To overcome the traditional issues of protein labeling, we report herein an effective approach for noncovalent conjugation of the biomolecule-derived fluorescent nanodots (biodot) to functional proteins without the addition of chemical linkers for biosensor development. The as-prepared fluorescent biodot-protein conjugates are very stable near physiological pH, exhibiting excellent photostability and thermal stability. More importantly, the native functions of proteins, including drug binding and enzymatic activities, are well-preserved after conjugating with biodots. The optimized protein conjugation strategy is then applied to prepare biodot-glucose oxidase (GOx) fluorescent sensing probes for sweat glucose detection. Results show that the as-prepared sensing probes could achieve better assay performance than those covalent conjugates as demonstrated herein. Specifically, GOx in the noncovalently bound conjugates are able to catalyze the oxidation of glucose effectively, which generates hydrogen peroxide as a byproduct. In the presence of Fe2+, Fenton reaction takes place to produce hydroxyl radicals and Fe3+, leading to significant fluorescence quenching of biodots on the conjugates. This simple one-step enzymatic assay in a single probe achieves a wide linear range of 25-1000 μM (R2 = 0.99) with a low detection limit of 25 μM. Furthermore, negligible interference is observed in the complex artificial sweat sample for accurate glucose quantification, achieving an excellent recovery rate of 100.5 ± 2.2%. This work provides a facile conjugation method that is generally applicable to a wide range of proteins, which will help to accelerate future development of multifunctional fluorescent probes to provide optical signals with unique protein functions (e.g., enzymatic, recognition, etc.) for biomedical sensing and imaging.

中文翻译：

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非共价荧光生物点蛋白与保留良好的天然功能结合，可改善汗液葡萄糖检测。

为了克服蛋白质标记的传统问题，我们在此报告了一种有效的方法，可将生物分子衍生的荧光纳米点（biodot）与功能蛋白进行非共价偶联，而无需添加用于生物传感器开发的化学接头。所制备的荧光生物点-蛋白质缀合物在生理pH附近非常稳定，表现出优异的光稳定性和热稳定性。更重要的是，与生物点缀合后，蛋白质的天然功能（包括药物结合和酶促活性）得到了很好的保留。然后将优化的蛋白质偶联策略应用于制备用于汗液葡萄糖检测的生物点葡萄糖氧化酶（GOx）荧光传感探针。结果表明，所制备的传感探针比本文所述的那些共价缀合物可实现更好的测定性能。具体地，非共价结合的缀合物中的GOx能够有效地催化葡萄糖的氧化，其产生副产物过氧化氢。在Fe2 +存在下，发生Fenton反应以产生羟基自由基和Fe3 +，从而导致缀合物上生物点的荧光猝灭。在单个探针中进行这种简单的一步酶法测定即可实现25-1000μM的宽线性范围（R2 = 0.99），检测限低至25μM。此外，在复杂的人工汗液样品中观察到的干扰可忽略不计，可进行准确的葡萄糖定量，从而实现了100.5±2.2％的极佳回收率。