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. 2023 Feb 9;13(2):251.
doi: 10.3390/bios13020251.

Analysis of the Interaction between DNA Aptamers and Cytochrome C on the Surface of Lipid Films and on the MUA Monolayer: A QCM-D Study

Marek Tatarko  1 , Sandro Spagnolo  1 , Martin Csiba  1 , Veronika Šubjaková  1 , Tibor Hianik  1
Affiliations

Analysis of the Interaction between DNA Aptamers and Cytochrome C on the Surface of Lipid Films and on the MUA Monolayer: A QCM-D Study

Marek Tatarko et al. Biosensors (Basel). .

Abstract

We analyzed the possibility of the detection of cytochrome c (cyt c) being physically adsorbed on lipid films or covalently bounded to 11-mercapto-1-undecanoic acid (MUA) chemisorbed on the gold layer using quartz crystal microbalance with dissipation monitoring (QCM-D). The negatively charged lipid film composed of a mixture of zwitterionic DMPC and negatively charged DMPG phospholipids at a molar ratio of 1:1 allowed the formation of a stable cyt c layer. Addition of DNA aptamers specific to cyt c, however, resulted in removal of cyt c from the surface. The interaction of cyt c with the lipid film and its removal by DNA aptamers were accompanied by changes in viscoelastic properties evaluated using the Kelvin-Voigt model. Cyt c covalently bound to MUA also provided a stable protein layer already at its relatively low concentrations (0.5 μM). A decrease in the resonant frequency following the addition of gold nanowires (AuNWs) modified by DNA aptamers was observed. The interaction of aptamers with cyt c on the surface can be a combination of specific and non-specific interactions due to electrostatic forces between negatively charged DNA aptamers and positively charged cyt c.

Keywords: DNA aptamers; QCM-D; cytochrome c; gold nanowires; lipid films.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The scheme of functionalization of AuNWs using amino-modified DNA aptamers specific to cyt c (NH2-Apt).
Figure 2
Figure 2
The scheme of preparation of the lipid film at the surface of the 1-dodecanethiol (DDT) layer chemisorbed at the gold surface of the piezocrystal by liposome fusion. Finally, cyt c was adsorbed.
Figure 3
Figure 3
The scheme of preparation of the MUA layer with covalently immobilized cyt c and the interaction with AuNWs modified by DNA aptamers. (a) MUA is chemisorbed at the thin gold layer of the piezocrystal; (b) terminal carboxyl groups of the MUA are activated by EDC/NHS; (c) cyt c is added and binds to the activated carboxyl groups of the MUA; (d) AuNWs modified by DNA aptamers specific to cyt c are added and interact with cyt c.
Figure 4
Figure 4
The kinetics of the changes in the resonant frequency divided by the number of harmonics, Δf/n, (a) and dissipation, ΔD, (b) for fundamental frequency, third and fifth harmonics following the addition of DMPC:DMPG (1:1) liposomes, 10 μM cyt c and 1 μM aptamer (apt) specific to cyt c on 1-dodecanthiol layer chemisorbed on the thin gold layer of piezocrystal. The moments of addition of liposomes, cyt c, DNA aptamer, and PBS wash are shown by arrows.
Figure 5
Figure 5
The kinetics of the changes in normalized resonant frequency (a) and dissipation (b) for third (24 MHz), fifth (40 MHz) and seventh (56 MHz) harmonic frequencies vs. time following addition to the MUA surface of 2 μM cyt c, 10 mg/mL BSA, and AuNW modified with DNA aptamers specific to cyt c (Apt-AuNW). The moments of addition of cyt c, BSA, Apt-AuNW, and PBS wash are shown by arrows.
Figure 6
Figure 6
The plot of the normalized third harmonic frequency vs. concentration of cyt c on the surface of MUA activated by EDC/NHS (curve 1) and following the addition of AuNWs modified by DNA aptamers (concentration of DNA aptamer was 1 μM before centrifugation and washing of AuNWs) (curve 2). Results are mean ± SD obtained from three independent experiments.
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