Apparatus and method for electrical detection of oligonucleotides through pore blockades

1. A method for detecting the presence of nucleic acids with a target sequence in a sample, the method comprising:

(a) providing a detection apparatus, said detection apparatus comprising:

(i) a first chamber;

(ii) a second chamber;

(iii) a membrane separating said first and second chambers, said membrane having one or more pores extending through the membrane between the first and second chambers;

(iv) a first electrode disposed in the first chamber; and

(v) a second electrode disposed in the second chamber;

(b) introducing a liquid into said first and second chambers;

(c) applying a direct electric current to said first and second electrodes with a negative polarity being connected to the first electrode;

(d) measuring electric current between the electrodes as a reference current;

(e) preparing at least one bead which is conjugated with one or more strands of capture probe nucleic acid that are complementary to a desired target sequence of a sample to be detected;

(f) capping charged groups on the prepared beads to render the beads with a neutral charge;

(g) introducing one or more of the capped beads and a sample into the first chamber to hybridize the capture probe to the desired target sequence; and

(h) detecting the target sequence by measuring electric current between the electrodes after introduction of the beads and sample and comparing the measured current with the reference current;

(i) wherein a decrease in measured electric current indicates the presence of one or more strands of target nucleic acid that are complementary to the probe sequence conjugated to the bead; and

(j) wherein said decrease in measured electric current results from electrophoretic movement of the beads bound to charged target nucleic acid strands to a pore of diameter less than the bead diameter or tapered to a lesser diameter and the beads causing sustained blockage of one or more pores.

2. A method as recited in claim 1 :

wherein each pore has a length between a first end and a second end;

wherein each pore has a diameter that decreases from said first end toward said second end;

wherein the beads have a diameter less than the diameter at the first end of the pore; and

wherein each pore has a diameter less than the diameter of the beads at a point along the length of the pore.

3. A method as recited in claim 1 , wherein each pore has a diameter ranging from approximately 10 nm to approximately 5 μm.

4. A method as recited in claim 1 , wherein each pore comprises an opening extending through said membrane.

5. A method as recited in claim 1 , wherein the pore comprises a pipette extending through said membrane.

6. A method as recited in claim 1 , wherein each bead comprises unmodified or functionalized polystyrene.

7. A method as recited in claim 6 , wherein each 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.

8. A method as recited in claim 1 , wherein said strands of capture probe nucleic acid comprise peptide nucleic acids.

9. A method as recited in claim 1 , wherein the liquid comprises an electrolyte solution comprising approximately 0.01 M to approximately 1.0 M of an electrolyte in buffered water.

10. A method as recited in claim 9 , wherein said electrolyte is selected from the group comprising of NaCl, KCl, and other strong electrolytes.

11. A method as recited in claim 1 , further comprising:

creating a fluid flow through said membrane from the second chamber to the first chamber, said flow generated osmotically, electroosmotically or hydrostatically.

12. A method as recited in claim 1 , further comprising detecting the presence of a single nucleic acid molecule of a desired target sequence.