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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.


Comparison of DNA amplification in uncoated and non-stick-coated metal micro-nanoliter fluidic chips.(a) Isothermal DNA amplification in the uncoated metal micro-nanoliter fluidic chip, which displays obvious differences among the five parallel bioreactors. (b) Isothermal DNA amplification in the non-stick-coated metal micro-nanoliter fluidic chip, where the amplification in the five parallel bioreactors displays good consistency and the time difference at the second derivative inflexions of the exponential DNA amplification curves for the five bioreactors are within 0.5 min. (c) Normalization processing of the isothermal DNA amplification curves in (b).
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f3: Comparison of DNA amplification in uncoated and non-stick-coated metal micro-nanoliter fluidic chips.(a) Isothermal DNA amplification in the uncoated metal micro-nanoliter fluidic chip, which displays obvious differences among the five parallel bioreactors. (b) Isothermal DNA amplification in the non-stick-coated metal micro-nanoliter fluidic chip, where the amplification in the five parallel bioreactors displays good consistency and the time difference at the second derivative inflexions of the exponential DNA amplification curves for the five bioreactors are within 0.5 min. (c) Normalization processing of the isothermal DNA amplification curves in (b).

Mentions: Using the same DNA template concentration of 1.3 fM (10−15 M) and 7-μL reaction mixtures in every bioreactor cell, isothermal DNA amplification in uncoated and non-stick-coated metal micro-nanoliter fluidic chips was performed using our portable confocal detector. The contrast between DNA isothermal amplified curves is shown in Figure 3. Figure 3 (a) corresponds to isothermal DNA amplification in the uncoated metal micro-nanoliter fluidic chip, where the times at the second derivative inflexions of the exponential DNA amplification curves for the five bioreactor cells were 24.85, 27.29, 27.80, 28.18, and 29.53 min. The maximum of the time difference at these inflexions was ~4.68 min. At the top of the amplification curves, the fluorescence intensities of the five bioreactor cells ranged from 3600–6900; the percent deviation of the fluorescence intensity was ~91.7%. Figure 3 (b) is isothermal DNA amplification in the non-stick-coated metal micro-nanoliter fluidic chip, where the times at the second derivative inflexions of the exponential DNA amplification curves for the five bioreactor cells corresponded to 19.42, 19.45, 19.59, 19.60, and 19.92 min. The maximum of the time differences at these inflexions was 0.5 min. At the top of the amplification curves, the fluorescence intensities of the five bioreactor cells ranged from 4000–4500; the percent deviation of the fluorescence intensity was ~11%. Figure 3 (c) shows the isothermal DNA amplification curves in Figure 3 (b) after normalization processing, which is generally used in commercial RT-PCR setups.


fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor
Comparison of DNA amplification in uncoated and non-stick-coated metal micro-nanoliter fluidic chips.(a) Isothermal DNA amplification in the uncoated metal micro-nanoliter fluidic chip, which displays obvious differences among the five parallel bioreactors. (b) Isothermal DNA amplification in the non-stick-coated metal micro-nanoliter fluidic chip, where the amplification in the five parallel bioreactors displays good consistency and the time difference at the second derivative inflexions of the exponential DNA amplification curves for the five bioreactors are within 0.5 min. (c) Normalization processing of the isothermal DNA amplification curves in (b).
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Related In: Results  -  Collection

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f3: Comparison of DNA amplification in uncoated and non-stick-coated metal micro-nanoliter fluidic chips.(a) Isothermal DNA amplification in the uncoated metal micro-nanoliter fluidic chip, which displays obvious differences among the five parallel bioreactors. (b) Isothermal DNA amplification in the non-stick-coated metal micro-nanoliter fluidic chip, where the amplification in the five parallel bioreactors displays good consistency and the time difference at the second derivative inflexions of the exponential DNA amplification curves for the five bioreactors are within 0.5 min. (c) Normalization processing of the isothermal DNA amplification curves in (b).
Mentions: Using the same DNA template concentration of 1.3 fM (10−15 M) and 7-μL reaction mixtures in every bioreactor cell, isothermal DNA amplification in uncoated and non-stick-coated metal micro-nanoliter fluidic chips was performed using our portable confocal detector. The contrast between DNA isothermal amplified curves is shown in Figure 3. Figure 3 (a) corresponds to isothermal DNA amplification in the uncoated metal micro-nanoliter fluidic chip, where the times at the second derivative inflexions of the exponential DNA amplification curves for the five bioreactor cells were 24.85, 27.29, 27.80, 28.18, and 29.53 min. The maximum of the time difference at these inflexions was ~4.68 min. At the top of the amplification curves, the fluorescence intensities of the five bioreactor cells ranged from 3600–6900; the percent deviation of the fluorescence intensity was ~91.7%. Figure 3 (b) is isothermal DNA amplification in the non-stick-coated metal micro-nanoliter fluidic chip, where the times at the second derivative inflexions of the exponential DNA amplification curves for the five bioreactor cells corresponded to 19.42, 19.45, 19.59, 19.60, and 19.92 min. The maximum of the time differences at these inflexions was 0.5 min. At the top of the amplification curves, the fluorescence intensities of the five bioreactor cells ranged from 4000–4500; the percent deviation of the fluorescence intensity was ~11%. Figure 3 (c) shows the isothermal DNA amplification curves in Figure 3 (b) after normalization processing, which is generally used in commercial RT-PCR setups.

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.