Method for separating nanoparticles and analyzing biological substance using microfluidic chip

Patent metadata

Field	Value
Publication number	US-9696301-B2
Application number	US-201314647876-A
Country	US
Kind code	B2
Filing date	Nov 20, 2013
Priority date	Nov 28, 2012
Publication date	Jul 4, 2017
Grant date	Jul 4, 2017

Abstract

The present invention relates to a method for separating nanoparticles and analyzing a biological substance using a microfluidic chip. The microfluidic chip of the present invention is effective in more sensitively and precisely detecting an analyte. According to the present invention, the use of the microfluidic chip enables the separation of nanoparticles through separation holes on the basis of size, achieving highly reliable analysis of a biological substance. In conclusion, the microfluidic chip of the present invention uses separation holes adapted to the size of nanoparticles to greatly increase the reliability of analysis of a biological substance, which will contribute to a marked improvement in the reliability of analysis based on microfluidics and a microfluidic system.

First claim

The invention claimed is:

1. A microfluidic chip comprising a sample inlet, a fluid inlet, and a fluidic channel wherein the fluidic channel consists of a reaction zone, a separation zone, and a discharge zone sequentially disposed in the downstream direction and the separation zone comprises a separation membrane formed with one or more separation holes through which nanoparticles pass,
wherein the separation zone of the fluidic channel has concave grooves formed in the lower layer of the fluidic channel and covered with the separation membrane, and
wherein the upper surface of the separation membrane is in contact with a barrier extending from the upper layer of the fluidic channel.

2. The microfluidic chip according to claim 1 , wherein a plurality of linear groups of the separation holes are arranged in a zigzag configuration.

3. The microfluidic chip according to claim 1 , wherein the barrier is in contact with a middle portion between the front end of the separation membrane toward the inlets and the rear end of the separation membrane toward the discharge zone.

4. The microfluidic chip according to claim 1 , wherein the separation holes are from 100 nm to 1000 nm in diameter.

5. The microfluidic chip according to claim 1 , wherein the separation membrane is produced by sequentially forming a silicon nitride film and a silicon oxide film on the surface of a substrate.

6. The microfluidic chip according to claim 1 , wherein a silicon oxide film is formed on the surface of the separation holes.

7. The microfluidic chip according to claim 1 , further comprising a magnetic force application unit provided between the reaction zone and the separation zone of the fluidic channel.

8. The microfluidic chip according to claim 7 , further comprising an outlet disposed between the magnetic force application unit and the separation zone to discharge unreacted samples therethrough.

9. A method for detecting a biological substance using the microfluidic chip according to claim 1 , the method comprising 1) introducing samples and a fluid into the fluidic channel of the microfluidic chip, 2) allowing the samples to react in the reaction zone to produce reaction products, 3) detecting the reaction products not passing through the separation holes present in the separation zone of the fluidic channel, and 4) analyzing the detected reaction products.

10. The method according to claim 9 , wherein magnetic nanoparticles, a biological substance, and a probe are introduced as the samples in step 1) and the samples are allowed to react to form magnetic nanoparticles-biological substance-probe complexes in step 2).

11. The method according to claim 10 , wherein a receptor bound to the magnetic nanoparticles and a receptor bound to the probe recognize the biological substance to form the magnetic nanoparticles-biological substance-probe complexes.

12. The method according to claim 9 , further comprising fixing and collecting unreacted magnetic nanoparticles and the magnetic nanoparticles-biological substance-probe complexes by a magnetic force application unit between steps 2) and 3).

13. The method according to claim 12 , further comprising discharging unreacted samples unfixed by the magnetic force application unit through an outlet after the fixing/collection step.

14. The method according to claim 13 , wherein after discharge of the unreacted samples, the application of the magnetic force by the magnetic force application unit is stopped to allow the unreacted magnetic nanoparticles and the magnetic nanoparticles-biological substance-probe complexes to move in the direction from the inlets of the fluidic channel toward the discharge zone.

15. The method according to claim 9 , wherein after movement of the unreacted magnetic nanoparticles and the magnetic nanoparticles-biological substance-probe complexes toward the discharge zone, the unreacted magnetic nanoparticles pass through the separation holes but the magnetic nanoparticles-biological substance-probe complexes do not pass through the separation holes.

16. A method for producing a separation membrane including separation holes for the microfluidic chip according to claim 1 , the method comprising 1) forming a silicon nitride film on a silicon substrate or a substrate comprising silicon by chemical vapor deposition (CVD), 2) forming 2 to 3 μm diameter separation holes in the substrate, and 3) forming a silicon oxide film on the substrate comprising the separation holes by chemical vapor deposition (CVD).

17. The method according to claim 16 , wherein, in step 3), the chemical vapor deposition (CVD) is continued until the diameter of the separation holes is reduced to a range of 100 nm to 1000 nm.

18. A method for detecting a biological substance using the microfluidic chip, wherein the microfluidic chip comprises a sample inlet, a fluid inlet, and a fluidic channel wherein the fluidic channel consists of a reaction zone, a separation zone, and a discharge zone sequentially disposed in the downstream direction and the separation zone comprises a separation membrane formed with one or more separation holes through which nanoparticles pass, the method comprising
1) introducing samples and a fluid into the fluidic channel of the microfluidic chip;
2) allowing the samples to react in the reaction zone to produce reaction products,
2-i) fixing and collecting unreacted magnetic nanoparticles and the magnetic nanoparticles-biological substance-probe complexes by a magnetic force application unit
2-ii) discharging unreacted samples unfixed by the magnetic force application unit through an outlet after the fixing/collection step;

3) detecting the reaction products not passing through the separation holes present in the separation zone of the fluidic channel; and
4) analyzing the detected reaction products,
wherein after discharge of the unreacted samples, the application of the magnetic force by the magnetic force application unit is stopped to allow the unreacted magnetic nanoparticles and the magnetic nanoparticles-biological substance-probe complexes to move in the direction from the inlets of the fluidic channel toward the discharge zone.

19. The method according to claim 18 , wherein magnetic nanoparticles, a biological substance, and a probe are introduced as the samples in step 1) and the samples are allowed to react to form magnetic nanoparticles-biological substance-probe complexes in step 2).

20. The method according to claim 19 , wherein a receptor bound to the magnetic nanoparticles and a receptor bound to the probe recognize the biological substance to form the magnetic nanoparticles-biological substance-probe complexes.

Assignees

• Snu R&Db Foundation
• Univ Seoul Nat R & Db Found

Inventors

• Kim Yong-Kweon
• Lee Yoon-Sik
• Koh Yul
• Kang Homan

Classifications

• B01L2300/0867

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

• C23C16/0245

  by etching with a plasma · CPC title

• B01L2400/043

  magnetic forces · CPC title

• C23C16/345

  Silicon nitride · CPC title

• C23C16/401

  containing silicon · CPC title