Limits...
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.


Contrast of the surface characteristics of the bioreactor with and without the silicone coating.(a) and (b) are the surface structure of the uncoated bioreactor at 40× and 2000× magnification, respectively; (c) is the contact angle of the liquid droplet on the surface of the uncoated bioreactor, ~71.3°. (d) and (e) are the surface structure of the non-stick-coated bioreactor at 40× and 2000× magnification, respectively; (f) corresponds the contact angle of the liquid droplet on the surface of the non-stick-coated bioreactor, ~103.1°.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5384283&req=5

f2: Contrast of the surface characteristics of the bioreactor with and without the silicone coating.(a) and (b) are the surface structure of the uncoated bioreactor at 40× and 2000× magnification, respectively; (c) is the contact angle of the liquid droplet on the surface of the uncoated bioreactor, ~71.3°. (d) and (e) are the surface structure of the non-stick-coated bioreactor at 40× and 2000× magnification, respectively; (f) corresponds the contact angle of the liquid droplet on the surface of the non-stick-coated bioreactor, ~103.1°.

Mentions: The surface adsorption of DNA molecules to a bioreactor has a significant effect on the dynamic detection of the nucleic acid amplification signal in micro-nanoliter assays and usually delays the detection response and renders the fluorescent signal of DNA amplification unsteady. The surface adsorption is mainly due to surface roughness and the surface not being inert. The surface structure of a bioreactor on metal micro-nanoliter fluidic chips is usually rough from machine fabrication. Thus, a non-stick silicone coating was used to coat the metal micro-nanoliter fluidic chips to make the surfaces of the bioreactor smooth and inert, yielding a high and steady DNA amplification signal. The surface structure of uncoated and non-stick-coated bioreactors was imaged using a scanning electron microscope (S-3000N, Hitachi Corporation, Japan) as shown in Figure 2. In Figure 2, (a) and (b) are the surface structure of the original bioreactor at 40× and 2000× magnification, respectively, displaying a rough microscopic structure from machine fabrication. (c) corresponds to the contact angle of a liquid droplet on the surface of the original bioreactor (71.3°) after the surface was washed with anhydrous ethanol (tested by the OCA-15plus contact angle system, DataPhysics Instruments GmbH, Germany). Figure 2 (d) and (e) are the surface structure of the non-stick-coated bioreactor at 40× and 2000× magnification, respectively, where the surfaces of the bioreactor were covered with an even silicone protective film of 10–20 μm in thickness. (f) corresponds to the contact angle of a liquid droplet on the surface of the non-stick-coated bioreactor (103.1°). The silicone protective film is inert to biological samples and general reagents, can withstand a carving stress of >2000 kg/mm2, and has a melting point 280°C.


fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor
Contrast of the surface characteristics of the bioreactor with and without the silicone coating.(a) and (b) are the surface structure of the uncoated bioreactor at 40× and 2000× magnification, respectively; (c) is the contact angle of the liquid droplet on the surface of the uncoated bioreactor, ~71.3°. (d) and (e) are the surface structure of the non-stick-coated bioreactor at 40× and 2000× magnification, respectively; (f) corresponds the contact angle of the liquid droplet on the surface of the non-stick-coated bioreactor, ~103.1°.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Contrast of the surface characteristics of the bioreactor with and without the silicone coating.(a) and (b) are the surface structure of the uncoated bioreactor at 40× and 2000× magnification, respectively; (c) is the contact angle of the liquid droplet on the surface of the uncoated bioreactor, ~71.3°. (d) and (e) are the surface structure of the non-stick-coated bioreactor at 40× and 2000× magnification, respectively; (f) corresponds the contact angle of the liquid droplet on the surface of the non-stick-coated bioreactor, ~103.1°.
Mentions: The surface adsorption of DNA molecules to a bioreactor has a significant effect on the dynamic detection of the nucleic acid amplification signal in micro-nanoliter assays and usually delays the detection response and renders the fluorescent signal of DNA amplification unsteady. The surface adsorption is mainly due to surface roughness and the surface not being inert. The surface structure of a bioreactor on metal micro-nanoliter fluidic chips is usually rough from machine fabrication. Thus, a non-stick silicone coating was used to coat the metal micro-nanoliter fluidic chips to make the surfaces of the bioreactor smooth and inert, yielding a high and steady DNA amplification signal. The surface structure of uncoated and non-stick-coated bioreactors was imaged using a scanning electron microscope (S-3000N, Hitachi Corporation, Japan) as shown in Figure 2. In Figure 2, (a) and (b) are the surface structure of the original bioreactor at 40× and 2000× magnification, respectively, displaying a rough microscopic structure from machine fabrication. (c) corresponds to the contact angle of a liquid droplet on the surface of the original bioreactor (71.3°) after the surface was washed with anhydrous ethanol (tested by the OCA-15plus contact angle system, DataPhysics Instruments GmbH, Germany). Figure 2 (d) and (e) are the surface structure of the non-stick-coated bioreactor at 40× and 2000× magnification, respectively, where the surfaces of the bioreactor were covered with an even silicone protective film of 10–20 μm in thickness. (f) corresponds to the contact angle of a liquid droplet on the surface of the non-stick-coated bioreactor (103.1°). The silicone protective film is inert to biological samples and general reagents, can withstand a carving stress of >2000 kg/mm2, and has a melting point 280°C.

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.