Nanobody-enhanced split-luciferase technology for innovative detection of the liver cancer biomarker alpha-fetoprotein

IF 3.7 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Biochemical Engineering Journal Pub Date : 2024-06-22 DOI:10.1016/j.bej.2024.109402

Mina Oliayi , Rahman Emamzadeh , Mahboobeh Nazari

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Abstract

Liver cancer is one of the most common cancers and the third leading cause of cancer deaths worldwide. Diagnosis and screening for liver cancer rely on the alpha-fetoprotein (AFP) biomarker. This study aimed to pioneer a novel assay for AFP detection utilizing a tri-part split-luciferase system in conjunction with nanobodies targeting AFP. The strategy involved fusing anti-AFP nanobodies P5 and P15 to the split-nanoluciferase components β9 and β10, respectively. Upon binding to AFP and in the presence of the third nanoluciferase component Δ11S, the proximity-induced reassembly of split-nanoluciferase components triggers luciferase activation and luminescence emission. Following expression in a bacterial system, purification, and assay implementation, the developed assay exhibited high sensitivity in detecting AFP within a linear range of 1–20 ng/ml, with a Limit of Detection (LOD) of 0.5 ng/ml. The assay results were in line with those obtained from ELISA, indicating its efficiency. This study highlights the specificity of the homogenous assay developed with nanobodies for AFP, offering a reliable and user-friendly test for AFP detection.

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用于创新性检测肝癌生物标记物甲胎蛋白的纳米体增强型分裂荧光素酶技术

肝癌是最常见的癌症之一，也是全球癌症死亡的第三大原因。肝癌的诊断和筛查依赖于甲胎蛋白（AFP）生物标志物。这项研究旨在开创一种新的甲胎蛋白检测方法，利用三部分分裂荧光素酶系统和靶向甲胎蛋白的纳米抗体。该策略包括将抗 AFP 纳米抗体 P5 和 P15 分别与分化荧光素酶成分 β9 和 β10 融合。与 AFP 结合后，在第三种纳米荧光素酶成分 Δ11S 存在的情况下，近距离诱导的分裂荧光素酶成分重新组合会引发荧光素酶活化和发光。在细菌系统中表达、纯化和检测实施后，所开发的检测方法在 1-20 纳克/毫升的线性范围内检测甲胎蛋白的灵敏度很高，检测限（LOD）为 0.5 纳克/毫升。检测结果与酶联免疫吸附法得出的结果一致，表明其效率很高。这项研究强调了用纳米抗体开发的甲胎蛋白同质检测方法的特异性，为甲胎蛋白检测提供了一种可靠且用户友好的检测方法。

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来源期刊

Biochemical Engineering Journal 工程技术-工程：化工

CiteScore

7.10

自引率

5.10%

发文量

380

审稿时长

34 days

期刊介绍： The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology. The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields: Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics Biosensors and Biodevices including biofabrication and novel fuel cell development Bioseparations including scale-up and protein refolding/renaturation Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells Bioreactor Systems including characterization, optimization and scale-up Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis Protein Engineering including enzyme engineering and directed evolution.