Novel signal amplification approach for HRP-based colorimetric genosensors using DNA binding protein tags

Aktas, Gülsen Betül; Skouridou, Vasso; Masip, Lluis

doi:10.1016/j.bios.2015.07.077

Cited by 11 publications

(1 citation statement)

References 27 publications

(24 reference statements)

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“…Primary antibodies selectively recognize and tightly bind to their target analytes to give sample specificity, whereas secondary antibodies bind to the Fc region of target-analyte-bound primary antibodies to amplify signals. For amplification of signals, a conventional secondary antibody is covalently linked with active enzymes, such as horseradish peroxidase (HRP), , that convert multiple copies of inactive substrates into signal-generating active forms, leading to the efficient detection of low-abundant target analytes. Although enzyme-linked secondary antibodies have been widely used in immunoassays, antibodies themselves are obtained from animals and further modified chemically in lengthy and complicated ways …”

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confidence: 99%

Development of Recombinant Immunoglobulin G-Binding Luciferase-Based Signal Amplifiers in Immunoassays

et al. 2020

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In general immunoassays, secondary antibodies are covalently linked with enzymes and bind to the Fc region of target-bound primary antibodies to amplify signals of low-abundant target molecules. The antibodies themselves are obtained from large mammals and are further modified with enzymes. In this study, we developed novel recombinant immunoglobulin G (IgG)-binding luciferase-based signal amplifiers (rILSAs) by genetically fusing luciferase (Nluc) with antimouse IgG1 nanobody (MG1Nb) and antibody-binding domain (ABD), individually or together, in a mix-and-match manner. We obtained three different highly pure rILSAs in large quantities using a bacterial overexpression system and one-step purification. Mouse-specific rILSA, MG1Nb-Nluc, and rabbit-specific rILSA, Nluc-ABD, selectively bound to target-molecule-bound mouse IgG1 and rabbit IgG primary antibodies, whereas the bispecific rILSA, MG1Nb-Nluc-ABD, mutually bound to both mouse IgG1 and rabbit IgG primary antibodies. All rILSAs exhibited an outstanding signal-amplifying capability comparable to those of conventional horseradish-peroxidase-conjugated secondary antibodies, regardless of the target molecules, in various immunoassay formats, such as enzyme-linked immunosorbent assay, Western blot, and lateral flow assays. Each rILSA was selected for its own individual purpose and applied to various types of target analytes, in combination with a variety of target-specific primary antibodies, effectively minimizing the use of animals as well as reducing the costs and time associated with the production and chemical conjugation of signal-amplifying enzymes.

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Development of Recombinant Immunoglobulin G-Binding Luciferase-Based Signal Amplifiers in Immunoassays

et al. 2020

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Nucleic Acid Amplification Strategy-Based Colorimetric Assays

Shi

Zheng

2019

Nucleic Acid Amplification Strategies for Biosensing, Bioimaging and Biomedicine

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Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures

2017

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A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the conjugates. Graphical abstract DNA sensing platform based on an immobilized ssDNA capture probe and a hybrid ssDNA-dsDNA detection probe that specifically hybridize with the ssDNA target. The hybrid ssDNA-dsDNA detection probe also provides the binding sites for the enzyme-DNA binding protein conjugates (HRP-scCro and GOx-dHP) that generate the colorimetric signal.

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Novel signal amplification approach for HRP-based colorimetric genosensors using DNA binding protein tags

Cited by 11 publications

References 27 publications

Development of Recombinant Immunoglobulin G-Binding Luciferase-Based Signal Amplifiers in Immunoassays

Development of Recombinant Immunoglobulin G-Binding Luciferase-Based Signal Amplifiers in Immunoassays

Nucleic Acid Amplification Strategy-Based Colorimetric Assays

Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures

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