{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,7]],"date-time":"2026-02-07T10:14:16Z","timestamp":1770459256999,"version":"3.49.0"},"reference-count":39,"publisher":"MDPI AG","issue":"14","license":[{"start":{"date-parts":[[2020,7,11]],"date-time":"2020-07-11T00:00:00Z","timestamp":1594425600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.<\/jats:p>","DOI":"10.3390\/s20143878","type":"journal-article","created":{"date-parts":[[2020,7,14]],"date-time":"2020-07-14T09:30:49Z","timestamp":1594719049000},"page":"3878","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF"],"prefix":"10.3390","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7172-0409","authenticated-orcid":false,"given":"Jinhee","family":"Lee","sequence":"first","affiliation":[{"name":"Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Atsuro","family":"Tatsumi","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaori","family":"Tsukakoshi","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2968-0268","authenticated-orcid":false,"given":"Ellie D.","family":"Wilson","sequence":"additional","affiliation":[{"name":"Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Koichi","family":"Abe","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Koji","family":"Sode","sequence":"additional","affiliation":[{"name":"Joint Department of Biomedical Engineering, The University of North Carolina at Chapel Hill and North Carolina State University, Chapel Hill, NC 27599, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kazunori","family":"Ikebukuro","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2020,7,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1126\/science.2200121","article-title":"Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase","volume":"249","author":"Tuerk","year":"1990","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1038\/346818a0","article-title":"In vitro selection of RNA molecules that bind specific ligands","volume":"346","author":"Ellington","year":"1990","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2901","DOI":"10.1081\/AL-200035778","article-title":"Electrochemical detection of protein using a double aptamer sandwich","volume":"37","author":"Ikebukuro","year":"2004","journal-title":"Anal. Lett."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1007\/s00216-019-02226-x","article-title":"Electrochemical biosensors based on nucleic acid aptamers","volume":"412","author":"Villalonga","year":"2020","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2436","DOI":"10.1110\/ps.051478705","article-title":"Structural characterization of the maytansinoid\u2013monoclonal antibody immunoconjugate, huN901\u2013DM1, by mass spectrometry","volume":"14","author":"Wang","year":"2005","journal-title":"Protein Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3731","DOI":"10.1073\/pnas.1120682109","article-title":"Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR","volume":"109","author":"Kazane","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2168","DOI":"10.1016\/j.bios.2004.09.002","article-title":"Novel electrochemical sensor system for protein using the aptamers in sandwich manner","volume":"20","author":"Ikebukuro","year":"2005","journal-title":"Biosens. Bioelectron."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.1002\/elan.200804555","article-title":"An aptamer-based bound\/free separation system for protein detection","volume":"21","author":"Fukasawa","year":"2009","journal-title":"Electroanalysis"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1068","DOI":"10.1177\/193229681100500507","article-title":"Review of glucose oxidases and glucose dehydrogenases: A bird\u2019s eye view of glucose sensing enzymes","volume":"5","author":"Ferri","year":"2011","journal-title":"J. Diabetes Sci. Technol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"250","DOI":"10.1038\/nchembio.1186","article-title":"Induction of innate and adaptive immunity by delivery of poly dA:dT to dendritic cells","volume":"9","author":"Barbuto","year":"2013","journal-title":"Nat. Chem. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"10850","DOI":"10.1021\/ja504711n","article-title":"A Modular approach for assembling aldehyde-tagged proteins on DNA scaffolds","volume":"136","author":"Liang","year":"2014","journal-title":"J. Am. Chem. Soc."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"8819","DOI":"10.1002\/anie.200701942","article-title":"A universal method for the preparation of covalent protein-DNA conjugates for use in creating protein nanostructures","volume":"46","author":"Duckworth","year":"2007","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"7491","DOI":"10.1039\/C8CC03268H","article-title":"Quantitative synthesis of protein\u2013DNA conjugates with 1:1 stoichiometry","volume":"54","author":"Yan","year":"2018","journal-title":"Chem. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"804","DOI":"10.1038\/nchem.2003","article-title":"Template-directed covalent conjugation of DNA to native antibodies, transferrin and other metal-binding proteins","volume":"6","author":"Rosen","year":"2014","journal-title":"Nat. Chem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5040","DOI":"10.1021\/ac300708r","article-title":"Detection of Antigens Using a Protein\u2013DNA Chimera Developed by Enzymatic Covalent Bonding with phiX Gene A*","volume":"84","author":"Akter","year":"2012","journal-title":"Anal. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"7030","DOI":"10.1021\/jacs.7b02572","article-title":"Sequence-directed covalent protein-DNA linkages in a single step using HUH-Tags","volume":"139","author":"Lovendahl","year":"2017","journal-title":"J. Am. Chem. Soc."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1007\/s10529-018-02641-7","article-title":"Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein","volume":"41","author":"Mie","year":"2019","journal-title":"Biotechnol. Lett."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"11467","DOI":"10.1039\/C5CC02906F","article-title":"Enzyme linking to DNA aptamers via a zinc finger as a bridge","volume":"51","author":"Abe","year":"2015","journal-title":"Chem. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1146\/annurev.biophys.29.1.183","article-title":"DNA recognition by Cys2His2 zinc finger proteins","volume":"29","author":"Wolfe","year":"2000","journal-title":"Annu. Rev. Biophys. Biomol. Struct."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/j.tibtech.2013.04.004","article-title":"ZFN, TALEN, and CRISPR\/Cas-based methods for genome engineering","volume":"31","author":"Gaj","year":"2013","journal-title":"Trends Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"636","DOI":"10.1038\/nrg2842","article-title":"Genome editing with engineered zinc finger nucleases","volume":"11","author":"Urnov","year":"2010","journal-title":"Nat. Rev. Genet."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"10782","DOI":"10.1021\/ja051969w","article-title":"DNA Sequence-Enabled Reassembly of the Green Fluorescent Protein","volume":"127","author":"Stains","year":"2005","journal-title":"J. Am. Chem. Soc."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3620","DOI":"10.1021\/bi0517032","article-title":"Sequence-Enabled Reassembly of \u03b2-Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA","volume":"45","author":"Ooi","year":"2006","journal-title":"Biochemistry"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"8028","DOI":"10.1021\/ac3018845","article-title":"Detection of pathogenic bacteria by using zinc finger protein fused with firefly luciferase","volume":"84","author":"Abe","year":"2012","journal-title":"Anal. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4991","DOI":"10.1039\/C4AY00977K","article-title":"Electrochemical detection of pathogenic bacteria by using a glucose dehydrogenase fused zinc finger protein","volume":"6","author":"Lee","year":"2014","journal-title":"Anal. Methods"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1210\/er.2003-0027","article-title":"Vascular endothelial growth factor: Basic science and clinical progress","volume":"25","author":"Ferrara","year":"2004","journal-title":"Endocr. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"7871","DOI":"10.1158\/1078-0432.CCR-08-0593","article-title":"VEGF as a marker for outcome among advanced breast cancer patients receiving anti-VEGF therapy with bevacizumab and vinorelbine chemotherapy","volume":"14","author":"Burstein","year":"2008","journal-title":"Clin. Cancer Res."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1038\/362841a0","article-title":"Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo","volume":"362","author":"Kim","year":"1993","journal-title":"Nature"},{"key":"ref_29","first-page":"103","article-title":"Dry age-related macular degeneration: A currently unmet clinical need","volume":"1","author":"Girmens","year":"2012","journal-title":"Intractable Rare Dis. Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1016\/S2213-2600(13)70090-0","article-title":"MILES Trial Group Serum VEGF-D a concentration as a biomarker of lymphangioleiomyomatosis severity and treatment response: A prospective analysis of the Multicenter International Lymphangioleiomyomatosis Efficacy of Sirolimus (MILES) trial","volume":"1","author":"Young","year":"2013","journal-title":"Lancet Respir. Med."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1016\/j.jad.2011.07.021","article-title":"Increased plasma VEGF levels in major depressive or manic episodes in patients with mood disorders","volume":"136","author":"Lee","year":"2012","journal-title":"J. Affect. Disord."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1055\/s-0042-106295","article-title":"Serum and plasma levels of vascular endothelial growth factors in relation to quality of glucose control, biomarkers of inflammation, and diabetic nephropathy","volume":"48","author":"Hanefeld","year":"2016","journal-title":"Horm. Metab. Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1469","DOI":"10.1093\/oxfordjournals.jbchem.a131836","article-title":"1-Methoxy-5-methylphenazinium methyl Sulfate. A photochemically stable electron mediator between NADH and various electron acceptors","volume":"82","author":"Hisada","year":"1977","journal-title":"J. Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"2255","DOI":"10.1007\/s10529-011-0694-5","article-title":"Screening of Aspergillus-derived FAD-glucose dehydrogenases from fungal genome database","volume":"33","author":"Mori","year":"2011","journal-title":"Biotechnol. Lett."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1171","DOI":"10.1016\/S0969-2126(96)00125-6","article-title":"Zif268 protein-DNA complex refined at 1.6 \u00c5 a model system for understanding zinc finger-DNA interactions","volume":"4","author":"Rould","year":"1996","journal-title":"Structure"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1016\/j.jbiotec.2015.08.011","article-title":"Improvement of the VEGF binding ability of DNA aptamers through in silico maturation and multimerization strategy","volume":"212","author":"Fukaya","year":"2015","journal-title":"J. Biotechnol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"215","DOI":"10.3390\/molecules15010215","article-title":"Screening and improvement of an anti-VEGF DNA aptamer","volume":"15","author":"Nonaka","year":"2010","journal-title":"Molecules"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"9524","DOI":"10.1093\/nar\/gkt697","article-title":"G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold","volume":"41","author":"Veedu","year":"2013","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1432","DOI":"10.1073\/pnas.98.4.1432","article-title":"Design of polyzinc finger peptides with structured linkers","volume":"98","author":"Moore","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/20\/14\/3878\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:50:19Z","timestamp":1760176219000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/20\/14\/3878"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,7,11]]},"references-count":39,"journal-issue":{"issue":"14","published-online":{"date-parts":[[2020,7]]}},"alternative-id":["s20143878"],"URL":"https:\/\/doi.org\/10.3390\/s20143878","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,7,11]]}}}