Serum total bile acids (TBA) level is used as a sensitive and reliable index for hepatobiliary diseases in clinics. Herein, a novel electrochemical biosensor was fabricated using enzymatic reaction coupling with the double oxidation circular amplification strategy for the detection of human serum TBA. With the catalysis of 3α-hydroxysteroid dehydrogenase (3α-HSD), 3α-bile acids reacted specifically with nicotinamide adenine dinucleotide (NAD⁺). And then, the reduced nicotinamide adenine dinucleotide (NADH) was produced. After that, the NADH reacted with the electron mediator of tris(2,2'-bipyridine) ruthenium(Ⅲ) (Ru(bpy)₃³⁺), which was then transformed to Ru(bpy)₃²⁺. Ultimately, Ru(bpy)₃²⁺ was further oxidized to Ru(bpy)₃³⁺ under a certain voltage, which was detected by the chronoamperometry assay. The detection was performed using a disposable unmodified screen-printed carbon electrode (SPCE) without sample preparation. The proposed biosensor showed high sensitivity and accuracy with the linear range from 5.0 to 150.0 pmol/L in 10⁶-fold dilution serum. The established method had a good correlation with the enzymatic cycling method (r = 0.9372, P < 0.001, n = 72) commonly used in clinic. The electrochemical biosensor is simple, ultrasensitive and without sample pretreatment, showing great potential for point-of-care testing (POCT) of serum TBA in clinical samples. In addition, the biosensor is cost-effective with a small volume of samples, especially suitable for those who have difficulties in blood collection, such as infants, children and some small animals.

血清总胆汁酸（TBA）水平在临床上被用作肝胆疾病的敏感而可靠的指标。在这里，一种新型的电化学生物传感器使用酶促反应与双氧化循环扩增策略耦合，用于检测人血清TBA。在3α-羟基类固醇脱氢酶（3α-HSD）的催化下，3α-胆汁酸与烟酰胺腺嘌呤二核苷酸（NAD ⁺）发生了特异性反应。然后，产生还原的烟酰胺腺嘌呤二核苷酸（NADH）。之后，NADH与三（2,2'-联吡啶）钌（Ⅲ）（Ru（bpy）₃ ³⁺）的电子介体反应，然后转化为Ru（bpy）₃ ²⁺。最终，Ru（bpy）₃ ²⁺在一定的电压下进一步被氧化成Ru（bpy）₃ ³⁺，通过计时电流分析法检测到。使用未经样品处理的一次性未修饰丝网印刷碳电极（SPCE）进行检测。所提出的生物传感器在10 ⁶倍稀释血清中具有5.0至150.0 pmol / L的线性范围，显示出高灵敏度和准确性。所建立的方法与酶循环法具有很好的相关性（r  = 0.9372，P  <0.001，n = 72）临床常用。电化学生物传感器简单，超灵敏且无需样品预处理，显示出在临床样品中进行血清TBA的即时检验（POCT）的巨大潜力。另外，该生物传感器具有少量样品的成本效益，特别适合于血液采集困难的人，例如婴儿，儿童和一些小动物。