Method and arrangement for calibrating a sensor element

• US 10,081,831 B2
• Filed: 10/29/2008
• Issued: 09/25/2018
• Est. Priority Date: 11/20/2007
• Status: Active Grant

• Current Assignee: Boehringer Ingelheim Vetmedica GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Original Assignee: Boehringer Ingelheim Vetmedica GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Inventors: Friedrich, Katja, Gumbrecht, Walter, Paulicka, Peter, Stanzel, Manfred, Weber, Renee
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US12/743,316
• Publication Number:
  US 20100248243A1
• Time in Patent Office: 3,618 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

A method is disclosed for calibrating a sensor element, which has an immobilized probe oligonucleotide, via which the bonding of a target nucleic acid (Z) can be detected by the sensor. In at least one embodiment, the method includes: a) bringing the sensor element into contact with a control nucleic acid (K), the melting temperature Tm(K) of which is less than the melting temperature Tm(Z) of the target nucleic acid (Z); b) hybridizing the control nucleic acid (K) to the probe oligonucleotide at a temperature T[p]<Tm(K), and detecting a positive control signal; and optionally c) modifying the stringent conditions such that T[n]>Tm(K) and detecting a negative control signal at a temperature T[n]. According to a refinement of at least one embodiment of the invention, a measuring signal of the target nucleic acid (Z) is measured at a measuring temperature T [mess], where Tm(K)<T[mess]<Tm(Z). The method is suited in particular for the calibration and quality control of microarrays.

Claims

1. A method for calibrating all of the elements of a sensor, via which the sensor can record binding of a target nucleic acid(Z), the method comprising:

contacting all of the elements of a sensor comprising an array of a plurality of oligonucleotide probes immobilized to an electrochemical sensor via a gold surface, each of which oligonucleotide probes includes a first nucleotide sequence that hybridizes to the target nucleic acid at a temperature up to Tm(Z) and a second nucleotide sequence that hybridizes to the control nucleic acid at a temperature up to Tm(K), the first nucleotide sequence and the second nucleotide sequence having a different number of binding base pairs, the second nucleotide sequence having fewer nucleotides than the first nucleotide sequence or both of fewer nucleotides than the first nucleotide sequence and a lower percentage of G and C nucleotides than the first nucleotide sequence, with a mixture having a control nucleic acid (K) and the target nucleic acid(Z), a melting temperature of a complex of the control nucleic acid and the immobilized probe Tm(K) being lower than a melting temperature of a complex of the target nucleic acid and the immobilized probe Tm(Z), Tm(K) being the temperature below which the control nucleic acid (K) hybridizes with an associated immobilized probe oligonucleotide, and Tm(Z) being the temperature below which the target nucleic acid (Z) hybridizes with an associated immobilized probe oligonucleotide;

hybridizing the control nucleic acid (K) to the all of the associated probe oligonucleotides at a temperature T[p]<

Tm(K), and recording an electrochemical positive control signal, T[p] being a temperature at which the electrochemical positive control signal is measured;

modifying stringency conditions at a measuring temperature T[mess] such that,

Tm(K)<

T[mess]<

Tm(Z), T[mess] is the temperature at which an electrochemical measurement signal is measured;

modifying the stringency conditions such that T[n]>

Tm(Z); and

recording an electrochemical negative control signal, T[n] is a temperature at which the electrochemical negative control signal is recorded.

2. The method as claimed in claim 1, wherein the stringency conditions are modified by increasing the temperature.

3. The method as claimed in claim 1, wherein an electrochemical measurement signal n is measured at a different temperature T[mess [[1−

]]n], where Tm(K)<

T[mess [[1−

]]n]<

Tm(Z).

4. The method as claimed in claim 1, wherein the target nucleic acid and the control nucleic acid are labeled with electrochemically detectable labels.

5. The method as claimed in claim 4, wherein at least one of the electrochemically detectable labels is an enzymatic label capable of converting a substrate into a product detectable by the electrochemical sensor element.

6. The method as claimed in claim 1, that further comprises contacting all of the sensor elements with an electrochemically detectable product in order to record a first calibration signal.

7. The method as claimed in claim 4, wherein additionally a temperature compensation signal is recorded from an additional element comprising the same electrochemically detectable label directly immobilized on an electrochemical sensor at each of the temperatures T[p], T[mess], T[mess n] and T[n], T[mess n] being the temperature at which an electrochemical measurement signal n is measured.

8. The method as claimed in claim 1, wherein the melting temperature Tm(K) of the complex of the control nucleic acid (K) and the immobilized probe is at least 5°

C. lower than the melting temperature Tm(Z) of the complex of the target nucleic acid (Z) and the immobilized probe.

9. The method of claim 1, in which the first and second nucleotide sequences are different nucleotide sequences.

10. The method of claim 1, in which the second nucleotide sequence has a lower GC content than the first nucleotide sequence.

11. The method of claim 6, in which the electrochemically detectable product is a product of an enzyme reaction, said enzyme being bound to said control nucleic acid.

12. The method of claim 6, in which the first and second nucleotide sequences are different nucleotide sequences.

13. The method of claim 6, in which the second nucleotide sequence has a lower GC content than the first nucleotide sequence.