Multi-biomarker biosensor

Abstract

Nanosubstrates as biosensors, methods of making such nanosubstrates, and methods of using such nanosubstrates to detect biomarkers are described.

1 . A method of preparing a nanosubstrate, comprising:
 providing a first substrate layer;   depositing an adhesion layer onto a surface of the first substrate layer;   depositing a second substrate layer onto the adhesion layer;   depositing a conductive layer onto the second substrate layer;   depositing an insulating layer onto the conducting layer;   creating a pattern of nanotrenches or nanowells in the insulating layer by lithography; and   removing the adhesion layer to release the nanosubstrate, the nanosubstrate comprising the second substrate layer, the conductive layer, and the insulating layer.   
     
     
         2 . The method of  claim 1 , wherein the second substrate layer comprises a photoresist material. 
     
     
         3 . The method of  claim 1 , wherein the steps of depositing the adhesion layer and depositing the conductive layer are carried out by chemical or physical vapor deposition. 
     
     
         4 . The method of  claim 1 , wherein the steps of depositing the second substrate layer and depositing the insulating layer are carried out by spin coating. 
     
     
         5 . The method of  claim 1 , wherein the lithography is electron beam lithography. 
     
     
         6 . The method of  claim 1 , wherein the nanosubstrate comprises an area of about 0.01 mm 2  to about 0.1 mm 2 . 
     
     
         7 . The method of  claim 1 , further comprising:
 contacting the substrate with an aqueous solution of nanoparticles; and   applying a DC voltage between an anode and a cathode, wherein the anode is the conductive layer of the nanosubstrate and the cathode is a conductor placed in the aqueous solution without contacting the nanosubstrate, thereby electrophoretically assembling the nanoparticles within the nanotrenches or nanowells of the nanosubstrate.   
     
     
         8 . The method of  claim 7 , wherein the nanoparticles are covalently bound to an antibody or antigen binding fragment thereof. 
     
     
         9 . The method of  claim 8 , further comprising attaching the nanosubstrate to a catheter. 
     
     
         10 . The method of  claim 9 , wherein the nanosubstrate is attached to the catheter with an adhesive. 
     
     
         11 . A method of detecting a biomarker, comprising
 (a) providing a nanosubstrate comprising:
 a first substrate layer; 
 a sacrificial layer deposited on the first substrate layer; 
 a second substrate layer deposited on the sacrificial layer, the second substrate layer comprising a photoresist material; 
 a conductive layer deposited on the second substrate layer; and 
 an insulating layer deposited on the conductive layer, the insulating layer interrupted by two or more nanotrenches or nanowells having different widths, each width at least about 20 nm; 
 two or more nanoparticles deposited in said two or more nanotrenches or nanowells, the nanoparticles belonging to two or more different size classes, wherein the nanoparticles in each size class have a diameter less than or equal to the width of the nanotrench or nanowell in which they are deposited, and wherein the nanoparticles of each size class are covalently bound to a different antibody or antigen binding fragment thereof; and 
   (b) contacting the nanosubstrate to a biological sample from a subject, the biological sample comprising a biomarker, wherein binding of the biomarker to the antibody or antigen binding fragment thereof results in a detectable signal, thereby detecting the biomarker.   
     
     
         12 . The method of  claim 11 , wherein the binding of the biomarker to the antibody or antigen binding fragment thereof is determined by ELISA. 
     
     
         13 . The method of  claim 11 , wherein the biomarker is a cancer biomarker. 
     
     
         14 . The method of  claim 13 , wherein detecting the biomarker is indicative of an increased risk of a disease or disorder in the subject.