Protein-Conjugated Quantum Dots for Detecting Trypsin and Trypsin Inhibitor Through Fluorescence Resonance Energy Transfer

We have developed quantum dot probes for detecting trypsin (Try) and trypsin inhibitor (TI) in aqueous solu- tions through fluorescence resonance energy transfer (FRET). Green-fluorescent CdTe quantum dots (QDs) served as the energy donors and rhodamine isothiocyanate (RITC) conjugated to bovine serum albumin (BSA-RITC) was the acceptor. By simply mixing the two fluorophores, FRET occurred when BSA-RITC bound to the CdTe QDs; as a result, the fluo- rescence intensity of the CdTe QDs at 520 nm decreased, while the fluorescence of RITC at 574 nm increased. When Try was used to digest BSA, the FRET efficiency decreased, allowing the detection of Try at concentrations as low as 110 pM. In the presence of TI, the digestion activity of Try was inhibited. As a result, the fluorescence intensity ratio IF574/IF520 of the QD-BSA-RITC solutions in the presence of a constant amount of Try increased upon increasing the concentration of TI; good linearity (R 2 = 0.99) existed over the range 0.3-5.0 nM. The LOD for TI was 250 pM. This simple and cost- effective probe was applied to determine the level of spiked TI (1.0 nM) in urine samples; the recoveries (95-110%) sug- gested low matrix interference and high sensitivity.