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Construction of Protein Probe with a His‐tag and an Electron‐transfer Peptide for a Target Protein Sensing
Electroanalysis ( IF 2.7 ) Pub Date : 2020-11-16 , DOI: 10.1002/elan.202060338 Kazuharu Sugawara 1 , Sora Ishizaki 1 , Soya Kikuchi 1 , Hideki Kuramitz 2 , Toshihiko Kadoya 1
Electroanalysis ( IF 2.7 ) Pub Date : 2020-11-16 , DOI: 10.1002/elan.202060338 Kazuharu Sugawara 1 , Sora Ishizaki 1 , Soya Kikuchi 1 , Hideki Kuramitz 2 , Toshihiko Kadoya 1
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A protein probe with an electron‐transfer peptide and a His‐tag was designed to electrochemically sense a target protein. We selected tyrosine‐rich (Y4C) and tryptophan‐rich (W4C) peptides for use as electron‐transfer agents. The peak for oxidation was based on the oxidations of the phenolic hydroxy groups in Y4C and on the indole rings in W4C. Asialofetuin (ASF) with galactose residues was the protein probe, and a galactose recognition protein, soybean agglutinin (SBA), was the target protein. A protein probe composed of an amino acid and carbohydrate residue was expected to be biocompatible. When voltammetric measurements were performed using a glassy carbon electrode, the oxidation peaks of H6Y4C and ASF‐H6Y4C appeared at the same potential. The peak current of ASF‐H6Y4C was 4‐fold that of H6Y4C because of the stronger adsorption of ASF‐H6Y4C onto the electrode. The electrode response of ASF‐H6Y4C with SBA was half that of ASF‐H6Y4C alone. By contrast, the peak current of ASF‐Y4CH6 was higher than that of ASF‐H6Y4C, which was the result of a greater degree of contact between the Y4C moieties and an electrode. On the other hand, the voltammetric behaviors of ASF with W4C and a His‐tag were similar to those with Y4C and a His‐tag. The sensitivity of SBA using ASF‐Y4CH6 was at the 10−13 M level. To confirm the function of the sensing system, measurements were performed in human serum with SBA and ASF‐Y4CH6. When SBA was added, the serum had a concentration that ranged between 5.0×10−13 and 4.0×10−12 M, and the amount of SBA that could be recovered ranged from 97 to 101%. Consequently, this system could be applied to the detection of SBA in serum.
中文翻译：
具有His标签和电子转移肽的目标蛋白传感蛋白探针的构建
具有电子转移肽和His-tag的蛋白质探针被设计用于电化学检测目标蛋白质。我们选择了富含酪氨酸的（Y 4 C）和富含色氨酸的（W 4 C）肽作为电子转移剂。氧化峰基于Y 4 C中酚羟基的氧化和W 4 C中吲哚环的氧化。带有半乳糖残基的Asiafefetuin（ASF）是蛋白质探针，半乳糖识别蛋白大豆凝集素（SBA） ），是目标蛋白。预期由氨基酸和碳水化合物残基组成的蛋白质探针具有生物相容性。使用玻璃碳电极进行伏安法测量时，H 6 Y的氧化峰4 C和ASF-H 6 Y 4 C的电位相同。ASF-H的峰值电流6 ý 4 C为4倍于H的6 ý 4 C，因为更强吸附ASF-H的6 ý 4下向电极。ASF-H的电极反应6 ý 4下用SBA是半ASF-H的那6 ý 4单独的C。相比之下，ASF‐Y 4 CH 6的峰值电流高于ASF‐H 6 Y 4 C的峰值电流，这是由于Y 4之间的接触程度更高的结果C部分和电极。另一方面，带有W 4 C和His-tag的ASF的伏安行为与带有Y 4 C和His-tag的ASF的伏安行为相似。使用ASF‐Y 4 CH 6的SBA的灵敏度为10 -13M 。为了确认传感系统的功能，在人血清中用SBA和ASF-Y 4 CH 6进行了测量。当添加SBA时，血清的浓度在5.0×10 -13和4.0×10 -12 M之间，并且可以回收的SBA的量在97％至101％的范围内。因此，该系统可用于血清中SBA的检测。
更新日期：2020-11-16
中文翻译：
具有His标签和电子转移肽的目标蛋白传感蛋白探针的构建
具有电子转移肽和His-tag的蛋白质探针被设计用于电化学检测目标蛋白质。我们选择了富含酪氨酸的（Y 4 C）和富含色氨酸的（W 4 C）肽作为电子转移剂。氧化峰基于Y 4 C中酚羟基的氧化和W 4 C中吲哚环的氧化。带有半乳糖残基的Asiafefetuin（ASF）是蛋白质探针，半乳糖识别蛋白大豆凝集素（SBA） ），是目标蛋白。预期由氨基酸和碳水化合物残基组成的蛋白质探针具有生物相容性。使用玻璃碳电极进行伏安法测量时，H 6 Y的氧化峰4 C和ASF-H 6 Y 4 C的电位相同。ASF-H的峰值电流6 ý 4 C为4倍于H的6 ý 4 C，因为更强吸附ASF-H的6 ý 4下向电极。ASF-H的电极反应6 ý 4下用SBA是半ASF-H的那6 ý 4单独的C。相比之下，ASF‐Y 4 CH 6的峰值电流高于ASF‐H 6 Y 4 C的峰值电流，这是由于Y 4之间的接触程度更高的结果C部分和电极。另一方面，带有W 4 C和His-tag的ASF的伏安行为与带有Y 4 C和His-tag的ASF的伏安行为相似。使用ASF‐Y 4 CH 6的SBA的灵敏度为10 -13M 。为了确认传感系统的功能，在人血清中用SBA和ASF-Y 4 CH 6进行了测量。当添加SBA时，血清的浓度在5.0×10 -13和4.0×10 -12 M之间，并且可以回收的SBA的量在97％至101％的范围内。因此，该系统可用于血清中SBA的检测。
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