Real-Time Monitoring of a Nucleic Acid Amplification Reaction Using a Mass Sensor Based on a Quartz-Crystal Microbalance

Abstract

Nucleic acid amplification reactions such as polymerase chain reaction (PCR), which uses a DNA polymerase to amplify individual double-stranded DNA fragments, are a useful technique for visualizing the presence of specific genomes. Although the fluorescent labeling method is mainly used with DNA amplification, other detection methods should be considered for further improvements, such as miniaturization and cost reduction, of reaction-monitoring devices. In this study, the quartz-crystal microbalance (QCM) method, which can measure nanogram-order masses, was applied for the real-time detection of DNA fragments in a solution with nucleic acids. This was combined with an isothermal nucleic acid amplification reaction based on the recombinase polymerase amplification (RPA) method, which allowed DNA amplification at a constant temperature. When the DNA amplification reaction was initiated on a QCM sensor plate with an immobilized primer DNA strand, a significant increase in mass was observed compared to when the primer DNA was not immobilized. QCM was shown to be sufficiently sensitive for the in situ detection of amplified DNA fragments. Combining a portable QCM device and RPA offers a sensitive point-of-care method for detecting nucleic acids.

Keywords: DNA fragment detection; isothermal nucleic acid amplification reaction; mass sensor; polymerase chain reaction; quartz-crystal microbalance; recombinase polymerase amplification.

Figure 1

Schematic illustrations of (a) a 27 MHz QCM device equipped with a temperature-control system and a QCM sensor cell, (b) the process of primer DNA immobilization on a QCM sensor surface, and (c) RPA reaction monitoring in a QCM cell.

Figure 2

Time courses of the frequency changes (∆F) of RPA reaction monitoring in response to the addition of magnesium acetate (Mg²⁺ ions) into the QCM cell filled with (1) RPA reaction solution and (2) the solution in the absence of DNA primers, and (3) in response to the addition of water instead of the Mg²⁺ ion solution. Repetition (n = 3) of curve (1) resulted in a mean of −5800 Hz and standard deviation of 210 Hz for the frequency response at 20 min.

Figure 3

Time courses of the frequency changes (∆F) of RPA reaction monitoring on the QCM plate (1) when non-primer DNA was immobilized and (2) when no primer DNA was immobilized, responding to the addition of Mg²⁺ ions into the QCM cell. The sequence of non-primer DNA: bio-5′-CGCCCCACGTAAAGCGACTAAAACCCCAGG-3′.

Figure 4

Time courses of the frequency changes (∆F) of RPA reaction monitoring on the QCM plate (1) when the primer DNA was immobilized and (2) when no primer DNA was immobilized in response to the addition of Mg²⁺ ions and the DNA primers were injected separately into the QCM cells.

Figure 5

Time courses of the frequency changes (∆F) of RPA reaction monitoring in RPA reaction solution diluted to (1) 1/2, (2) 1/4, and (3) 1/8 using Milli-Q-water. The experimental conditions: (1) [Mg²⁺] = 7 mM, [Primer] = 0.24 µM, (2) [Mg²⁺] = 3.5 mM, [Primer] = 0.12 µM, and (3) [Mg²⁺] = 1.75 mM, [Primer] = 0.06 µM at 35 °C.

Figure 6

Time courses of the frequency changes (∆F) of RPA reaction monitoring with the addition of Mg²⁺ ions and after resetting monitoring with the addition of EDTA solution on the QCM plate (1) when primer DNA was immobilized and (2) when no primer DNA was immobilized. The experimental condition: [EDTA] = 100 mM.

Figure 7

(a) Time courses of the frequency changes (∆F) of RPA reaction monitoring in response to the addition of Mg²⁺ ions using pipetting rather than stirring in the QCM device. Repetition (n = 3) of the curve resulted in a mean of −4900 Hz and standard deviation of 130 Hz for the frequency response at 20 min. (b) Plots of fluorescence intensity when the dsDNA staining agent was mixed with the reaction solution and sampled from a QCM cell every 2 min and (c) an image from the fluorescence analysis. The relative fluorescence intensity of the samples was calculated using the fluorescence intensity of 81 ng of dsDNA amount as 1.

Figure 8

Time courses of the frequency changes (∆F) in RPA reaction monitoring in response to the addition of Mg²⁺ ions without using pipetting rather than stirring in the custom-made QCM device.