An integrated ion-exchange membrane-based microfluidic device for irreversible dissociation and quantification of miRNA from ribonucleoproteins.
Lab Chip
; 23(2): 285-294, 2023 01 17.
Article
in En
| MEDLINE
| ID: mdl-36524732
ABSTRACT
Ribonucleoproteins (RNPs), particularly microRNA-induced silencing complex (miRISC), have been associated with cancer-related gene regulation. Specific RNA-protein associations in miRISC complexes or those found in let-7 lin28A complexes can downregulate tumor-suppressing genes and can be directly linked to cancer. The high protein-RNA electrostatic binding affinity is a particular challenge for the quantification of the associated microRNAs (miRNAs). We report here the first microfluidic point-of-care assay that allows direct quantification of RNP-associated RNAs, which has the potential to greatly advance RNP profiling for liquid biopsy. Key to the technology is an integrated cation-anion exchange membrane (CEM/AEM) platform for rapid and irreversible dissociation (k = 0.0025 s-1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The CEM-induced depletion front is used to concentrate the RNP at the depletion front such that the high electric field (>100 V cm-1) within the concentration boundary layer induces irreversible dissociation of the low KD (â¼0.5 nM) complex, with â¼100% dissociation even though the association rate (kon = 6.1 s-1) is 1000 times higher. The high field also electrophoretically drives the dissociated RNA out of the concentrated zone without reassociation. A detection limit of 1.1 nM is achieved for Cy3 labelled miR-21.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
MicroRNAs
/
Microfluidics
/
Neoplasms
Limits:
Humans
Language:
En
Journal:
Lab Chip
Journal subject:
BIOTECNOLOGIA
/
QUIMICA
Year:
2023
Document type:
Article
Affiliation country:
Estados Unidos