Electrochemical detection of nucleic acid sequences

• US 7,169,358 B2
• Filed: 02/25/2002
• Issued: 01/30/2007
• Est. Priority Date: 03/18/1997
• Status: Expired due to Fees

• Current Assignee: Magellan Diagnostics, Inc. (Meridian Bioscience, Inc.)
• Original Assignee: ESA Biosciences, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Zhang, Honghua, Roe, R. Michael, Henkens, Robert W., Wegner, Steven E., Thompson, Deborah M., Stewart, Thomas N., Sundseth, Rebecca, Wojciechowski, Marek, Naser, Najih, O'Daly, John P.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/082,714
• Publication Number:
  US 20040072158A1
• Time in Patent Office: 1,800 Days
• Field of Search: 435/6, 422/52, 422/55, 422/61, 422/68.1, 422/82.1, 422/53, 536/23.1
• US Class Current: 422/68.1
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

An electrochemical detection system which specifically detects selected nucleic acid segments is described. The system utilizes biological probes such as nucleic acid or peptide nucleic acid probes which are complementary to and specifically hybridize with selected nucleic acid segments in order to generate a measurable current when an amperometric potential is applied. The electrochemical signal can be quantified.

Claims

1. A battery or AC-powered portable biosensor apparatus comprising:

a circuit board with a plurality of working and reference electrodes formed

thereon;

a plurality of nucleic acid segments attached to the surface of said plurality of working electrodes that capture nucleic acid targets by hybridization; and

a monitor for measuring a current produced following hybridization of targets with the nucleic acid segments, said monitor being operably connected so that it controls the electrical potential of the electrodes and records, analyzes and displays the currents generated at the individual electrodes.

2. The portable biosensor apparatus of claim 1 wherein the circuit board is a printed circuit board or a screen printed circuit board or has a combination of printed and screen printed elements.

3. The portable biosensor apparatus of claim 2 further comprising a multiplexed potentiostat to apply electrical potential and to measure the current generated by each of said nucleic acid segments following hybridization to different target nucleic acids.

4. The portable biosensor apparatus of claim 1 wherein the monitor is a continuous or pulse amperometric monitor.

5. The portable biosensor apparatus of claim 4 wherein the amperometric monitor measures the currents generated by the elements of a sensor array with one or more working electrodes associated with a single reference electrode and simultaneously detects and quantitates a plurality of targets.

6. The portable biosensor apparatus of claim 5 wherein the amperometric monitor comprises pulse and intermittent pulse modes.

7. The portable biosensor apparatus of claim 1 further comprising an amperometric monitor operably linked to said plurality of electrodes so that currents at individual working electrodes are independent of each other.

8. The portable biosensor apparatus of claim 1 further comprising a touch memory chip to upload calibration curves and controlling parameters to the monitor.

9. The portable biosensor apparatus of claim 1 further comprising a liquid crystal display and an output port that enables data transfer to an external device.

10. The portable biosensor apparatus of claim 1 further comprising an integrated microprocessor for changing parameters, saving and analyzing data.

11. The portable biosensor apparatus of claim 1 further comprising a single-key start operation.

12. The portable biosensor apparatus of claim 1 wherein the nucleic acid segments are hybridized with single strand DNA generated from an amplified RNA or genomic DNA sample digested with an exonuclease.

13. A battery or AC-powered portable biosensor apparatus comprising:

a circuit board with a plurality of working and reference electrodes formed thereon;

a plurality of nucleic acid segments attached to the surface of said plurality of working electrodes that capture nucleic acid targets by hybridization, wherein each hybrid formed has one or more labels attached that catalytically enhance the current generated following hybridization; and

a monitor for measuring the currents produced following hybridization of targets with the nucleic acid segments, said monitor being operably connected so that it controls the electrical potential of the electrodes and records, analyzes and displays the currents generated at the individual electrodes.

14. The biosensor apparatus of claim 13, wherein the nucleic acid segments are hybridized with single strand DNA generated from an amplified RNA or genomic DNA sample digested with an exonuclease.