Detection of purine and pyrimidine nucleotides and underivatized nucleic acids by sinusoidal voltammetry

• US 5,958,215 A
• Filed: 07/18/1997
• Issued: 09/28/1999
• Est. Priority Date: 09/18/1995
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Kuhr, Werner G., Singhal, Pankaj
• Primary Examiner(s): Tung, T.
• Application Number:
  US08/896,548
• Time in Patent Office: 802 Days
• Field of Search: 204/400, 204/434, 205/787, 205/792, 205/775, 205/794
• US Class Current: 205/787
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48

Abstract

Sinusoidal voltammetry was employed to detect both purine and pyrimidine-based nucleic acids. Adenine and cytosine, representing these two classes of nucleic acids, could be detected with nanomolar detection limits at a copper electrode under these conditions, where the sensitivity for adenine was much higher than that for cytosine. Detection limits for purine-containing nucleotides (e.g., adenosine 5'"'"'-monophosphate (AMP), adenosine 5'"'"'-diphosphate (ADP), and adenosine 5'"'"'-triphosphate (ATP)) were on the order of 70-200 nM using this method. These detection limits are achieved for native nucleotides and are over two orders of magnitude lower than those found with UV absorbance detection. Pyrimidine-based nucleotides could also be detected with high sensitivity due to the presence of a sugar backbone which is electroactive at the copper surface. This detector is not fouled by the nucleotides and can be used for the sensitive detection of analyses eluting continuously from a chromatography column or a electrophoresis capillary. Entire nucleic acid molecules can be readily detected. For example, both single stranded and double stranded DNA is detected with a detection limit in the picomolar concentration range (i.e., 10^-12 moles/L). In the present invention, the signal from a double stranded DNA is roughly twice that arising from a single stranded DNA strand of the same length.

Claims

1. A method for detecting nucleotides and nucleic acid polymers in a sample comprising the steps of:

contacting a surface of an electrocatalytic metal electrode with the sample dissolved in a high pH aqueous buffer;

applying a sinusoidal voltage having an amplitude and a fundamental frequency to the electrode, the amplitude being sufficient to elicit a redox reaction of at least one form of nucleotides and nucleic acid polymers in contact with the electrode;

measuring a current at the electrode, the current resulting from the elicited redox reaction;

producing a frequency spectrum of the measured current; and

detecting the nucleotides and nucleic acid polymers by reference to a harmonic of the frequency spectrum greater than the fundamental frequency.

2. The method of claim 1, wherein the electrocatalytic metal is selected from the group consisting of copper, lead, gold and nickel.

3. The method of claim 1, wherein the fundamental frequency is between 1 and 5 Hz.

4. The method of claim 3, wherein the fundamental frequency is 2 Hz.

5. The method of claim 1, wherein signal at a fifth harmonic of the frequency spectrum is used to detect nucleotides.

6. The method of claim 1, wherein signal at a sixth harmonic of the frequency spectrum is used to detect nucleic acid polymers.

7. The method of claim 1, wherein the amplitude of the sinusoidal voltage ranges from about 0.05V to 0.55 V, measured against a silver/silver chloride electrode.

8. The method of claim 1 further comprising the step of comparing signal magnitude and phase angle at each harmonic of the frequency spectrum to identify a chemical type of each nucleotide detected.

9. A device for detecting nucleotides and nucleic acid polymers in a sample comprising:

an electrocatalytic metal electrode;

means for bringing a sample into contact with the electrode;

means for applying a sinusoidal voltage having an amplitude and a fundamental frequency to the electrode, the amplitude being sufficient to elicit a redox reaction of at least one form of nucleotides and nucleic acid polymers at the electrode;

means for measuring a current at the electrode, the current resulting from the elicited redox reaction;

means for producing a frequency spectrum of the measured current; and

means for analyzing signal at of least one harmonic of the frequency spectrum greater than the fundamental frequency to detect nucleotides and nucleic acid polymers.

10. The device of claim 9, wherein the fundamental frequency is between 1 and 5 Hz.

11. The device of claim 10, wherein the fundamental frequency is 2 Hz.

12. The device of claim 9, wherein the means for analyzing signal analyzes the signal at a fifth harmonic of the frequency spectrum to detect nucleotides.

13. The device of claim 9, wherein the means for analyzing signal analyzes the signal at a sixth harmonic of the frequency spectrum to detect nucleic acid polymers.

14. The device of claim 9, wherein the amplitude of the sinusoidal voltage ranges from about 0.05V to 0.55 V, measured against a silver/silver chloride electrode.

15. The device of claim 9, wherein the electrocatalytic metal is selected from the group consisting of copper, lead, gold and nickel.

16. The device of claim 9 further comprising means for comparing signal magnitude and phase angle at each harmonic of the frequency spectrum to identify a chemical type of each nucleotide detected.

17. A method for detecting nucleotides and nucleic acid polymers in a sample comprising the steps of:

contacting a surface of a copper electrode with the sample dissolved in an aqueous buffer with a pH of more than 7.0;

applying a sinusoidal voltage having an amplitude of from about 0.05 V to about 0.55 V, measured against a silver/silver chloride electrode, and a fundamental frequency of 2 Hz. to the copper electrode to elicit a redox reaction of at least one form of nucleotides and nucleic acid polymers at the copper electrode;

measuring a current at the copper electrode, the current resulting from the elicited redox reaction;

producing a frequency spectrum of the measured current; and

detecting the nucleotides and nucleic acid polymers by reference to a harmonic of the frequency spectrum greater than the fundamental frequency.

18. The method of claim 17, wherein nucleotides are detected by reference to a fifth harmonic of the frequency spectrum.

19. The method of claim 17, wherein nucleic acid polymers are detected by reference to a sixth harmonic of the frequency spectrum.

20. The method of claim 17 further comprising the step of comparing signal magnitude and phase angle at each harmonic of the frequency spectrum to identify a chemical type of each nucleotide detected.

21. A device for detecting nucleotides and nucleic acid polymers in a sample comprising:

a copper electrode;

means for contacting the copper electrode with the sample dissolved in a high pH aqueous buffer;

means for applying a sinusoidal voltage having an amplitude of from about 0.05 V to about 0.55 V, measured against a silver/silver chloride electrode, and a fundamental frequency of 2 Hz. to the copper electrode to elicit a redox reaction of nucleotides and nucleic acid polymers at the copper electrode;

means for measuring a current at the copper electrode, the current resulting from the elicited redox reaction;

means for producing a frequency spectrum of the measured current; and

means for detecting the nucleotides and nucleic acid polymers by reference to a harmonic of the frequency spectrum greater than the fundamental frequency.

22. The device of claim 21, wherein the means for detecting the nucleotides detects the nucleotides by reference to a fifth harmonic of the frequency spectrum.

23. The device of claim 21, wherein the means for detecting the nucleic acid polymers detects the nucleotides by reference to a sixth harmonic of the frequency spectrum.

24. The device of claim 21 further comprising means for comparing signal magnitude and phase angle at each harmonic of the frequency spectrum to identify a chemical type of each nucleotide detected.