I. Introduction
The-state-of-the-art bio- and nanotechnology have opened up an avenue to non-invasive liquid biopsy for identifying diseases from biomolecules in bloodstream, especially DNA. In this work, we combined sequence-specific-labeling scheme using mutated Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 without endonuclease activity (CRISPR/dCas9) and ion concentration polarization (ICP) phenomenon as a mechanism to selectively preconcentrate targeted DNA molecules for rapid and direct detection.
II. Results
Theoretical analysis on ICP phenomenon figured out a critical mobility, elucidating two distinguishable concentrating behaviors in the vicinity of a nanojunction; a stacking and a propagating behavior (Figure 1(a), 1(b)). Through the modulation of the critical mobility to shift those behaviors, the C-C chemokine receptor type 5 (CCR5) sequences were optically detected without PCR amplification (Figure 1(c), 1(d), 1(e)).
III. Conclusion
The proposed dCas9-mediated genetic detection methodology based on ICP would provide rapid and accurate micro/nanofluidic platform of liquid biopsies for disease diagnostics, especially PCR-free gene detection.
Acknowledgements
This work was supported by Korean Health Technology RND Project (HI13C1468 and HI14C0559).
References
- S. J. Kim, Y.-A. Song and J. Han, Chem. Soc. Rev., 2010, 39, 912-922.
- S. J. Kim, Y.-C. Wang, J. H. Lee, H. Jang and J. Han, Phys. Rev. Lett., 2007, 99, 044501.
- J. Choi, K. Huh, D. J. Moon, H. Lee, S. Y. Son, K. Kim, H. C. Kim, J.-H. Chae, G. Y. Sung, H.-Y. Kim, J. W. Hong and S. J. Kim, RSC Advances, 2015, 5, 66178-66184.
Fig. 1. Time-lapse images of each concentrating behavior of (a) Sulforhodamine B molecule (stacking regime, convection dominant case) and (b) Alexa 532 molecule (propagating regime, electromigration dominant case). (c) Target genetic sequence binding process of dCas9:sgRNA complex. Schematic for detecting target sequence by dCas9-mediated ICP platform (d) with target gene (positive) and (e) without target gene (negative).