Methods and compositions for paper-based and hybrid microfluidic devices integrated with nucleic acid amplification for disease diagnosis

Patent metadata

Field	Value
Publication number	US-10875024-B2
Application number	US-201514796127-A
Country	US
Kind code	B2
Filing date	Jul 10, 2015
Priority date	Jul 10, 2014
Publication date	Dec 29, 2020
Grant date	Dec 29, 2020

Abstract

Certain embodiments are directed to paper and its hybrid microfluidic devices integrated with nucleic acid amplification for simple, cost-effective, rapid, and sensitive pathogen detection, especially in low-resource settings.

First claim

The invention claimed is:

1. A biochip for pathogen detection comprising a polymer/paper hybrid microfluidic device comprising at least four separate layers, wherein the at least four separate layers comprise:
a top polymer layer comprising an inlet reservoir and microchannels connected to the inlet reservoir;
a middle paper layer positioned below the top polymer layer, said middle paper layer comprising:
(a) an amplification layer having at least one cylindrical amplification well forming a cylindrical hole in the amplification layer having an open top portion being in fluid communication with the inlet reservoir of the top layer, a closed bottom portion positioned 1 millimeter (mm) to 4 mm below the top layer, and a horizontal cross-sectional diameter 0.5 mm to 3 mm, the at least one amplification well is configured to receive an amplification mixture and perform a nucleic acid amplification reaction during use to produce an amplified product;
(b) a detection layer in fluid communication with the amplification layer, the detection layer comprising at least one cylindrical detection well forming a cylindrical hole in the detection layer having a top portion and a closed bottom portion positioned 1 mm to 4 mm below the top layer, and a horizontal cross-sectional diameter 0.5 mm to 3 mm, said at least one detection well is in fluid communication with the at least one amplification well and is configured to receive an amplified product from the amplification well during use;

a paper insert layer comprising an absorbed detection reagent, said paper insert layer having a thickness of 0.05 to 0.25 mm and a pore diameter of 5 micrometers (μm) to 15 μm and is positioned inside the at least one detection well and is disposed directly on the closed bottom part of the at least one cylindrical detection well, said paper insert layer forms a separate layer within the cylindrical detection well and is thinner than the detection layer; and
a polymer or glass support layer positioned below the detection layer,
wherein the top polymer layer, the middle paper layer, the paper insert layer and the polymer or glass support layer form the at least four separate layers of the polymer/paper hybrid microfluidic device.

2. The biochip of claim 1 , wherein the polymer/paper hybrid microfluidic device comprises a plurality of amplification wells.

3. The biochip of claim 1 , wherein the polymer is polydimethysiloxane (PDMS).

4. The biochip of claim 1 , wherein the absorbed detection reagent on the paper insert layer comprises DNA primers.

5. The biochip of claim 1 , wherein the polymer or glass support layer is glass.

6. The biochip of claim 1 , wherein the amplification layer further comprises multiple sublayers.

7. The biochip of claim 6 , wherein the multiple sublayers are cut to form amplification wells.

8. The biochip of claim 1 , wherein the detection layer comprises the at least one cylindrical detection well linked to reagent delivery channels.

9. The biochip of claim 1 , further comprising a sequence specific probe in the at least one cylindrical detection well.

10. The biochip of claim 1 , wherein the top polymer layer further comprises a filter configured to remove components of a sample and to provide a filtered sample to the at least one cylindrical amplification well.

Assignees

• Li Xiujun
• Dou Maowei
• Dominguez Delfina
• Univ Texas

Inventors

• Li Xiujun
• Dou Maowei
• Dominguez Delfina

Classifications

• C12Q1/689 Primary

  for bacteria · CPC title

• C12Q1/6867

  Replicase-based amplification, e.g. using Q-beta replicase · CPC title

• C12Q1/6865

  Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS] · CPC title

• B01L7/52

  with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples · CPC title

• B01L2300/0867

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