Strong enzyme immobilization associated by anatase TiO2 sputtered on platinum black nanoclusters to improve sensitivity and long-term stability of electrochemical cholesterol sensor

Abstract

Enzyme immobilization is one of the essential features to achieve a high-performance, enzyme-based electrochemical sensor. Here, we developed a simple and strong immobilization strategy that provides a sensitive, selective, and stable cholesterol sensor fabricated by conventional MEMS processes. As the immobilization strategy, pure anatase TiO2 film was sputtered on platinum black (BPt) nanoclusters as a working electrode for large effective surface areas, since TiO2 has various surface OH groups, including Ti − OH, that can be used as linkers to immobilize enzyme molecules. We prepared BPt, TiO2/BPt, and annealed TiO2/BPt and analyzed using quantitative XPS and FTIR measurement. It was demonstrated that TiO2/BPt achieved solid enzyme immobilization (our quantified score = 1.23) compared to BPt (score = 0.04) and annealed TiO2/BPt (score = −0.02) electrodes. This is due to its large amount of reactive OH groups (reactive IOH/ITi-O ∼ 23.8 % of TiO2/BPt vs. inactive IO1s/IPt4f ∼ 17.7 % of BPt) and high surface energy. Accordingly, TiO2/BPt exhibited superior sensitivity (81.2 μA∙μM−1 cm−2; LOD = 0.96 μM) and a low KM value (9.21 μM) compared to other electrodes. Notably, TiO2/BPt provided great stability (∼96.4 %) for 30 days as well as selectivity and reusability. We believe this study provides new insights that could allow simpler and better immobilization methods for industrial applicable biosensors.