Human serum albumin (HSA) is not electroactive, because the electroactive amino acids have been buried in its structure. Therefore, direct electrochemical determination of the HSA as the most abundant protein in human plasma cannot be performed. In this work, a novel electrochemical biosensor has been developed based on synthesis of dual templates molecularly imprinted polymers (DTMIPs) having trypsin (TRP) and HSA as template molecules onto the surface of a glassy carbon electrode (GCE) modified with graphene-ionic liquid (Gr-IL). By incubation of the biosensor with TRP, and HSA, tryptic hydrolysis of the HSA is occurred which breaks the HSA down into free amino acids. The HSA involves five electroactive amino acids including cysteine, tryptophan, tyrosine, methionine and histidine whose differential normal pulse voltammetric (DNPV) responses are overlapped and generated a single peak. In order to increase the sensitivity of the DTMIP/Gr-IL/GCE for determination of the HSA, hydrodynamic DNPV (HDNPV) data were generated, and used for analytical purposes. The second-order HDNPV data were generated at different pulse amplitudes and used to develop four second-order calibration models by multivariate curve resolution-alternating least squares (MCR-ALS), parallel factor analysis2 (PARAFAC2), multi-way partial least squares/residual bilinearization (N-PLS/RBL), and unfolded partial least squares/residual bilinearization (U-PLS/RBL) to select the best procedure for determination of the HSA in the presence of gamma-globulin and glucose as uncalibrated interferences. The results confirmed the best performance for the biosensor assisted by MCR-ALS for ultrasensitive and selective determination of the HSA in both synthetic and real matrices which was comparable with HPLC-UV as a reference method.

人血清白蛋白（HSA）不具有电活性，因为电活性氨基酸已埋藏在其结构中。因此，无法对人血浆中最丰富的蛋白质 HSA 进行直接电化学测定。在这项工作中，基于在石墨烯离子液体修饰的玻碳电极（GCE）表面上合成以胰蛋白酶（TRP）和HSA为模板分子的双模板分子印迹聚合物（DTMIP），开发了一种新型电化学生物传感器（Gr-IL）。通过将生物传感器与 TRP 和 HSA 一起孵育，HSA 发生胰蛋白酶水解，将 HSA 分解成游离氨基酸。HSA 涉及五种电活性氨基酸，包括半胱氨酸、色氨酸、酪氨酸、蛋氨酸和组氨酸的微分正常脉冲伏安 (DNPV) 响应重叠并产生单个峰。为了提高 DTMIP/Gr-IL/GCE 测定 HSA 的灵敏度，生成了流体动力学 DNPV (HDNPV) 数据，并将其用于分析目的。二阶 HDNPV 数据在不同的脉冲幅度下生成，并用于通过多元曲线分辨率交替最小二乘法（MCR-ALS）、平行因子分析2（PARAFAC2）、多路偏最小二乘法/残差双线性化 (N-PLS/RBL) 和展开偏最小二乘/残差双线性化 (U-PLS/RBL)，以选择在存在 γ-球蛋白和葡萄糖作为未校准干扰的情况下测定 HSA 的最佳程序。