dCas9-mediated Nanoelectrokinetic Direct Detection of Target Gene for Liquid Biopsy

Abstract

The-state-of-the-art bio-and nanotechnology have opened up an avenue to noninvasive 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. Theoretical analysis on ICP phenomenon figured out a critical mobility, elucidating two distinguishable concentrating behaviors near a nanojunction, a stacking and a propagating behavior. 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. Conclusively, the proposed dCas9-mediated genetic detection methodology based on ICP would provide rapid and accurate micro/nanofluidic platform of liquid biopsies for disease diagnostics.

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…As an alternative, Lee et al developed a nano-electrokinetic chip where DNA and DNA-dCas9 complexes were separated via electrophoresis ( Lee et al, 2018 ). A charge selective junction in the chip formed a concentration boundary layer, creating an electric field gradient, and therefore a spatial gradient in electrophoretic force which works against the drag force from the flow in the channel.…”

CRISPR/Cas9-Based Lateral Flow and Fluorescence Diagnostics

“…CRISPR/Cas12 and Cas13 have nucleic acid cleavage abilities such that target nucleic acid interrogation and recognition leads to ectopic strand breaks of a secondary reporter molecule that can be detected by lateral flow assays (LFA) or fluorometry [8][9][10][11][12][13]. CRISPR/Cas9 has also been developed for diagnostics using solid-state nanopores, microfluidics, and LFA [14][15][16][17][18]. Some of these Cas9-based methodologies can be cumbersome to employ and can require specialized starting material, formulations, or instrumentation [14,15,[18][19][20][21][22][23][24].…”

A Multiscale‐Porous Anion Exchange Membrane for Convenient and Scalable Electrokinetic Concentration of Cationic Species

“…[ 6–8 ] Most electrokinetic concentrators rely on cation exchange membranes (CEM) such as Nafion that induce intensive electric fields at a region of ion depletion that forms near the anodic side of CEM. [ 9–15 ] We can selectively concentrate anionic species (negatively charged species) at this region by countering the electrophoretic velocity they experience with a counter hydraulic drag velocity induced by an electro–osmotic flow or hydraulic pump. [ 16 ] Ko et al.…”