Methods for manufacturing an electrochemical sensor for effective diagnostic oligonucleotide detection

Abstract

The present invention features methods for manufacturing an electrochemical sensor for detecting a diagnostic target oligonucleotide. The methods described herein provide for an electrochemical sensor with a higher level of coverage of the probes on its surface, thus allowing for more sensitive detection of a target oligonucleotide. The methods may feature first mixing disulfide terminated oligonucleotides having a free thiol moiety at the 3′ end with a gold substrate and subsequently introducing to the gold substrate a composition for reducing thiol moieties to cause the oligonucleotides to bind to the surface of the gold substrate. In some embodiments, the method comprises removing excess thiol and oligonucleotides, which may help with non-competitive binding. In some embodiments, the method comprises rinsing the gold substrate with water and drying with nitrogen.

What is claimed is: 
     
         1 . A method of producing an electrochemical sensor for detecting a target oligonucleotide, the method comprising:
 a) mixing disulfide terminated oligonucleotides having a free thiol moiety at a 3′ end with a gold substrate; and   b) subsequent to (a) introducing to the gold substrate a composition for reducing thiol moieties of the oligonucleotides, thereby causing the oligonucleotides to bind to a surface of the gold substrate.   
     
     
         2 . The method of  claim 1  further comprising removing excess thiol and oligonucleotides. 
     
     
         3 . The method of  claim 2  further comprising adding a back-filler additive to the surface of the gold substrate, the back filler binds to a portion of space on the gold substrate not occupied by the oligonucleotides. 
     
     
         4 . The method of  claim 3 , wherein the back-filler additive is organic. 
     
     
         5 . The method of  claim 3 , wherein the back-filler additive comprises a thiol moiety at a first end which binds to the surface of the gold substrate. 
     
     
         6 . The method of  claim 3 , wherein the back-filler additive further comprises a carbon chain linked to the thiol moiety at the first end of the back-filler additive. 
     
     
         7 . The method of  claim 3 , wherein the back-filler additive is mercaptohexanol. 
     
     
         8 . The method of  claim 3 , wherein the back-filler additive is nonreactive. 
     
     
         9 . The method of  claim 3  further comprising rinsing the gold substrate with water and drying with nitrogen. 
     
     
         10 . The method of  claim 1 , further comprising preparing the gold substrate, wherein preparing the gold substrate comprises electrochemically cleaning a surface of the gold substrate. 
     
     
         11 . The method of  claim 1 , further comprising clean-up steps, wherein the clean-up steps comprise adding an additive to the gold substrate. 
     
     
         12 . The method of  claim 1 , wherein the additive is a buffer, a salt solution, or a combination thereof. 
     
     
         13 . The method of  claim 12 , wherein the salt solution comprises sodium (Na + ), sodium chloride (NaCl), potassium (K + ), potassium chloride (KCl), lithium (Li + ), lithium chloride (LiCl), magnesium (Mg +2 ), magnesium chloride (MgCl 2 ), calcium (Ca +2 ), chloride (Cl − ), phosphate (PO 4   −3 ), nitrate (NO − ), acetate (C 2 H 3 O 2   − ), carbonate (CO 3   −2 ), bicarbonate (HCO 3   − ), or a combination thereof. 
     
     
         14 . The method of  claim 12 , wherein the buffer comprises a phosphate buffer, a carbonate buffer, a MOPS buffer, a MOPSO buffer, a BES buffer, a TES buffer, a HEPES buffer, a DIPSO buffer, a MOBS buffer, a PBS buffer, or a TRIS buffer. 
     
     
         15 . The method of  claim 12 , wherein the additive has a pH of 7 to 7.4. 
     
     
         16 . The method of  claim 12 , wherein the additive comprises a surfactant. 
     
     
         17 . A method of producing an electrochemical sensor for detecting a target oligonucleotide, the method comprising:
 a) mixing disulfide terminated oligonucleotides having a free thiol moiety at a 3′ end with to a gold substrate;   b) subsequent to (a) introducing to the gold substrate a composition for reducing the thiol moieties or the oligonucleotides, thereby causing the oligonucleotides to bind to a surface of the gold substrate;   c) removing excess thiol and oligonucleotides from the surface of the gold substrate; and   d) adding a back-filler additive to the surface of the gold substrate, the back filler binds to a portion of space on the gold substrate not occupied by the oligonucleotides; and   e) rinsing the gold substrate with water and drying with nitrogen.   
     
     
         18 . The method of  claim 17 , wherein the back-filler additive is organic. 
     
     
         19 . The method of  claim 17 , wherein the back-filler additive comprises a thiol moiety at a first end that binds to the surface of the gold substrate. 
     
     
         20 . The method of  claim 17 , wherein the back-filler additive further comprises a carbon chain linked to the thiol moiety at the first end of the back-filler additive. 
     
     
         21 . The method of  claim 17 , wherein the back-filler additive is nonreactive. 
     
     
         22 . The method of  claim 17 , furthering comprising preparing the gold substrate, wherein preparing the gold substrate comprises electrochemically cleaning a surface of the gold substrate. 
     
     
         23 . The method of  claim 17 , further comprising clean-up steps, wherein the clean-up steps comprise adding an additive to the gold substrate. 
     
     
         24 . The method of  claim 23 , wherein the additive is a buffer, a salt solution, or a combination thereof. 
     
     
         25 . The method of  claim 24 , wherein the additive has a pH of 7 to 7.4.