Modified carbon nitride nanozyme as bifunctional glucose oxidase-peroxidase for metal-free bioinspired cascade photocatalysis

Nanomaterials-based biomimetic catalysts with multiple functions are necessary to address challenges in artificial enzymes mimicking physiological processes. Here we report a metal-free nanozyme of modified graphitic carbon nitride and demonstrate its bifunctional enzyme-mimicking roles. With oxidase mimicking, hydrogen peroxide is generated from the coupled photocatalysis of glucose oxidation and dioxygen reduction under visible-light irradiation with a near 100% apparent quantum efficiency. Then, the in situ generated hydrogen peroxide serves for the subsequent peroxidase-mimicking reaction that oxidises a chromogenic substrate on the same catalysts in dark to complete the bifunctional oxidase-peroxidase for biomimetic detection of glucose. The bifunctional cascade catalysis is successfully demonstrated in microfluidics for the real-time colorimetric detection of glucose with a low detection limit of 0.8 μM within 30 s. The artificial nanozymes with physiological functions provide the feasible strategies for mimicking the natural enzymes and realizing the biomedical diagnostics with a smart and miniature device.

Binding energy (eV)    2L denotes adsorption site on the second layer. ΔErelative represent the relative energy based on the most stable energy (ΔErelative = 0.00 eV) of adsorption configuration. For K-GCN model, we firstly calculated the doping of K atom in (1 1) slab model to expand its optimized structure to (2 2) slab model. After (1 1) calculation (a~c), the K atom migrated a lot from the initial adsorption site, especially hollow site, since the small cell size restricts the reconstruction of the first layer. Therefore, we further calculated (2 2) slab model (d~e), as a result, we found that the first layer was reconstructed flexibly and most stable K doping site is at the hollow site. It revealed that the reactive profile of H2O2 generation followed the zero-order kinetics as a function of the catalyst amount in batch system in (a). Before 20 mg, the reaction kinetics exhibited firstorder trend from the linearly catalysis, revealing the gradually increase of active sites for sufficient reaction with saturated O2. From the observation of moderate increase, the ratelimiting step was shifted to the process of dissolving and diffusion of H2O2 from catalyst surface to solution when the catalyst larger than 20 mg. Source data are provided as a Source Data file.

Supplementary Notes
The comparison of active efficiency between batch and flow model: the whole reactive efficiency of cascade glucose detection was potentially determined from the dominated process of H2O2 production with respect to the limitations of photon transfer and mass transfer. The resultant microfluidics was capable of receding the unexpected above limitations to support the sustaining H2O2 for the subsequent TMB oxidation in cascade reaction.
In The mass transfer efficiency is mostly determined by the capability of the regents movement to the catalyst surface and the products remove through the desorption and diffusion 21 . In this case, the microfluidics with advective flow exhibits the remarkable improvement of mass transfer in catalysis because of their short molecular diffusion distances, high mass transfer rates, and large surface-to-volume ratios 22 . To help in understanding above points, we performed the series of calculation to explore the main parameters for the mass transfer predomination in chemical kinetics.
It is well known that the overall rate constant was determined by two parts, an external mass transfer from the coating catalyst to liquid phase and an intrinsic reaction rate (Supplementary   Equation 1). Additionally, the diffusion also has a higher effect on fractional conversion than intrinsic rate constant in overall rate constant as batch case (Supplementary Figure 24a). Upon the generated H2O2 on the surface, the graphitic structure could confine the slow diffusion of H2O2 out of the reaction region as a result of the large diffusion length (~200 µm) of H2O2 for GCN 23 . However, the shorter molecular diffusion distances (44 µm in depth) in the microfluidic system can improve the mass transfer coefficient (km) around 5 times larger than batch case (~200 µm) from the Supplementary Equation 2. In fluidic chip, its surface to-volume ratio (αv) around 45454 m -1 was larger than that of 392 m -1 in batch case. Therefore, such higher surface area-to-volume ratio and mass transfer efficiency would significantly contribute for H2O2 generation within a few seconds than batch system, leading to the larger effect of fractional conversion on overall rate constant 24 .
where ki is the intrinsic reaction rate constant, K the Langmuir adsorption coefficient, km the mass transfer coefficient, αv the surface to the reactor volume, and D and  the diffusion coefficient of H2O2 (1.71×10 -9 m 2 s -1 ) and the thickness of diffusion layer.
where ki is the intrinsic rate constant (0.17 mM h -1 ), km the mass-transfer coefficient, α the interfacial area per unit volume, K the Langmuir adsorption coefficient (1. where Np is the total incident photons, Ne is the total reactive electrons, nH2O2 is the amount of H2O2 molecules in batch (10.1 µmol) and flow system (0.7 µmol), NA is Avogadro constant, h is the Planck constant, c is the speed of light, S is the marked irradiation area (3 cm × 1 cm), P is the intensity of irradiation light (0.9 mW, however the average intensity for the whole activating region in bulk reactor is 0.1 mW), t is the photoreaction time (5 min), and λ is the wavelength of the monochromatic light (420 nm). k is the constant parameter in equation.