REQUIMTE/LAQV, ISEP, Polytechnic of Porto, Rua Dr. António Bernardino de Almeida, 4249-015, Porto, Portugal.
REQUIMTE/LAQV, Departamento de Engenharia Química, Faculdade de Engenharia, Universidade Do Porto, Rua Dr. Roberto Frias, 4200-465, Porto, Portugal.
Talanta. 2023 Nov 1;264:124692. doi: 10.1016/j.talanta.2023.124692. Epub 2023 Jun 1.
Cardiovascular diseases are among the major causes of mortality and morbidity. Warfarin is often prescribed for these disorders, an anticoagulant with inter and intra-dosage variability dose required to achieve the target international normalized ratio. Warfarin presents a narrow therapeutic index, and due to its variability, it can often be associated with the risk of hemorrhage, or in other patients, thromboembolism. Single-nucleotide polymorphisms are included in the causes that contribute to this variability. The Cytochrome P450 (CYP) 2C93 genetic polymorphism modifies its enzymatic activity, and hence warfarin's plasmatic concentration. Thus, the need for a selective, rapid, low-cost, and real-time detection device is crucial before prescribing warfarin. In this work, a disposable electrochemical DNA-based biosensor capable of detecting CYP2C93 polymorphism was developed. By analyzing genomic databases, two specific 78 base pairs DNA probes; one with the wild-type adenine (Target-A) and another with the cytosine (Target-C) single-nucleotide genetic variation were designed. The biosensor implied the immobilization on screen-printed gold electrodes of a self-assembled monolayer composed by mercaptohexanol and a linear CYP2C93 DNA-capture probe. To improve the selectivity and avoid secondary structures a sandwich format of the CYP2C93 allele was designed using complementary fluorescein isothiocyanate-labeled signaling DNA probe and enzymatic amplification of the electrochemical signal. Chronoamperometric measurements were performed at a range of 0.015-1.00 nM for both DNA targets achieving limit of detection of 42 p.m. The developed DNA-based biosensor was able to discriminate between the two synthetic target DNA targets, as well as the targeted denatured genomic DNA, extracted from volunteers genotyped as non-variant homozygous (A/A) and heterozygous (A/C) of the CYP2C9*3 polymorphism.
心血管疾病是主要的死亡和发病原因之一。华法林通常用于治疗这些疾病，它是一种抗凝剂，需要根据个体剂量变化来达到目标国际标准化比值。华法林的治疗指数较窄，由于其变异性，常常与出血风险相关，而在其他患者中，则与血栓栓塞风险相关。单核苷酸多态性是导致这种变异性的原因之一。细胞色素 P450（CYP）2C93 基因多态性改变了其酶活性，进而影响华法林的血浆浓度。因此，在开出处方之前，需要一种选择性、快速、低成本和实时的检测设备。在这项工作中，开发了一种基于 DNA 的一次性电化学生物传感器，用于检测 CYP2C93 多态性。通过分析基因组数据库，设计了两个特定的 78 个碱基对 DNA 探针；一个带有野生型腺嘌呤（靶标-A），另一个带有胞嘧啶（靶标-C）单核苷酸遗传变异。该生物传感器涉及将巯基己醇组成的自组装单层和线性 CYP2C93 DNA 捕获探针固定在丝网印刷金电极上。为了提高选择性并避免二级结构，设计了 CYP2C93 等位基因的三明治结构，使用互补的荧光素异硫氰酸酯标记的信号 DNA 探针和电化学信号的酶放大。对两种 DNA 靶标进行了 0.015-1.00 nM 范围的计时电流测量，达到了 42 pM 的检测限。所开发的基于 DNA 的生物传感器能够区分两种合成靶标 DNA 以及从志愿者中提取的靶向变性基因组 DNA，志愿者的 CYP2C9*3 多态性基因型为非变异纯合子（A/A）和杂合子（A/C）。
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