(504a) An Ion-Exchange Membrane-Based Microfluidic Device for Field-Induced Enrichment, Irreversible Dissociation and Quantification of miRNA from Ribonucleoproteins

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, between the negatively charged RNA and the positively charged protein, is a particular challenge for the quantification of the associated microRNAs (miRNAs). Significant pretreatment is necessary to dissociate the RNP, which often leads to high analyte loss. We have developed 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 concentration and irreversible dissociation (k_off = 0.0025 s−1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The field-induced ion depletion action of the ion-selective membrane creates a depletion front that concentrates the RNPs at the boundary of the front and subject them to the high field there. The high field induces a dissociation rate much higher than the field-free value and renders the dissociation irreversible, with a 10³ higher dissociation rate. The irreversible and non-equilibrium dissociation is further amplified by opposite electrophoretic migration of the dissociated charged molecules.

In the microfluidic design, RNP sample is pumped across a depletion front induced between two CEMs and the resulting high electric field (>100 V cm^-1) in the concentration boundary layer is used to concentrate and hold the RNP in the channel. As the RNP is held in this boundary layer it undergoes rapid dissociation, where the positively charged RNA-binding protein, Cas9, and negatively charged microRNA, miR21, are electrophoretically driven apart faster than their rate of reassociation. The dissociated Cas9 is driven downstream past the depletion region and can be collected and measured as the RNA is held in the concentration boundary layer. The miR21 concentration is measured overtime using an integrated anion exchange membrane sensor located over its concentrated region in the channel. We are able to achieve irreversible dissociation of the low K_D (∼0.5 nM) complex, with ∼100% dissociation even though the association rate (k_on /[RNP]= 6.1 s−1) is 1000 times higher at our RNP concentration [RNP]. A detection limit of 1.1 nM is achieved for Cy3 labeled miR-21 used to visualize the concentration and dissociation processes. This technology allows for the dissociation and quantification of RNA from strongly bound RNP and is a great step towards use of RNP in diagnostics as a potential disease biomarker.

McCarthy, K., Go, D. B., Senapati, S. and Chang, H.-C., “An Integrated Ion-Exchange Membrane based microfluidic chip for irreversible dissociation and quantification of miRNA from ribonucleoproteins”, LabChip, 23:285-294(2022).