A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System
Abstract In this study, a versatile method is developed to create an antibody/split‐enzyme complex for use in electrochemical immunosensors. SpyCatchers are genetically fused at each terminus of flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) and a protease recognition sequence...
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| Format: | Article |
| Language: | English |
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Wiley-VCH
2025-01-01
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| Series: | Advanced Sensor Research |
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| Online Access: | https://doi.org/10.1002/adsr.202400112 |
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| author | Yuka Tobita Kensuke Hirano Daimei Miura Yuma Hatano Wakako Tsugawa Kazunori Ikebukuro Koji Sode Ryutaro Asano |
| author_facet | Yuka Tobita Kensuke Hirano Daimei Miura Yuma Hatano Wakako Tsugawa Kazunori Ikebukuro Koji Sode Ryutaro Asano |
| author_sort | Yuka Tobita |
| collection | DOAJ |
| description | Abstract In this study, a versatile method is developed to create an antibody/split‐enzyme complex for use in electrochemical immunosensors. SpyCatchers are genetically fused at each terminus of flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) and a protease recognition sequence is inserted into a loop region to prepare a soluble protein and the split GDH. SpyTag‐fused single‐chain variable fragments (scFvs) are employed, which leads to the simple preparation of antibody‐split enzyme complexes (split AECs). The addition of pentameric C‐reactive protein (CRP), as a representative multimeric protein, restored the GDH activity of the split AECs, as the two split AEC fragments formed active GDH when in close proximity. CRP in human serum is electrochemically detected within 5 min without any washing steps with a clinically relevant CRP detection range (0.03–1 mg dL−1). The detection system is expanded to detect hemoglobin, SARS‐CoV‐2 spike protein, and inactivated SARS‐CoV‐2 by exchanging the scFvs. These results show that the convenient preparation method of the split GDH and the split AEC by the insertion of the protease recognition sequence and the use of SpyCatcher/SpyTag can realize a rapid, homogeneous, and universal electrochemical detection system that can be integrated into a commercially available electrochemical glucose sensor. |
| format | Article |
| id | doaj-art-f5ad6cd33d21470fa02891b48c539461 |
| institution | Kabale University |
| issn | 2751-1219 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Wiley-VCH |
| record_format | Article |
| series | Advanced Sensor Research |
| spelling | doaj-art-f5ad6cd33d21470fa02891b48c5394612025-01-09T21:16:46ZengWiley-VCHAdvanced Sensor Research2751-12192025-01-0141n/an/a10.1002/adsr.202400112A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing SystemYuka Tobita0Kensuke Hirano1Daimei Miura2Yuma Hatano3Wakako Tsugawa4Kazunori Ikebukuro5Koji Sode6Ryutaro Asano7Department of Biotechnology and Life Science Graduate School of Engineering Tokyo University of Agriculture and Technology 2‐24‐16 Naka‐cho Koganei Tokyo 184–8588 JapanDepartment of Industrial Technology and Innovation Graduate School of Engineering Tokyo University of Agriculture and Technology 2‐24‐16 Naka‐cho Koganei Tokyo 184–8588 JapanDepartment of Biotechnology and Life Science Graduate School of Engineering Tokyo University of Agriculture and Technology 2‐24‐16 Naka‐cho Koganei Tokyo 184–8588 JapanDepartment of Biotechnology and Life Science Graduate School of Engineering Tokyo University of Agriculture and Technology 2‐24‐16 Naka‐cho Koganei Tokyo 184–8588 JapanDepartment of Biotechnology and Life Science Graduate School of Engineering Tokyo University of Agriculture and Technology 2‐24‐16 Naka‐cho Koganei Tokyo 184–8588 JapanDepartment of Biotechnology and Life Science Graduate School of Engineering Tokyo University of Agriculture and Technology 2‐24‐16 Naka‐cho Koganei Tokyo 184–8588 JapanJoint Department of Biomedical Engineering University of North Carolina at Chapel Hill and North Carolina State University Chapel Hill NC 27599 USADepartment of Biotechnology and Life Science Graduate School of Engineering Tokyo University of Agriculture and Technology 2‐24‐16 Naka‐cho Koganei Tokyo 184–8588 JapanAbstract In this study, a versatile method is developed to create an antibody/split‐enzyme complex for use in electrochemical immunosensors. SpyCatchers are genetically fused at each terminus of flavin adenine dinucleotide‐dependent glucose dehydrogenase (FAD‐GDH) and a protease recognition sequence is inserted into a loop region to prepare a soluble protein and the split GDH. SpyTag‐fused single‐chain variable fragments (scFvs) are employed, which leads to the simple preparation of antibody‐split enzyme complexes (split AECs). The addition of pentameric C‐reactive protein (CRP), as a representative multimeric protein, restored the GDH activity of the split AECs, as the two split AEC fragments formed active GDH when in close proximity. CRP in human serum is electrochemically detected within 5 min without any washing steps with a clinically relevant CRP detection range (0.03–1 mg dL−1). The detection system is expanded to detect hemoglobin, SARS‐CoV‐2 spike protein, and inactivated SARS‐CoV‐2 by exchanging the scFvs. These results show that the convenient preparation method of the split GDH and the split AEC by the insertion of the protease recognition sequence and the use of SpyCatcher/SpyTag can realize a rapid, homogeneous, and universal electrochemical detection system that can be integrated into a commercially available electrochemical glucose sensor.https://doi.org/10.1002/adsr.202400112C‐reactive proteinelectrochemical sensorshomogeneous detectionsingle‐chain fragment variablessplit glucose dehydrogenases |
| spellingShingle | Yuka Tobita Kensuke Hirano Daimei Miura Yuma Hatano Wakako Tsugawa Kazunori Ikebukuro Koji Sode Ryutaro Asano A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System Advanced Sensor Research C‐reactive protein electrochemical sensors homogeneous detection single‐chain fragment variables split glucose dehydrogenases |
| title | A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System |
| title_full | A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System |
| title_fullStr | A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System |
| title_full_unstemmed | A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System |
| title_short | A Versatile Method to Create Antibody/Split‐Enzyme Complexes and Its Application to a Rapid, Homogeneous, and Universal Electrochemical Immunosensing System |
| title_sort | versatile method to create antibody split enzyme complexes and its application to a rapid homogeneous and universal electrochemical immunosensing system |
| topic | C‐reactive protein electrochemical sensors homogeneous detection single‐chain fragment variables split glucose dehydrogenases |
| url | https://doi.org/10.1002/adsr.202400112 |
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