In this study, an electrochemical biosensor composed of a horseradish peroxidase (HRP)-encapsulated protein nanoparticles (HEPNP) was fabricated for the sensitive and selective detection of H2O2. The HEPNP has a three-dimensional structure that can contain a large amount of HRP; therefore, HEPNP can amplify the electrochemical signals necessary for the detection of H2O2. Furthermore, reduced graphene oxide (rGO) was used to increase the efficiency of electron transfer from the HEPNP to an electrode, which could enhance the electrochemical signal. This biosensor showed a sensitive electrochemical performance for detection of H2O2 with signals in the range from 0.01–100 μM, and it could detect low concentrations up to 0.01 μM. Furthermore, this biosensor was operated against interferences from glucose, ascorbic acid, and uric acid. In addition, this fabricated H2O2 biosensor showed selective detection performance in human blood serum. Therefore, the proposed biosensor could promote the sensitive and selective detection of H2O2 in clinical applications.

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Enzyme-based electrochemical biosensors have been widely used because of their sensitivity, rapidity, and highly target-specific reactions. However, the immobilized enzymes can hinder electron transfer on the surface of the electrode and can decrease the sensitivity of electrochemical biosensors. To overcome this problem, we synthesized conductive gold nanoclusters with two sets of embedded enzyme nanoparticles, which are called gold nanocluster-embedded dual-enzyme nanoparticles (AuNC-DENPs) composed of a glucose oxidase-stabilized gold nanocluster (GOX-AuNC) and a horseradish peroxide-stabilized gold nanocluster (HRP-AuNC). Moreover, the application of these nanoparticles as an enzyme-based highly sensitive electrochemical sensor was investigated. Owing to the effect of AuNCs, these nanoparticles have good conductivity compared to bare protein nanoparticles. In addition, the synthesized AuNC-DENPs enabled the combination of a two-enzyme cascade reaction, in which the GOX-AuNC component of the nanoparticle oxidized glucose to generate hydrogen peroxide, which then reacted with the adjacent HRP-AuNC component on the nanoparticle. Given the proximity of the two enzyme components in a single nanoparticle, the AuNC-DENPs markedly reduced the diffusion and decomposition of H2O2 during the cascade reaction and showed an enhanced catalytic reaction compared to a mixture of enzymes. As a result, the biosensor exhibited high sensitivity (18,944 μA/mM cm2) and could detect very low concentrations of glucose ranging from 5 to 320 nM and a LOD of 2.58 nM. In addition, by analyzing nontarget materials and serum together with glucose, it was confirmed that the sensor has good selectivity for glucose.

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Hydrogen peroxide (H2O2) has been a fascinating target in various chemical, biological, clinical, and industrial fields. Several types of fluorescent protein-stabilized gold nanoclusters (protein-AuNCs) have been developed for sensitive and easy detection of H2O2. However, its low sensitivity makes is difficult to measure negligible concentrations of H2O2. Therefore, to overcome this limitation, we developed a horseradish peroxidase-encapsulated fluorescent bio-nanoparticle (HEFBNP), comprising bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) and horseradish peroxidase-stabilized gold nanoclusters (HRP-AuNCs). The fabricated HEFBNP can sensitively detect H2O2 owing to its two properties. The first is that HEFBNPs have a continuous two-step fluorescence quenching mechanism, which comes from the heterogenous fluorescence quenching mechanism of HRP-AuNCs and BSA-AuNCs. Second, the proximity of two protein-AuNCs in a single HEFBNP allows a reaction intermediate (•OH) to rapidly reach the adjacent protein-AuNCs. As a result, HEFBNP can improve the overall reaction event and decrease the loss of intermediate in the solution. Due to the continuous quenching mechanism and effective reaction event, a HEFBNP-based sensing system can measure very low concentrations of H2O2 up to 0.5 nM and show good selectivity. Furthermore, we design a glass-based microfluidic device to make it easier use HEFBNP, which allowed us to detect H2O2 with the naked eye. Overall, the proposed H2O2 sensing system is expected to be an easy and highly sensitive on-site detection tool in chemistry, biology, clinics, and industry fields.