Apparatus and method for electrical detection of oligonucleotides through pore blockades

Abstract

Systems and methods for specific nucleic acid (NA) sequence detection that do not rely on polymerase chain reaction (PCR) for target sequence amplification and do not require any special reagents other than a complementary sequence capture probe conjugated to spherical beads.

1 - 3 . (canceled) 
     
     
         4 . An apparatus for sequence-specific nucleic acid detection in a sample, the apparatus comprising:
 a first chamber;   a second chamber;   a membrane separating said first and second chambers, said membrane having a pore extending through the membrane between the first and second chambers;   a first electrode disposed in the first chamber; and   a second electrode disposed in the second chamber;   wherein when a liquid is introduced into said first and second chambers, wherein when a source of direct electric current is connected to said electrodes with a negative polarity being connected to the first electrode, and wherein when a bead which is conjugated with one or more strands of capture probe nucleic acid that is complementary to a desired target sequence to be detected is subsequently introduced into the liquid in the first chamber, the presence of said target nucleic acid is indicated by a decrease in electric current between the electrodes in relation to electric current between the electrodes prior to introduction of the beads into the first chamber, said decrease in electric current resulting from movement of the bead and the bead causing blockage of the pore.   
     
     
         5 . An apparatus as recited in  claim 4 :
 wherein the pore has a length between a first end and a second end;   wherein the pore has a diameter that decreases from said first end toward said second end;   wherein the bead has a diameter less than the diameter at the first end of the pore; and   wherein the pore has a diameter less than the diameter of the bead at a point along the length of the pore.   
     
     
         6 . An apparatus as recited in  claim 4 , wherein the pore has a diameter ranging from approximately 10 nm to approximately 5 μm. 
     
     
         7 . An apparatus as recited in  claim 4 , wherein the pore comprises an opening extending through said membrane. 
     
     
         8 . An apparatus as recited in  claim 4 , wherein the pore comprises a pipette extending through said membrane. 
     
     
         9 . An apparatus as recited in  claim 4 , wherein the bead comprises unmodified or functionalized polystyrene. 
     
     
         10 . An apparatus as recited in  claim 9 , wherein the bead has a surface functionalized with amine or carboxyl groups to facilitate cross-linking to a terminal carboxyl or amine group, respectively, added to the terminus of nucleic acid capture probes. 
     
     
         11 . An apparatus as recited in  claim 4 , wherein the strands of capture probe nucleic acid comprise a peptide nucleic acid. 
     
     
         12 . An apparatus as recited in  claim 4 , wherein the liquid comprises an electrolyte solution comprising approximately 0.01 M to approximately 1.0 M of an electrolyte in buffered water. 
     
     
         13 . An apparatus as recited in  claim 12 , wherein said electrolyte is selected from the group comprising of NaCl, KCl, and other electrolytes. 
     
     
         14 . An apparatus as recited in  claim 4 , wherein a single molecule of a desired target sequence can be detected. 
     
     
         15 . An apparatus for sequence-specific nucleic acid detection in a sample, the apparatus comprising:
 a first chamber;   a second chamber;   a membrane separating said first and second chambers, said membrane having a pore extending through the membrane between the first and second chambers;   wherein the pore has a length between a first end and a second end;   wherein the pore has a diameter that decreases from said first end toward said second end;   wherein the bead has a diameter less than the diameter at the first end of the pore;   wherein the pore has a diameter less than the diameter of the beads at a point along the length of the pore;   a first electrode disposed in the first chamber; and   a second electrode disposed in the second chamber.   
     
     
         16 . An apparatus as recited in  claim 15 , wherein when a liquid is introduced into said first and second chambers, wherein when a source of direct electric current is connected to said electrodes with a negative polarity being connected to the first electrode, and wherein when a bead which is conjugated with one or more strands of capture probe nucleic acid that is complementary to a desired target sequence to be detected is subsequently introduced into the liquid in the first chamber, the presence of said target nucleic acid is indicated by a decrease in electric current between the electrodes in relation to electric current between the electrodes prior to introduction of the beads into the first chamber, said decrease in electric current resulting from movement of the bead and the bead causing blockage of the pore. 
     
     
         17 . An apparatus as recited in  claim 15 , wherein the pore has a diameter ranging from approximately 10 nm to approximately 5 μm. 
     
     
         18 . An apparatus as recited in  claim 15 , wherein the pore comprises an opening extending through said membrane. 
     
     
         19 . An apparatus as recited in  claim 15 , wherein the pore comprises a pipette extending through said membrane. 
     
     
         20 . An apparatus as recited in  claim 15 , wherein the bead comprises unmodified or functionalized polystyrene. 
     
     
         21 . An apparatus as recited in  claim 20 , wherein the bead has a surface functionalized with amine or carboxyl groups to facilitate cross-linking to a terminal carboxyl or amine group, respectively, added to the terminus of nucleic acid capture probes. 
     
     
         22 . An apparatus as recited in  claim 16 , wherein said strands of capture probe nucleic acid comprise a peptide nucleic acid. 
     
     
         23 . An apparatus as recited in  claim 16 , wherein the liquid comprises an electrolyte solution comprising approximately 0.01 M to approximately 1.0 M of an electrolyte in buffered water. 
     
     
         24 . An apparatus as recited in  claim 23 , wherein said electrolyte is selected from the group comprising of NaCl, KCl, and other electrolytes. 
     
     
         25 . An apparatus as recited in  claim 15 , wherein a single molecule of a desired sequence can be detected.