Electrochemical aptamer-based biosensors (E-ABs) are attractive candidates for use in biomarker detection systems due to their sensitivity, rapid response, and design flexibility. There are only several redox probes that were employed previously for this application, and a combination of redox probes affords some advantages in target detection. Thus, it would be advantageous to study new redox probes in an E-AB system. In this study, we report the use of amine-reactive phenazine ethosulfate (arPES) for E-AB through its conjugation to the terminus of thrombin-binding aptamer. The constructed E-AB can detect thrombin by square-wave voltammetry (SWV), showing peak current at −0.15 V vs. Ag/AgCl at pH 7, which differs from redox probes used previously for E-ABs. We also compared the characteristics of PES as a redox probe for E-AB to methylene blue (MB), which is widely used. arPES showed stable signal at physiological pH. Moreover, the pH profile of arPES modified thrombin-binding aptamer revealed the potential application of arPES for a simultaneous multianalyte detection system. This could be achieved using different aptamers with several redox probes in tandem that harbor various electrochemical peak potentials. Our findings present a great opportunity to improve the current standard of biological fluid monitoring using E-AB.
Introduction: We propose an innovated semisynthetic biosensing molecule, the Abtamer, an antibody-aptamer one-to-one complex. An important consideration in the design of electrochemical biosensors is the molecular recognition element (MRE) from which the signal to be transduced is generated. Among MREs utilized for affinity principle based electrochemical biosensors, antibodies are commonly used due to their high sensitivity and selectivity towards specific targets. However, antibodies’ structural rigidity proves limiting in biosensing applications. In contrast, DNA/RNA aptamers are easily-synthesized, versatile biomolecules, which are able to exhibit a high affinity and sensitivity towards their selected target [1]. Owing to their structure, aptamers are inherently flexible which allows for pronounced conformational changes upon binding. Furthermore, their small size allows for the immobilization of a larger density of molecules on the sensor surface, aiding in their sensitivity [2]. However, these same characteristics put aptamers at a disadvantage in terms of their selectivity towards targets. Without various post-synthesis modifications, DNA/RNA oligonucleotides can exhibit nonspecific electrostatic interactions with other analytes in solution. To this end, we propose a new biosensing molecule for biomedical sensor applications based on antibody-aptamer conjugates, designated as “Abtamers,” which includes advantages of both antibodies and aptamers; e.g. high sensitivity, selectivity with molecular dynamics upon target binding, facilitating signals for sensing. We propose to use insulin as a model target for an electrochemical sensor employing this novel biorecognition element. The electrochemical detection of insulin levels in persons with diabetes would provide another useful metric in diabetes technology, as insulin measurements are typically done via ELISA. Methods: The antibody-aptamer conjugate constructed utilized an insulin single chain variable fragment (scFv) and an insulin aptamer. To circumvent the non-optimal binding of polypeptides to oligonucleotides, we utilized the SpyCatcher/SpyTag complex as the bioconjugation system to couple the scFv to the aptamer in a one-to-one manner [3-4]. We created a recombinant fusion molecule of the insulin scFv and SpyCatcher, and created a SpyTag-insulin aptamer fusion molecule. The combination of these two fusion molecules results in an insulin antibody-insulin aptamer conjugate. We aim to employ this new MRE to detect insulin via square wave voltammetry. Results and Discussion: Through the binding of the two fusion molecules, we have created an irreversibly bound conjugate, conserving the structures of both the antibody and aptamer. The modification of a redox probe to the insulin aptamer domain in the Abtamer conjugate allows for a simple and facile method to create an electroactive biomolecule which can be immobilized to an electrode surface. We expect the binding affinity of this molecule to be higher towards specified targets due to both an antibody and aptamer binding event. Furthermore, the flexibility of the aptamer allows for conformational changes upon binding which can be exploited in electrochemical sensing applications. The developed insulin antibody-insulin aptamer conjugate can be applied as a biorecognition element for an electrochemical sensing platform, thereby realizing a unique biosensing principle, the “Abta-sensor” References: [1] A. Ravalli, D. Voccia, I. Palchetti, and G. Marrazza, Biosensors, 6(3) (2016). [2] M. A. Morales, and J. M. Halpern, Bioconjug. Chem., 29(10), 3231–3239 (2018). [3] H. Kimura, R. Asano, N. Tsukamoto, W. Tsugawa, and K. Sode, Anal. Chem., 90(24), 14500–14506 (2018). [4] B. Zakeri, J. O. Fierer, E. Celik, E. C. Chittock, U. Schwarz-Linek, V. T. Moy, and M. Howarth, Proc. Natl. Acad. Sci. USA., 109(12) E690-E697 (2012).
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