Combined extraction and PCR systems

Patent metadata

Field	Value
Publication number	US-11220706-B2
Application number	US-201916448713-A
Country	US
Kind code	B2
Filing date	Jun 21, 2019
Priority date	Dec 23, 2016
Publication date	Jan 11, 2022
Grant date	Jan 11, 2022

Abstract

The disclosure provides methods and systems for analyzing fluid samples comprising obtaining fluid samples in at least one cavity of a substrate and introducing also buffers and/or reagents in the cavity, performing nucleic acid extraction and/or purification in the cavity, and performing nucleic acid amplification in the same cavity.

First claim

What is claimed is:

1. A method for analyzing a fluid sample, the method comprising:
inserting a fluid sample into a first fluid reservoir of a substrate and buffers or reagents into a second fluid reservoir of the substrate, wherein the first fluid reservoir and the second fluid reservoir are fluidly coupled to a first end of a meandering microfluidic channel that is fluidly coupled on a second end to at least one cavity of the substrate, wherein the fluid sample and the buffers or reagents mix together as they pass through the meandering microfluidic channel and into the at least one cavity;
performing nucleic acid extraction or purification in the cavity; and
subsequently performing nucleic acid amplification in the cavity, wherein the cavity comprises a silicon-based pillar filter.

2. The method of claim 1 , wherein the silicon-based pillar filter comprises a plurality of micropillar structures.

3. The method of claim 1 , wherein the silicon-based pillar filter comprises silicon pillars.

4. The method of claim 1 , further comprising dividing the fluid sample over a multitude of cavities.

5. The method of claim 1 , further comprising performing nucleic acid amplification after performing nucleic acid extraction without performing an elution step in between.

6. The method of claim 1 , wherein the second end of the meandering microfluidic channel is fluidly coupled to a plurality of cavities, wherein the method further comprises performing, for each cavity, nucleic acid extraction in the cavity and consequently performing nucleic acid amplification in the cavity.

7. The method of claim 1 , wherein the second end of the meandering microfluidic channel is fluidly coupled to a plurality of cavities, wherein the method further comprises:
for each cavity, performing nucleic acid extraction in the cavity and subsequently performing nucleic acid amplification in the cavity, and
applying a digital PCR analysis for the plurality of cavities for determining a concentration of a target in the fluid sample.

8. The method of claim 6 , further comprising providing a predetermined volume of fluid containing nucleic acid carriers per cavity, for obtaining an average of less than a nucleic acid carrier per cavity.

9. The method of claim 8 , further comprising providing a volume of less than 10 nanoliters per cavity.

10. A microfluidics system for analyzing a fluid sample, the microfluidics system comprising
a substrate comprising:
a first fluid reservoir configured to receive the fluid sample;
a second fluid reservoir configured to receive buffers or reagents;
a meandering microfluidic channel having a first end that is fluidly coupled to the first fluid reservoir and the second fluid reservoir, wherein the meandering microfluidic channel facilitates mixing of the fluid sample and the buffers or reagents as the fluid sample and the buffers or reagents pass through the meandering microfluidic channel; and
at least one cavity fluidly coupled to a second end of the meandering microfluidic channel, wherein the at least one cavity comprises a surface configured to facilitate nucleic acid extraction, and a silicon-based pillar filter; and

a controller configured to control conditions of the cavity to facilitate nucleic acid extraction in the cavity and to subsequently control conditions of the cavity to facilitate nucleic acid amplification within the cavity.

11. The microfluidics system of claim 10 , wherein the silicon-based pillar filter comprises a plurality of micropillar structures.

12. The microfluidics system of claim 10 , wherein the silicon-based pillar filter comprises silicon pillars.

13. The microfluidics system of claim 10 , wherein the controller is configured to facilitate performance of nucleic acid amplification after performance of nucleic acid extraction without performing an elution step in between.

14. The microfluidics system of claim 10 , wherein the second end of the meandering microfluidic channel is fluidly coupled to a plurality of cavities and wherein the controller is configured to induce nucleic acid extraction and subsequently nucleic acid amplification in each of the cavities.

15. The microfluidics system of claim 14 , wherein the microfluidics system further comprises a processor configured to perform a digital PCR analysis based on the plurality of cavities.

16. The microfluidics system of claim 14 , wherein each of the plurality of cavities has a maximum capacity of 10 nL.

17. The microfluidics system of claim 10 , wherein the microfluidics surface of the at least one cavity comprises silicon oxide or wherein the substrate comprises at least one trench for thermally isolating the at least one cavity.

18. The microfluidics system of claim 10 , wherein the system is further configured to provide capillary pumping, comprises a mixer for mixing buffers or reagents, or comprises a heater for adjusting a temperature of the cavity.

19. A diagnostic device comprising the microfluidics system of claim 10 , the diagnostic device being a lab-on-chip device.

20. A polymerase chain reaction (PCR) method comprising:
inserting into a microfluidics system a fluid sample and one or more buffers or reagents, wherein the microfluidics system comprises:
a substrate comprising:
a first fluid reservoir configured to receive the fluid sample;
a second fluid reservoir configured to receive the buffers or reagents;
a meandering microfluidic channel having a first end that is fluidly coupled to the first fluid reservoir and the second fluid reservoir, wherein the meandering microfluidic channel facilitates mixing of the fluid sample and the buffers or reagents as the fluid sample and the buffers or reagents pass through the meandering microfluidic channel; and
at least one cavity fluidly coupled to a second end of the meandering microfluidic channel, wherein the at least one cavity comprises a surface configured to facilitate nucleic acid extraction, and a silicon-based pillar filter; and

a controller configured to control conditions of the cavity to facilitate nucleic acid extraction in the cavity and to subsequently control conditions of the cavity to facilitate nucleic acid amplification within the cavity;

extracting nucleic acid from the at least one cavity; and
processing the extracted nucleic acid to screen for antibiotic resistance, identify relative changes in gene expression, or link multiple nucleic acids targets to a single genetic carrier.

Assignees

• Imec Vzw

Inventors

• Stakenborg Tim
• Fiorini Paolo

Classifications

• B01L3/502746

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

• 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

• B01L2300/0867

  Multiple inlets and one sample wells, e.g. mixing, dilution · CPC title

• B01L3/502715

  characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title