In Situ Generation and Consumption of H₂O₂ by Bienzyme–Quantum Dots Bioconjugates for Improved Chemiluminescence Resonance Energy Transfer

Exploration of quantum dots (QDs) as energy acceptors revolutionizes the current chemiluminescence resonance energy transfer (CRET), since QDs possess large Stokes shifts and high luminescence efficiency. However, the strong and high concentration of oxidant (typically H₂O₂) needed for luminol chemiluminescence (CL) reaction could cause oxidative quenching to QDs, thereby decreasing the CRET performance. Here we proposed the use of bienzyme–QDs bioconjugate as the energy acceptor for improved CRET sensing. Two enzymes, one for H₂O₂ generation (oxidase) and another for H₂O₂ consumption (horseradish peroxidase, HRP), were bioconjugated onto the surface of QDs. The bienzyme allowed fast in situ cascaded H₂O₂ generation and consumption, thus alleviating fluorescence quenching of QDs. The nanosized QDs accommodate the two enzymes in a nanometric range, and the CL reaction was confined on the surface of QDs accordingly, thereby amplifying the CL reaction rate and improving CRET efficiency. As a result, CRET efficiency of 30–38% was obtained; the highest CRET efficiency by far was obtained using QDs as the energy acceptor. The proposed CRET system could be explored for ultrasensitive sensing of various oxidase substrates (here exemplified with cholesterol, glucose, and benzylamine), allowing for quantitative measurement of a spectrum of metabolites with high sensitivity and specificity. Limits of detection (LOD, 3σ) for cholesterol, glucose, and benzylamine were found to be 0.8, 3.4, and 10 nM, respectively. Furthermore, multiparametric blood analysis (glucose and cholesterol) is demonstrated.