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fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor

View Article: PubMed Central - PubMed

ABSTRACT

A sensitive DNA isothermal amplification method for the detection of DNA at fM to aM concentrations for pathogen identification was developed using a non-stick-coated metal microfluidic bioreactor. A portable confocal optical detector was utilized to monitor the DNA amplification in micro- to nanoliter reaction assays in real-time, with fluorescence collection near the optical diffraction limit. The non-stick-coated metal microfluidic bioreactor, with a surface contact angle of 103°, was largely inert to bio-molecules, and DNA amplification could be performed in a minimum reaction volume of 40 nL. The isothermal nucleic acid amplification for Mycoplasma pneumoniae identification in the non-stick-coated microfluidic bioreactor could be performed at a minimum DNA template concentration of 1.3 aM, and a detection limit of three copies of genomic DNA was obtained. This microfluidic bioreactor offers a promising clinically relevant pathogen molecular diagnostic method via the amplification of targets from only a few copies of genomic DNA from a single bacterium.

No MeSH data available.


Fabrication of the microfluidic chip.(a) The micro-nanoliter fluidic chip consists of four independent reaction areas; each reaction area has one buffer cell and five bioreactor cells. The inlet hole is 1.2 mm in diameter, the same size as an Eppendorf tip. The bioreactor cell is 3.0 mm in diameter and 1.0 mm in depth, yielding a 7-μl bioreactor cell. The channel is 0.5 mm in both width and depth. (b) The processes of micro-nanoliter fluidic chip fabrication, where the primary chip was first fabricated by the Computer Numerical Control Machining Center and then coated with the non-stick coating to decrease the machining roughness and make the surface inert. After the chip was washed and dried, a thin polycarbonate film was affixed to the surface for encapsulation.
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f7: Fabrication of the microfluidic chip.(a) The micro-nanoliter fluidic chip consists of four independent reaction areas; each reaction area has one buffer cell and five bioreactor cells. The inlet hole is 1.2 mm in diameter, the same size as an Eppendorf tip. The bioreactor cell is 3.0 mm in diameter and 1.0 mm in depth, yielding a 7-μl bioreactor cell. The channel is 0.5 mm in both width and depth. (b) The processes of micro-nanoliter fluidic chip fabrication, where the primary chip was first fabricated by the Computer Numerical Control Machining Center and then coated with the non-stick coating to decrease the machining roughness and make the surface inert. After the chip was washed and dried, a thin polycarbonate film was affixed to the surface for encapsulation.

Mentions: The structure of the micro-nanoliter fluidic chips was designed as shown in Figure 7(a), where the chip is 60 mm in diameter and 2.5–3.0 mm in thickness. The fixed hole in the center of the chip was used to fasten the chip to the rotation stage of the detection system. Both the inlet and outlet holes are 1.2 mm in diameter to load reagents and sample by the Eppendorf head. The bioreactor cell was produced with different micro-nanoliter volumes of 40 nL to 7 μL, and the channel was carved with different sizes (0.5–0.1 mm) in both width and depth to decrease reagent consumption. The buffer cell was 3.0 mm in diameter and 0.5 mm in depth, which is used to collect air from the injected sample to eliminate bubbles from the bioreactor cells. The inlet hole was connected to the buffer cell by the channel on the back of the chip. Micro-nanoliter fluidic chips were fabricated by the process shown in Figure 7(b). First, the bioreactor cell, channel, buffer cell, inlet hole, outlet hole, and fixed hole were produced on the Al plate with a Computer Numerical Control Machining Center JT-M960L (Jiatai Numerical Control Co., Ltd, Quanzhou, China), and the primary micro-nanoliter fluidic chip was obtained. Second, the surface of the micro-nanoliter fluidic chip was covered with the non-stick coating layer to approximately 10–20 μm thickness to make the surface of the chip smooth and inert to DNA molecules. Then, the chip was washed using anhydrous ethanol and dried by nitrogen. Finally, a thin polycarbonate film (~0.1 mm) from ABI Corporation was tightly affixed to the surface of the micro-nanoliter fluidic chip for encapsulation.


fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor
Fabrication of the microfluidic chip.(a) The micro-nanoliter fluidic chip consists of four independent reaction areas; each reaction area has one buffer cell and five bioreactor cells. The inlet hole is 1.2 mm in diameter, the same size as an Eppendorf tip. The bioreactor cell is 3.0 mm in diameter and 1.0 mm in depth, yielding a 7-μl bioreactor cell. The channel is 0.5 mm in both width and depth. (b) The processes of micro-nanoliter fluidic chip fabrication, where the primary chip was first fabricated by the Computer Numerical Control Machining Center and then coated with the non-stick coating to decrease the machining roughness and make the surface inert. After the chip was washed and dried, a thin polycarbonate film was affixed to the surface for encapsulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384283&req=5

f7: Fabrication of the microfluidic chip.(a) The micro-nanoliter fluidic chip consists of four independent reaction areas; each reaction area has one buffer cell and five bioreactor cells. The inlet hole is 1.2 mm in diameter, the same size as an Eppendorf tip. The bioreactor cell is 3.0 mm in diameter and 1.0 mm in depth, yielding a 7-μl bioreactor cell. The channel is 0.5 mm in both width and depth. (b) The processes of micro-nanoliter fluidic chip fabrication, where the primary chip was first fabricated by the Computer Numerical Control Machining Center and then coated with the non-stick coating to decrease the machining roughness and make the surface inert. After the chip was washed and dried, a thin polycarbonate film was affixed to the surface for encapsulation.
Mentions: The structure of the micro-nanoliter fluidic chips was designed as shown in Figure 7(a), where the chip is 60 mm in diameter and 2.5–3.0 mm in thickness. The fixed hole in the center of the chip was used to fasten the chip to the rotation stage of the detection system. Both the inlet and outlet holes are 1.2 mm in diameter to load reagents and sample by the Eppendorf head. The bioreactor cell was produced with different micro-nanoliter volumes of 40 nL to 7 μL, and the channel was carved with different sizes (0.5–0.1 mm) in both width and depth to decrease reagent consumption. The buffer cell was 3.0 mm in diameter and 0.5 mm in depth, which is used to collect air from the injected sample to eliminate bubbles from the bioreactor cells. The inlet hole was connected to the buffer cell by the channel on the back of the chip. Micro-nanoliter fluidic chips were fabricated by the process shown in Figure 7(b). First, the bioreactor cell, channel, buffer cell, inlet hole, outlet hole, and fixed hole were produced on the Al plate with a Computer Numerical Control Machining Center JT-M960L (Jiatai Numerical Control Co., Ltd, Quanzhou, China), and the primary micro-nanoliter fluidic chip was obtained. Second, the surface of the micro-nanoliter fluidic chip was covered with the non-stick coating layer to approximately 10–20 μm thickness to make the surface of the chip smooth and inert to DNA molecules. Then, the chip was washed using anhydrous ethanol and dried by nitrogen. Finally, a thin polycarbonate film (~0.1 mm) from ABI Corporation was tightly affixed to the surface of the micro-nanoliter fluidic chip for encapsulation.

View Article: PubMed Central - PubMed

ABSTRACT

A sensitive DNA isothermal amplification method for the detection of DNA at fM to aM concentrations for pathogen identification was developed using a non-stick-coated metal microfluidic bioreactor. A portable confocal optical detector was utilized to monitor the DNA amplification in micro- to nanoliter reaction assays in real-time, with fluorescence collection near the optical diffraction limit. The non-stick-coated metal microfluidic bioreactor, with a surface contact angle of 103°, was largely inert to bio-molecules, and DNA amplification could be performed in a minimum reaction volume of 40 nL. The isothermal nucleic acid amplification for Mycoplasma pneumoniae identification in the non-stick-coated microfluidic bioreactor could be performed at a minimum DNA template concentration of 1.3 aM, and a detection limit of three copies of genomic DNA was obtained. This microfluidic bioreactor offers a promising clinically relevant pathogen molecular diagnostic method via the amplification of targets from only a few copies of genomic DNA from a single bacterium.

No MeSH data available.