Freeze-thaw induced co-assembly of multi-enzyme immobilized AuNPs probes for fast detection of glucose and hypoxanthine

Multi-enzymatic cascade reaction for analyte has the advantage of volume-confined environment and high-performance. Herein, a freeze-thaw induced co-assembly (FTIA) of multi-enzyme-AuNPs hybrids for high performance cascade reaction was developed for the first time. This preparation method does not require extra reagents and complex coupling steps, and has the superiority of simplicity, rapidity, low cost and environmental friendliness. Given the close proximity of glucose oxidase (GOx) and horseradish peroxidase (HRP) on the surface of AuNPs, this new detector greatly reduced the diffusion and decomposition of H2O2 and improved the sensitivity of glucose detection. Hence, the acquired AuNPs/GOx/HRP can undoubtedly be used to construct a sensitive and speedy colorimetric sensor for the assay of glucose (linear range of 42-2670 mu M with an LOD of 10 mu M). In addition, it was proved that AuNPs can be used as versatile scaffolds by simply replacing GOx with xanthine oxidase (XOD) to detect hypoxanthine (linear range of 20-1330 mu M with an LOD of 20 mu M). The constructed glucose detection method can be completed in 10 min and has been applied to the assay of glucose in human blood samples.