Simultaneous sensitive and precise determination of multibiomarkers is of great significance for improving detection efficiency, reducing diagnosis and treatment expenses, and elevating survival rates. However, the development of simple and portable biosensors for simultaneous determination of multiplexed targets in biological fluids still faces challenges. Herein, a unique and versatile immobilization-free dual-target electrochemical biosensing platform, which combines distinguishable magnetic signal reporters with buoyancy-magnetism separation, was designed and constructed for simultaneous detection of carcinoembryonic (CEA) and α-fetoprotein (AFP) in intricate biological fluids. To construct such distinguishable magnetic signal reporters with signal transduction, amplification, and output, secondary antibodies of CEA and AFP were respectively functionalized on methylene blue (MB) and 6-(ferrocenyl)hexanethiol (FeC) modified Fe₃O₄@Au magnetic nanocomposites. Meanwhile, a multifunctional flotation probe with dual target recognition, capture, and isolation capability was prepared by conjugating primary antibodies (Ab₁-CEA, Ab₁-AFP) to hollow buoyant microspheres. The target antigens of CEA and AFP can trigger a flotation-mediated sandwich-type immunoreaction and capture a certain amount of the distinguishable magnetic signal reporter, which enables the conversion of the target CEA and AFP quantities to the signal of the potential-resolved MB and FeC. Thus, the MB and FeC currents of magnetically adsorbed distinguishable magnetic reporters can be used to determine the CEA and AFP targets simultaneously and precisely. Accordingly, the proposed strategy exhibited a delightful linear response for CEA and AFP in the range of 100 fg·mL^–1–100 ng·mL^–1 with detection limits of 33.34 and 17.02 fg·mL^–1 (S/N = 3), respectively. Meanwhile, no significant nonspecific adsorption and cross-talk were observed. The biosensing platform has shown satisfactory performance in the determination of real clinical samples. More importantly, the proposed approach can be conveniently extended to universal detection just by simply substituting biorecognition events. Thus, this work opens up a new promising perspective for dual and even multiple targets and offers promising potential applications in clinical diagnosis.

中文翻译：

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可区分的磁性报告基因与浮力磁分离的协调用于无固定化双靶点电化学免疫传感

同时灵敏、精确地测定多种生物标志物对于提高检测效率、降低诊断和治疗费用、提高生存率具有重要意义。然而，开发用于同时测定生物液体中多重目标的简单便携式生物传感器仍然面临挑战。在此，设计和构建了一种独特且多功能的免固定双靶点电化学生物传感平台，该平台将可区分的磁信号报告器与浮磁分离相结合，用于同时检测复杂生物中的癌胚（CEA）和甲胎蛋白（AFP）。液体。为了构建具有信号转导、放大和输出功能的可区分磁信号报告基因，CEA 和 AFP 的二抗分别在亚甲基蓝 (MB) 和 6-(二茂铁基)己硫醇 (FeC) 修饰的 Fe ₃ O ₄ @Au 磁性纳米复合材料上进行功能化。同时，通过将一抗（Ab ₁ -CEA、Ab ₁ -AFP）与空心浮力微球结合，制备了具有双重目标识别、捕获和分离能力的多功能浮选探针。 CEA和AFP的目标抗原可以触发浮选介导的夹心式免疫反应，并捕获一定量的可区分的磁信号报告基因，从而能够将目标CEA和AFP的量转换为潜在可分辨的MB和B的信号。 FeC。因此，磁吸附的可区分磁性报告分子的 MB 和 FeC 电流可用于同时精确地确定 CEA 和 AFP 目标。因此，所提出的策略在 100 fg·mL ^–1 –100 ng·mL ^–1范围内对 CEA 和 AFP 表现出良好的线性响应，检测限为 33.34 和 17.02 fg·mL ^–1 ( S / N = 3) ， 分别。同时，没有观察到明显的非特异性吸附和串扰。该生物传感平台在实际临床样本的测定中表现出了令人满意的性能。更重要的是，所提出的方法只需简单地替换生物识别事件就可以方便地扩展到通用检测。因此，这项工作为双重甚至多重目标开辟了新的有前景的前景，并在临床诊断中提供了有希望的潜在应用。