extraction reagent at the first contact region;   (f) applying an electrical field to the immobilized complex formed between the exosome and the exosome extraction reagent at the first contact region; and   (e) detecting an interaction between the biomarker and the detection reagent.   
     
     
         27 . (canceled) 
     
     
         30 . The method according to  claim 26 , wherein the biological fluid is selected from the group consisting of saliva, whole blood, blood plasma, blood serum, amniotic fluid, bile, colostrum, breast milk, cerebrospinal fluid (CSF), lymph, gastric acid, nasal mucus, pleural fluid, semen, tears, and urine. 
     
     
         31 . The method of  claim 30 , wherein the biological fluid is saliva. 
     
     
         32 . The method of  claim 30 , wherein the biological fluid is whole blood, blood plasma, or blood serum. 
     
     
         33 . The method according to  claim 26 , wherein the surface-exposed exosome marker is CD63. 
     
     
         34 . The method according to  claim 26 , wherein the exosome extraction reagent comprises an antibody. 
     
     
         35 . The method according to  claim 26 , wherein the exosome extraction reagent comprises an exosome-specific lectin. 
     
     
         36 . The method according to  claim 26 , wherein the second affinity moiety comprises an antibody. 
     
     
         37 . The method according to  claim 26 , wherein the second affinity moiety comprises a nucleic acid. 
     
     
         38 . The method according to  claim 26 , wherein the solid phase comprises an electrochemical sensor. 
     
     
         39 . The method according to  claim 26 , wherein the absolute value of the maximum voltage of the electric field does not exceed 1 volt. 
     
     
         40 . The method of  claim 39 , wherein the absolute value of the maximum voltage of the electric field does not exceed 500 millivolt (mV). 
     
     
         41 . The method according to  claim 26 , wherein the electric field is applied for less than 300 seconds. 
     
     
         42 . The method of  claim 41 , wherein the electric field is applied for 200±30 seconds. 
     
     
         43 . The method according to  claim 26 , wherein the electric field is a non-uniform electric field. 
     
     
         44 . The method of  claim 43 , wherein the non-uniform electric field is a cyclic square wave electrical field (csw E-field). 
     
     
         45 . The method of  claim 44 , wherein the csw E-field comprises 15 cycles of −300±50 mV for 9±2 seconds followed by 200±50 mV for 1±0.5 second. 
     
     
         46 . The method of  claim 45 , wherein the csw E-field comprises 20 cycles of −300 mV for 9 seconds followed by 200 mV for 1 second. 1.