Department of Experimental Medicine, Sapienza University of Rome, Viale Regina Elena 324, 00166 Rome, Italy.
Department of Biological and Ecological Sciences, University of Tuscia, 01100 Viterbo, Italy.
Biosensors (Basel). 2022 Jul 12;12(7):519. doi: 10.3390/bios12070519.
The regulation of nervous and cardiovascular systems and some brain-related behaviors, such as stress, panic, anxiety, and depression, are strictly dependent on the levels of the main catecholamines of clinical interest, dopamine (DA), epinephrine (EP), and norepinephrine (NEP). Therefore, there is an urgent need for a reliable sensing device able to accurately monitor them in biological fluids for early diagnosis of the diseases related to their abnormal levels. In this paper, we present the first tyrosinase (Tyr)-based biosensor based on chitosan nanoparticles (ChitNPs) for total catecholamine (CA) detection in human urine samples. ChitNPs were synthetized according to an ionic gelation process and successively characterized by SEM and EDX techniques. The screen-printed graphene electrode was prepared by a two-step drop-casting method of: (i) ChitNPS; and (ii) Tyr enzyme. Optimization of the electrochemical platform was performed in terms of the loading method of Tyr on ChitNPs (nanoprecipitation and layer-by-layer), enzyme concentration, and enzyme immobilization with and without 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linking agents. The Tyr/EDC-NHS/ChitNPs nanocomposite showed good conductivity and biocompatibility with Tyr enzyme, as evidenced by its high biocatalytic activity toward the oxidation of DA, EP, and NEP to the relative o-quinone derivatives electrochemically reduced at the modified electrode. The resulting Tyr/EDC-NHS/ChitNPs-based biosensor performs interference-free total catecholamine detection, expressed as a DA concentration, with a very low LOD of 0.17 μM, an excellent sensitivity of 0.583 μA μM cm, good stability, and a fast response time (3 s). The performance of the biosensor was successively assessed in human urine samples, showing satisfactory results and, thus, demonstrating the feasibility of the proposed biosensor for analyzing total CA in physiological samples.
神经系统和心血管系统的调节以及一些与大脑相关的行为，如应激、恐慌、焦虑和抑郁，严格依赖于具有临床意义的主要儿茶酚胺的水平，即多巴胺（DA）、肾上腺素（EP）和去甲肾上腺素（NEP）。因此，迫切需要一种可靠的传感装置，能够在生物流体中准确监测这些物质，以便早期诊断与它们异常水平相关的疾病。在本文中，我们提出了第一个基于酪氨酸酶（Tyr）的生物传感器，用于检测人尿液样本中的总儿茶酚胺（CA）。ChitNPs 是通过离子凝胶化过程合成的，并通过 SEM 和 EDX 技术进行了表征。通过两步滴铸法制备了丝网印刷石墨烯电极：（i）ChitNPs；（ii）Tyr 酶。从 Tyr 在 ChitNPs 上的加载方法（纳米沉淀和层层）、酶浓度以及有无 1-乙基-3-（3-二甲基氨基丙基）-碳化二亚胺（EDC）和 N-羟基琥珀酰亚胺（NHS）作为交联剂固定酶等方面对电化学平台进行了优化。Tyr/EDC-NHS/ChitNPs 纳米复合材料表现出良好的导电性和与 Tyr 酶的生物相容性，这可以通过其对 DA、EP 和 NEP 氧化为相对 o-醌衍生物的高生物催化活性来证明，该衍生物在修饰电极上电化学还原。基于 Tyr/EDC-NHS/ChitNPs 的生物传感器可进行无干扰的总儿茶酚胺检测，表现为 DA 浓度，具有非常低的检出限 0.17 μM、出色的灵敏度 0.583 μA μM cm、良好的稳定性和快速的响应时间（3 s）。该生物传感器在人尿液样本中的性能进行了评估，结果令人满意，从而证明了该生物传感器用于分析生理样本中总 CA 的可行性。
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