A new specific fullerene-based fluorescent probe for trypsin

Abstract

A novel fluorescent probe (C₆₀-FL) was designed and synthesized for the direct determination of trypsin, based on photo-induced electron transfer (PET). The probe consists of two functional moieties: fluorescein which performs as a fluorophore and an electron donor, and fullerene (C₆₀) which acts as an electron acceptor and trypsin substrate analogue. In the presence of trypsin, the probe exhibited fluorescence increase due to the inhibition of electron transfer by the combination of C₆₀-FL with trypsin. The response of the probe to trypsin was direct and rapid. Experimental results showed that the increase in fluorescence intensity is proportional to the concentration of trypsin within the range of 4.40 × 10⁻⁷ to 7.04 × 10⁻⁵ g mL⁻¹ under the optimized experimental conditions. The detection limit of the proposed method was 40 ng mL⁻¹. The method had high selectivity for trypsin over other enzymes and proteins, such as lipase, α-amylase, bovine serum albumin, zinc metallothionein, glutathione reductase, thioredoxin and α-chymotrypsin etc. The remarkable properties of C₆₀-FL help to extend the development of fluorescent probes for investigating enzymes in a biological context.