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

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.

Figure 1. Sequence-specific molecular identification of DNA at fM to aM concentrations via isothermal amplification in a non-stick-coated metal microfluidic bioreactor with a minimum reagent consumption of 40 nL and detection limit of three genomic copies.

Figure 2. 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°.

Figure 3. 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).

Figure 4. Comparison of the isothermal nucleic acid amplification reactions in an Eppendorf tube vs. the non-stick-coated metal micro-nanoliter fluidic bioreactor.

(a) Real-time curves of common 25-μL isothermal nucleic acid amplification reactions in the Eppendorf tube using an ABI 7700 Fast Real-Time PCR System, where the DNA template concentrations were 1.3 × 10², 1.3 × 10¹, 1.3 × 10⁰, 1.3 × 10⁻¹, 1.3 × 10⁻², and 1.3 × 10⁻³ fM and the times at the second derivative inflexions of the exponential DNA amplification curves were 16.3, 18.4, 20.8, 23.7, and 28.9 min and 45.0 min without amplification. (b) Real-time curves of 7-μL nucleic acid amplification reactions in the non-stick-coated metal micro-nanoliter fluidic bioreactor measured using our portable real-time fluorescent confocal detector, where the DNA template concentrations were 1.3 × 10², 1.3 × 10¹, 1.3 × 10⁰, 1.3 × 10⁻¹, 1.3 × 10⁻², and 1.3 × 10⁻³ fM and the times at the second derivative inflexions of the exponential DNA amplification curves were 14.8, 17.4, 19.6, 21.5, 24.5, and 28.7 min.

Figure 5. Comparison of DNA amplification in the non-stick-coated metal micro-nanoliter fluidic bioreactors at a DNA template concentration of 1.3 × 10¹ fM and different reaction volumes (from microliter to nanoliter).

The horizontal axis corresponds to the time of DNA amplification, and the vertical axis is the relative signal intensity. The six curves correspond to the six different micro-nanoliter bioreactors: 7 μL, 3 μL, 785 nL, 392 nL, 98 nL, and 40 nL. Each of the reactions was repeated five times. The times at the second derivative inflexions of the exponential DNA amplification curves for the six different micro-nanoliter reaction assays were 17.32, 17.48, 16.73, 17.14, 17.22, and 16.54 min, and the time CV at these inflexions was ~1.98%.

Figure 6. Sensitivity analysis of isothermal DNA amplification reactions in the non-stick-coated metal micro-nanoliter fluidic bioreactor.

The horizontal axis corresponds to the time of the DNA amplification process, and the vertical axis is the relative fluorescent intensity. The six curves correspond to the six different copy numbers of DNA template used: 5.6 × 10⁴, 5.6 × 10³, 5.6 × 10², 5.6 × 10¹, 6, and 3 copies. The times at the second derivative inflexions of the exponential DNA amplification curves for the six different template concentrations were 17.32, 19.42, 21.34, 23.26, 28.71, and 30.62 min, respectively.

Figure 7. 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.