Manipulating the hydrophobicity of DNA as a universal strategy for visual biosensing

Abstract

Current visual biosensing methods, including colorimetric-based, fluorescence-based and chemiluminescence-based methods, are inappropriate for the hundreds of millions of people affected by color blindness and color weakness. Compared with these available methods, a droplet motion-based strategy might be a promising protocol for extension to a wider user base. Here we report a protocol for manipulating the hydrophobicity of DNA, which offers a droplet motion-based biosensing platform for the visual detection of small molecules (ATP), nucleic acids (microRNA) and proteins (thrombin). The protocol starts with target-triggered rolling-circle amplification that can readily generate short single-stranded DNA (ssDNA) fragments or long ssDNA. By exploiting macroscopic wetting behavior and molecular interaction, one can tailor the conformation of ssDNA on the water–oil interface to control the relevant DNA hydrophobicity. The wettability of DNA can be translated into visual signals via reading the sliding speed or the critical sliding angle. The time range for the entire protocol is 1 d, and the detection process takes 1 min.

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Fig. 1: Target-dependent RCA.
Fig. 2: Molecular dynamics simulations of ssDNA with variable lengths at the water–n-decane interface.
Fig. 3: Molecular contact mechanism at the water–oil interface.
Fig. 4: Confocal laser-scanning microscopy characterization of the droplet wetting behavior at the water–oil interface.
Fig. 5: Droplet motion controlled by ATP-dependent RCA.
Fig. 6: Sensitivity, selectivity and practicability of the detection of miR-21 and thrombin.

Data availability

The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (21535002) to S.Z.; the National Natural Science Foundation of China (21525523, 21722507, 21574048 and 21874121), the National Basic Research Program of China (973 Program, 2015CB932600) and the National Key R&D Program of China (2017YFA0208000) to F.X.; the National Natural Science Foundation of China (31800829), the Natural Science Foundation of Shandong Province (ZR2018BB054) and PhD Research Foundation of Linyi University (LYDX2018BS005) to Z.F.G.; and the National Natural Science Foundation of China (21703210) and the China Postdoctoral Science Foundation (2017M610492) to R.L.

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Authors

Contributions

Z.F.G., F.X. and L.J. designed the research. Z.F.G., R.L., J.W. and J.D. performed the research. J.D., W.-H.H., M.L. and S.Z. contributed new reagents and analytic tools. Z.F.G., R.L., J.W., S.W., F.X., S.Z. and L.J. analyzed the data. L.J. supervised the project. Z.F.G., R.L., F.X. and S.Z. wrote the paper.

Corresponding authors

Correspondence to Fan Xia or Shusheng Zhang.

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The authors declare no competing interests.

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Related links

Key references using this protocol

Gao, Z. F. et al. Chem 4, 2929–2943 (2018): https://doi.org/10.1016/j.chempr.2018.09.028

Gao, Z. F. et al. NPG Asia Mater. 10, 177–189 (2018): https://doi.org/10.1038/s41427-018-0024-7

Zhan, S., Pan, Y., Gao, Z. F., Lou, X. D. & Xia, F. TrAC Trends Anal. Chem. 108, 183–194 (2018): https://doi.org/10.1016/j.trac.2018.09.001

Integrated supplementary information

Supplementary Fig. 1. Gel electrophoresis image after RCA.

(1) DNA size marker; (2) padlock probe; (3) padlock probe circularization with ATP; (4) RCA reaction with ATP; (5) padlock probe circularization without ATP; (6) RCA reaction without ATP. The ATP concentration is 500 μM. Adapted with permission from ref. 28, Cell Press.

Supplementary Fig. 2. Control experiment and practical performance of ATP detection.

(a) The recorded surface tensions (black) and CSAs (red) at various ATP concentrations. The CSA and surface tension of pure ATP solutions barely changed with varying concentrations of ATP. Error bars represent the standard deviation from at least six individual measurements. Arrows refer to the black and red data corresponding to the black and red axes on the left and right, respectively. (b) Response of CSAs to 0 nM, 0.5 nM, 50 nM, and 5000 nM ATP in buffer and human serum solution, respectively. A standard addition method was next conducted in spiked human serum samples (diluted 200-fold before use) to verify the accuracy of the system55. Based on the highly specific binding of ATP to the T4 DNA ligase, the CSA recorded in human serum was comparable to that in buffer solution at all ATP concentrations, indicating this protocol could be effectively applied in complicated samples. The control experiments, diluted serum sample and buffer solution without ATP, proved that the serum sample containing sugars, proteins, lipids, etc., has a negligible effect on the CSA. Error bars represent the standard deviation from at least six individual measurements. Adapted with permission from ref. 28, Cell Press.

Supplementary Fig. 3. Control experiments for miR-21 and thrombin detection.

The recorded surface tensions (black) and CSAs (red) at various (a) miR-21, (b) thrombin concentrations. The CSA and surface tension barely changed with varying concentrations of miRNA or thrombin. Error bars represent the standard deviation from at least six individual measurements. Arrows refer to the black and red data corresponding to the black and red axes on the left and right, respectively.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Supplementary Table 1

Reporting Summary

Supplementary Video 1

The distribution of the water–oil interface between the RCA droplet without ATP and the organogel surface, which was studied by an inverted fluorescence microscope tilted from 0° to 8.1°. The bottom-view video was recorded in the grayscale oil channel. The movie playback accelerated ten times. Adapted with permission from ref. 1, Cell Press. This movie shows that the RCA droplet without ATP barely moved on the organogel surface because the oil layer was damaged due to the strong hydrophobic interaction between short DNA fragments and oil molecules.

Supplementary Video 2

The distribution of the water–oil interface between the RCA droplet with ATP and the organogel surface, which was studied by an inverted fluorescence microscope tilted from 0° to 8.1°. The bottom-view video is recorded at grayscale oil channel. The shifts at ~60 s and 105 s result from camera movement. The movie playback is accelerated ten times. This movie shows the RCA droplet with ATP sliding easily on the organogel surface because the oil layer was almost intact due to the weak hydrophobic interaction between long ssDNA and oil molecules. Adapted with permission from ref. 1, Cell Press.

Supplementary Video 3

Sliding behavior of RCA droplets with ATP on the organogel surface. The droplet is stained with methylene blue to display clearly. The droplet size is 2 μL. Adapted with permission from ref. 1, Cell Press.

Supplementary Video 4

Sliding behavior of RCA droplets without ATP on the organogel surface. The droplet is stained with rhodamine B to display clearly. The droplet size is 2 μL. The movie playback is accelerated eight times. Adapted with permission from ref. 1, Cell Press.

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Gao, Z.F., Liu, R., Wang, J. et al. Manipulating the hydrophobicity of DNA as a universal strategy for visual biosensing. Nat Protoc 15, 316–337 (2020). https://doi.org/10.1038/s41596-019-0235-6

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