Treffer: An integrated microfluidic system with shear force control for an automatically modified mRNA display technique for screening high-specificity peptide probes.

We developed a low-cost, integrated microfluidic system (IMS) featuring micropumps, microvalves, micromixers, and thermoelectric modules that fully automated a modified-mRNA display technique for the screening of high-specificity peptide probes in under a day. Peptides targeting KIF2C, a protein overexpressed in various cancers, which has been extensively explored for the development of biomolecular therapeutics, were screened on this new platform. The IMS enabled precise control of shear force (ranging from 0.96 to 9.6 nN) during washing, eliminating off-target peptides under physiologically relevant conditions. As a result, the total screening time was reduced from several days to less than one day. The overall process of the mRNA display could be divided into two stages: (i) mRNA preparation and (ii) mRNA display selection. To the best of our knowledge, it is the first work of integrating mRNA display selection into a single microfluidic chip. Among the screened candidates, one peptide, OCTRD 14, demonstrated both high binding affinity and high target specificity in three independent in vitro assays. In the dissociation constant ( K<subscript>d</subscript> ) measurements, OCTRD14 exhibited a K<subscript>d</subscript> of 2.4 μM. Although this K<subscript>d</subscript> value was not particularly low, the binding affinity observed in the in vitro assay was comparable to that of the positive control ( i.e. antibody). Since the compared signal was detected by using an antibody, this suggests that OCTRD14 may behave similarly to antibody-like candidates. Overall, this high-efficiency peptide screening IMS shows promise for future biomedical applications.