Distinguishing Steric Occlusion from Enzyme–Surface Interactions in Nanoparticle-Based CRISPR/Cas Biosensing

Nanoparticle (NP)-bound reporters for CRISPR/Cas biosensing can improve signal, simplify readout, and expand multiplexing capabilities; however, the proximity to surfaces can simultaneously hinder enzymatic activity through steric occlusion, reduced reporter accessibility, or enzyme interactions with the surface itself. Such effects are typically observed indirectly through comparison of limit of detection (LoD) or end-point signal against a standardized reporter in solution, and steric occlusion at surfaces has yet to be mechanistically isolated. To distinguish steric occlusion from other effects, we compared a Cy3/Cy5 hairpin reporter free in solution vs displayed on CdSe/CdS/ZnS quantum dots (QDs) or CdSe/CdZnS nanoplatelets (NPLs). Substrate proximity effects were distinguished from direct surface interactions by selective surface passivation, and multiacceptor FRET measurements placed the loop apex at 3.1 ± 0.5 nm from the QD surface, representing the upper bound on the distance of any cleavage site from the surface. For passivated QDs, we found that the on-surface and off-surface specificity constants were indistinguishable for both LbCas12a and LwCas13a; similar qualitative results were observed for LbCas12a and NPLs. In the absence of surface passivation, apparent kcat,obs/KM,obs decreased by approximately 2.3-fold on QDs and 4.5-fold on NPLs for LbCas12a, in line with enzyme adsorption or inactivation on insufficiently blocked surfaces. For surface-passivated QD and NPL chemistries tested here, we found that steric hindrance did not play a detectable role in enzyme inhibition, even for substrates within approximately 3 nm of the surface.