Since p53 protein has become recognized biomarker for both diagnostic and therapeutic purposes in oncological diseases with particular relevance for bladder cancer, it is highly desirable to search for a novel sensing tool for detecting the patient's p53 level at the early stage. Here we report the first study on the development and validation of a novel disposable competitive amperometric immunosensor for determination of p53 protein at subnanomolar levels, based on p53 immobilization on gold nanoparticles/carbon nanotubes modified screen-printed carbon electrodes. The assay protocol requires the use of single anti-p53 mouse monoclonal antibody (DO-7 clone), able to recognize both wild-type and mutant p53. The developed immunosensor as well as the protocol of the electrochemical immunoassay were optimized by means of an experimental design procedure to assess the suitability of the device to be validated and applied for the determination of p53 in untreated and undiluted urine samples. It was found that the developed competitive immunodevice was able to achieve wide linear range detection of wild-type p53 from 20 pM to 10 nM with a low detection limit of 14 pM in synthetic urine samples, suggesting the sensor's capability of working in a complex sample matrix. The excellent performance results also in terms of selectivity, trueness and precision, coupled with the advantages of an easy preparation and low-cost assay in contrast to other methods which require very complex, time-consuming and costly nanostructured architectures, makes the developed competitive immunosensor an analytically robust diagnostic tool, valuable for implementation of screening and follow-up programs in patients with urologic malignancies.

中文翻译：

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用碳纳米管/金纳米颗粒丝网印刷电极测定尿液中 p53 蛋白的竞争性电流免疫传感器：一种用于尿路癌早期诊断的潜在快速无创筛查工具

由于 p53 蛋白已成为公认的用于肿瘤疾病诊断和治疗目的的生物标志物，尤其与膀胱癌相关，因此非常需要寻找一种新的传感工具来在早期检测患者的 p53 水平。在这里，我们报告了基于 p53 固定在金纳米粒子/碳纳米管修饰的丝网印刷碳电极上的第一项关于开发和验证新型一次性竞争性安培免疫传感器的研究，该传感器用于测定亚纳摩尔水平的 p53 蛋白。检测协议需要使用单个抗 p53 小鼠单克隆抗体（DO-7 克隆），能够识别野生型和突变型 p53。开发的免疫传感器以及电化学免疫分析的协议通过实验设计程序进行了优化，以评估要验证的设备的适用性，并将其应用于未处理和未稀释的尿样中 p53 的测定。结果发现，开发的竞争性免疫装置能够实现对野生型 p53 从 20 pM 到 10 nM 的宽线性范围检测，在合成尿样中检测限低至 14 pM，表明该传感器具有处理复杂样品的能力矩阵。与其他需要非常复杂、耗时和昂贵的纳米结构结构的方法相比，在选择性、真实性和精确度方面也具有出色的性能，加上易于制备和低成本测定的优势，