Label free detection of nucleic acid amplification

Patent metadata

Field	Value
Publication number	US-9376713-B2
Application number	US-88891710-A
Country	US
Kind code	B2
Filing date	Sep 23, 2010
Priority date	Sep 23, 2009
Publication date	Jun 28, 2016
Grant date	Jun 28, 2016

Abstract

Provided are methods and devices for label-free detection of nucleic acids that are amplified by polymerase chain reaction. A solution containing the components necessary for a PCR is introduced to a microfluidic amplification chamber and an electric field applied to a confined region in which PCR occurs. PCR product generated in the confined region is detected by measuring an electrical parameter that is, for example, solution impedance. The devices and methods provided herein are used, for example, in assays to detect one or more pathogens or for point-of-care tests. In an aspect, the PCR product is confined to droplets and the assay relates to detecting an electrical parameter of a flowing droplet, thereby detecting PCR product without a label. In an aspect, the PCR occurs in the droplet.

First claim

We claim:

1. A method of detecting a nucleic acid amplification product in solution, said method comprising:
providing a microfluidic amplification chamber comprising an electrode array on at least one surface of said microfluidic amplification chamber;
introducing a solution that may contain a template to be amplified by nucleic acid amplification in said microfluidic amplification chamber;
performing a nucleic acid amplification on said solution, wherein said nucleic acid amplification is performed in a confined region or said nucleic acid amplification product is contained in a confined region, to generate an amplified nucleic acid amplification product in solution, wherein said confined region has a volume selected from a range that is greater than or equal to 0.5 pL and less than or equal to 1 nL;
applying an electric potential to said electrode array to produce an electric field in said confined region; and
measuring an electrical parameter of said solution in said confined region, wherein said electrical parameter is solution impedance, thereby detecting said nucleic acid amplification product in solution;
wherein said step of introducing the solution comprises:
providing a droplet of the solution surrounded by a suspending fluid comprising an ionic liquid, and the droplet corresponds to the confined region; and
electrically contacting the droplet with the electrode array;
wherein said detection of nucleic acid amplification is label-free.

2. The method of claim 1 , wherein said confined region has a volume that is less than or equal to:
1 pL for detecting said nucleic acid amplification product from a bacterial cell; and
10 pL for detecting said nucleic acid amplification product from a mammalian cell.

3. The method of claim 1 , wherein said electrode array is positioned on one surface of said microfluidic amplification chamber.

4. The method of claim 1 , wherein said electrode array comprises an interdigitated array of electrodes having a width selected from a range that is greater than or equal to 10 nm and less than or equal to 100 pm and a separation distance between adjacent electrodes selected from a range that is greater than or equal to 10 nm and less than or equal to 100 μm.

5. The method of claim 1 , wherein said microfluidic amplification chamber comprises side walls formed of PDMS and a bottom surface of SiO 2 , wherein said bottom surface supports said electrode array.

6. The method of claim 1 , wherein said microfluidic amplification chamber is controllably isolated by one or more fluid control valves.

7. The method of claim 6 , wherein said fluid control valve is operably connected to a reservoir containing said solution for performing nucleic acid amplification or to a reservoir containing said solution that may contain template to be amplified by nucleic acid amplification reaction.

8. The method of claim 1 , wherein said confined region has a minimum concentration of template in said confined region that is greater than or equal to 10 template molecules per pico liter of volume.

9. The method of claim 1 , wherein said method has a detection limit of as low as 0.1 μg DNA templates in said confined volume.

10. The method of claim 1 , wherein said detecting is performed continuously or after each nucleic acid amplification cycle.

11. The method of claim 10 , wherein said nucleic acid amplification is by polymerase chain reaction (PCR) and said detecting is capable of resolving a PCR product concentration difference between consecutive PCR cycles.

12. The method of claim 11 , wherein said consecutive cycle is for a PCR cycle number that is selected from a range that is greater than or equal to 4 and less than or equal to 9.

13. The method of claim 1 , wherein said nucleic acid amplification is performed on a sample for which the detection comprises a presence or an absence of said nucleic acid amplification product.

14. The method of claim 13 , wherein said sample comprises one or more target cells, said method further comprising lysing said target cells and contacting said lysate with said solution.

15. The method of claim 14 wherein said nucleic acid amplification product is a contiguous sequence of bacterial DNA.

16. The method of claim 14 , wherein said target cells comprise a plurality cell populations, and said method is for detecting a food-borne pathogen.

17. The method of claim 1 , wherein said ionic liquid is a hydrophobic liquid that is immiscible with an aqueous solution.

18. The method of claim 17 , further comprising the step of:
introducing the suspending fluid to a collecting conduit at a suspending fluid flow-rate;
introducing the solution to the collecting conduit at a solution flow rate; and
forming the droplet having a user-selected droplet volume or droplet spacing by adjusting an inlet flow ratio, said inlet flow ratio corresponding to the ratio of suspending fluid flow-rate to solution flow-rate.

19. The method of claim 18 , wherein the formed droplet flows past said electrode array and said measured electrical parameter is a dielectric capacitance of said droplet.

20. The method of claim 1 , wherein said detection has a sensitivity that is as low as 1 template molecule/ 10 pL.

21. The method of claim 1 , wherein said microfluidic amplification chamber has a volume that is selected from a range of 10 μL to 150 μL.

22. The method of claim 1 , wherein prior to said nucleic acid amplification step, said solution comprises DNA having a concentration that is selected from a range that is greater than or equal to 5*10 8 DNA molecules/μL and less than or equal to 10 10 DNA molecules/μL.

23. The method of claim 1 , wherein said nucleic acid amplification product is DNA or RNA.

24. The method of claim 23 , wherein said nucleic acid amplification product is DNA having a length that is selected from a range that is greater than or equal to 50 base pairs and less than or equal to 5000 base pairs.

25. The method of claim 1 , wherein said detection is performed in a point-of-care detection assay.

26. The method of claim 1 , further comprising identifying the presence or absence of nucleic acid amplification product.

27. The method of claim 1 , further comprising determining the concentration of the nucleic acid amplification product or the number of nucleic acid amplification product.

28. The method of claim 1 , wherein the nucleic acid amplification comprises isothermal amplification of RNA.

29. The method of claim 1 , wherein the detection of said nucleic acid amplification product is for the presence or absence of a pathogen or a genetic defect that predisposes an individual to a disease state.

30. The method of claim 29 , wherein the pathogen is a bacterial cell selected from the group consisting of: Listeria moncytogenes; Escherichia coli; Campylobacter jejune; Listeria innocua; and Lactobacillus acidophilus .

31. A method of detecting a nucleic acid amplification product in solution from a sample, said method comprising:
obtaining a sample comprising cells;
introducing said sample to an integrated biochip, wherein said integrated biochip comprises a microfluidic amplification chamber having an electrode array on one or more surfaces of said microfluidic amplification chamber, and performing the following steps on said integrated biochip:
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Assignees

• Bashir Rashid
• Liu Yi-Shao
• Salm Eric
• Chang Woo-Jin
• Watkins Nicholas N
• Univ Illinois

Inventors

• Bashir Rashid
• Liu Yi-Shao
• Salm Eric
• Chang Woo-Jin
• Watkins Nicholas N

Classifications

• B01L3/502761

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title

• B01L3/5027

  by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip · CPC title

• B03C5/026

  using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC] · CPC title

• B01L3/502746

  characterised by the means for controlling flow resistance, e.g. flow controllers, baffles or throttle valves · CPC title

• B01L3/0241

  Drop counters; Drop formers · CPC title