Sensing apparatus and method

• US 20080032295A1
• Filed: 03/02/2007
• Published: 02/07/2008
• Est. Priority Date: 03/09/2001
• Status: Active Grant

• Current Assignee: DNAE Group Holdings Limited (Genting Bhd.)
• Original Assignee: DNA Electronics Limited (Genting Malaysia Berhad)
• Inventors: Toumazou, Christofer, Purushothaman, Sunil
• Granted Patent: US 7,888,015 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Abstract

Use of a pH sensor comprising an ion-sensitive field effect transistor (ISFET) to perform real time detection/quantification of nucleic acid amplification, e.g. polymerase chain reaction (PCR) nucleic acid amplification, based on detection of protons released during the primer extension phase.

Claims

1. A method of monitoring nucleic acid amplification in a sample comprising a buffered nucleic acid amplification mixture for amplification of target sequence if present in the sample, characterised in that said monitoring is by means of detecting change of pH resulting from proton release in the presence of target sequence as amplification proceeds beyond a threshold number of cycles for buffering capacity of the sample to be overcome, said detecting employing a sensing apparatus comprising an ISFET having a sensing surface exposed to the sample and arranged to generate an electrical output signal in response to change of pH at said transistor surface and means for detecting an electrical output signal from the ISFET.

2. The method of claim 1 wherein said nucleic acid amplification is polymerase chain reaction (PCR) amplification.

3. The method of claim 1 wherein the ISFET is provided with a layer of silicon nitride.

4. The method of claim 3 wherein a DNA polymerase enzyme linked layer is provided over the layer of silicon nitride.

5. The method of claim 1 wherein a reference electrode is employed.

6. The method of claim 1 wherein the target nucleic acid for amplification is captured on beads and nucleic acid amplification occurs on the beads which are brought into the vicinity of the ISFET sensing surface.

7. The method of claim 1 wherein target nucleic acid for amplification is captured by probe immobilised on the ISFET.

8. The method of claim 1 wherein said ISFET sensing surface is exposed to said sample in a low volume reaction chamber of 1 pl to about 10 μ

l.

9. The method of claim 8 wherein said reaction chamber is in a microfluidic device comprising one or more further identical chambers containing said ISFET whereby nucleic acid amplification can be monitored simultaneously in more than one sample.

10. The method of claim 8 wherein said reaction chamber is on a chip which includes resistive on-chip heating elements and temperature sensors whereby said chamber is capable of being heated and cooled for PCR thermocycling.

11. The method of claim 10 wherein said chip additionally includes on-chip temperature control circuitry.

12. The method of claim 2 wherein the sample for PCR monitoring is caused to flow through a channel or chamber of a microfluidic device and as it flows is subjected consecutively to different temperatures whereby thermocycling for PCR is achieved.

13. The method of claim 12 wherein said sample is caused to flow through a chamber or channel which passes consecutively through different temperature zones suitable for said thermocycling.

14. The method of claim 13 wherein said sample is caused to flow through a channel which passes consecutively through different temperature zones provided in the base of said microfluidic device, said zones being suitable for successive repeats along the channel of the PCR stages of denaturing, primer annealing and primer extension.

15. The method of claim 14 wherein more than one ISFET is employed for sensing of pH change of the sample at different positions along said channel.

16. The method of claim 12 wherein said sample is moved backwards and forwards in a microchamber between required temperature zones for thermocycling and said ISFET is provided in a wall of said chamber.

17. A sensing apparatus for monitoring nucleic acid amplification in a sample in accordance claim 1 wherein said apparatus comprises a chamber or channel to receive said sample in or on a base and one or more pH sensitive ISFETs arranged for monitoring of nucleic acid amplification in said chamber or channel, said base as presented in said chamber of channel having a coating to improve amplification efficiency.

18. A sensing apparatus for monitoring nucleic acid amplification in a sample in accordance with claim 1 wherein said apparatus comprises a microfluidic device in which a chamber or channel is provided to receive the sample in or on a base and one or more pH sensitive ISFETs are arranged for monitoring of nucleic acid amplification in said chamber or channel.

19. The apparatus of claim 17 wherein said base is a silicon substrate.

20. The apparatus of claim 18 wherein said base is a silicon substrate.

21. The apparatus of claim 17 wherein a low volume reaction chamber of 1 pl to about 10 μ

l containing said sample overlies a pH sensitive ISFET.

22. The apparatus of claim 18 wherein a low volume reaction chamber of 1 pl to about 10 μ

l containing said sample overlies a pH sensitive ISFET.

23. The apparatus of claim 17 wherein the sample for PCR monitoring is caused to flow through a channel or chamber of a microfluidic device and as it flows is subjected consecutively to different temperatures whereby thermo-cycling for PCR is achieved.

24. The apparatus of claim 23 wherein said sample is caused to flow through a chamber or channel which passes consecutively through different temperature zones suitable for said thermocycling.

25. The apparatus of claim 24 wherein said sample is caused to flow through a channel which passes consecutively through different temperature zones provided in the base of said microfluidic device, said zones being suitable for successive repeats along the channel of the PCR stages of denaturing, primer annealing and primer extension.

26. The apparatus of claim 25 wherein more than one ISFET is employed for sensing of pH change of the sample at different positions along said channel.

27. The apparatus of claim 18 wherein the sample for PCR monitoring is caused to flow through a channel or chamber of a microfluidic device and as it flows is subjected consecutively to different temperatures whereby thermocycling for PCR is achieved.

28. The apparatus of claim 27 wherein said sample is caused to flow through a chamber or channel which passes consecutively through different temperature zones suitable for said thermocycling.

29. The apparatus of claim 28 wherein said sample is caused to flow through a channel which passes consecutively through different temperature zones provided in the base of said microfluidic device, said zones being suitable for successive repeats along the channel of the PCR stages of denaturing, primer annealing and primer extension.

30. The apparatus of claim 29 wherein more than one ISFET is employed for sensing of pH change of the sample at different positions along said channel.

31. The apparatus of claim 23 wherein said sample is moved backwards and forwards in a microchamber between required temperature zones for thermocycling and said ISFET is provided in a wall of said chamber.

32. The apparatus of claim 28 wherein said sample is moved backwards and forwards in a microchamber between required temperature zones for thermocycling and said ISFET is provided in a wall of said chamber.

33. A kit for detecting a target nucleic acid sequence in a sample by nucleic acid amplification, which comprises a sensing device comprising a reaction chamber or channel and a pH sensitive ISFET for monitoring nucleic amplification in said chamber or channel in accordance with claim 1, wherein primers for said amplification are either provided in the reaction chamber or channel or separately within the kit.

34. The kit of claim 33 wherein said sensing device is in the form of a microfluidic device.