Loading of hyaluronic acid–conjugated MoS2 nanosheets with rhodamine B for sensing hyaluronidase activity in human urine and live cells

Efficient molecule loading onto nanomaterials is a key step for advanced biosensing and imaging because of improved sensitivity and enhanced targeting of the resultant nanoprobe. In contrast to other nanomaterials, MoS₂ nanosheets (NSs) possess unique advantageous of large surface area and abundant sulfur defects. Herein, we explored the conjugation of MoS₂ NSs with thiol-conjugated hyaluronic acid (HA-SH) for selective detection of hyaluronidase (HAase) and specific binding to CD44 receptor-positive cancer cells. Rhodamine B (RhB) molecules were electrostatically and hydrophobically coupled to the HA–SH–MoS₂ NS surface with a binding constant of 7.14 × 10¹⁶ M⁻¹. The HA–SH–MoS₂ NS-mediated fluorescence quenching of the adsorbed RhB molecules proceeds via a combination of static and dynamic quenching in the ratio of 10:1. Upon the addition of HAase, the adsorbed RhB molecules are liberated from the RB-HA–SH–MoS₂ NSs via HAase-mediated hydrolysis of HA-SH. As a result, the restored fluorescence of RhB molecules is proportional to the HAase concentration. The RhB-HA–SH–MoS₂ NSs achieve a detection limit of 0.37 U/mL for HAase with a linear response range from 0.05 to 4.5 U/mL and satisfactory selectivity against potential interferents. The RhB-HA–SH–MoS₂ NSs not only effectively quantify HAase in urine sample but also enable selective imaging of intracellular HAase activity through CD44 receptor-mediated endocytosis.