Enzyme linking to DNA aptamers <i>via</i> a zinc finger as a bridge

Abe, Koichi; Murakami, Yoshiyuki; Tatsumi, Atsuro; Sumida, K.; Kezuka, Aki; Fukaya, Takahiro; Kumagai, Tomohisa; Osawa, Yuko; Sode, Koji

doi:10.1039/c5cc02906f

Cited by 6 publications

(4 citation statements)

References 22 publications

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“…Using the nanoprobes containing 22 Nluc, we further investigate the sensitivity of the assay. As low as 2.5 nM thrombin was easily detected (Figure S6D); this detection limit is in line with other reported enzyme-based ELISA assay for thrombin. − …”

Section: Resultssupporting

confidence: 90%

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly

et al. 2015

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Context driven biosensor assembly with modular targeting and detection moieties is gaining significant attentions. Although protein-based nanoparticles have emerged as an excellent platform for biosensor assembly, current strategies of decorating bionanoparticles with targeting and detection moieties often suffer from unfavorable spacing and orientation as well as bionanoparticle aggregation. Herein, we report a highly modular post-translational modification approach for biosensor assembly based on sortase A-mediated ligation. This approach enables the simultaneous modifications of the Bacillus stearothermophilus E2 nanoparticles with different functional moieties for antibody, enzyme, DNA aptamer, and dye decoration. The resulting easy-purification platform offers a high degree of targeting and detection modularity with signal amplification. This flexibility is demonstrated for the detection of both immobilized antigens and cancer cells.

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Section: Resultssupporting

confidence: 90%

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly

et al. 2015

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“…Thus, it will form a logarithmic trend in Raman intensity, when the concentration is more than 1500 ppt. As shown in Figures 3B and 3C, the sensitivity of our assay for Zn(II) can be as low as 100 ppt, which is about 1–2 orders of magnitude higher than reported method 7,9,18–19 . To determine the sensitivity, we have used SERS response at 1380 cm −1 .…”

Section: Resultsmentioning

confidence: 59%

Development of a SERS Probe for Selective Detection of Healthy Prostate and Malignant Prostate Cancer Cells Using Zn^II

Pramanik

Chavva

Nellore

et al. 2017

Chemistry An Asian Journal

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Even in 21st century, prostate cancer remains the second leading cause of cancer death for men. Since normal prostate gland contains the most Zn(II) and there are huge differences in Zn(II) content between the healthy and malignant prostate cancer cells, mobile zinc can be used as a biomarker for prostate cancer prediction. Current article reports the design of novel and highly efficient surface enhanced Raman spectroscopy (SERS) probe using p-(imidazole)azo) benzenethiol attached gold nanoparticle as a Raman reporter, which has the capability to identify prostate cancer cells based on Zn(II) sensing. A facile synthesis, characterization and evaluation as Zn(II) sensing Raman probe has been reported. Reported data indicate that after binding with Zn(II), Raman reporter attached gold nanoparticle forms assembly structure, which allows selective detection of Zn(II) even at 100 ppt concentration. Theoretical full-wave finite-difference time-domain (FDTD) simulation has been used to understand huge enhancement of SERS signal. Reported SERS probe is highly promising for in-vivo sensing of cancer, where near IR light can be easily used to avoid tissue auto-fluorescence and to enhance tissue penetration depth. Reported data show that SERS probe can distinguish metastatic cancer cells from normal prostate cells very easily with sensitivity as low as 5 cancer cells/mL. Designed SERS probe has the capability to be used as chemical toolkit for determining mobile Zn(II) concentrations in the biological sample.

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“…However, the method still suffered from low bioactivity of the conjugated enzyme. Inspired by our previous study [ 18 ], we have focused on the zinc finger protein (ZF), a monomeric double-stranded DNA-binding protein, to create a GDH-labelled aptamer. ZF is a DNA-binding protein which is capable of recognizing and binding to a specific sequence of double-stranded DNA [ 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF

Lee

Tatsumi

Tsukakoshi

et al. 2020

Sensors

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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.

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Enzyme linking to DNA aptamers via a zinc finger as a bridge

Abstract: We propose a novel enzyme-labeling method for DNA aptamers using enzyme-fused zinc finger proteins. We achieved thrombin detection and vascular endothelial growth factor detection using zinc finger-fused firefly luciferase.

Cited by 6 publications

References 22 publications

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly

Development of a SERS Probe for Selective Detection of Healthy Prostate and Malignant Prostate Cancer Cells Using Zn^II

Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF

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Enzyme linking to DNA aptamers via a zinc finger as a bridge

Abstract: We propose a novel enzyme-labeling method for DNA aptamers using enzyme-fused zinc finger proteins. We achieved thrombin detection and vascular endothelial growth factor detection using zinc finger-fused firefly luciferase.

Cited by 6 publications

References 22 publications

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly

Post-Translational Modification of Bionanoparticles as a Modular Platform for Biosensor Assembly

Development of a SERS Probe for Selective Detection of Healthy Prostate and Malignant Prostate Cancer Cells Using ZnII

Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF

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Product

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Development of a SERS Probe for Selective Detection of Healthy Prostate and Malignant Prostate Cancer Cells Using Zn^II