The Abtamer: A Novel Antibody-Aptamer Conjugate for Electrochemical Biosensing Applications (2020)

Introduction: We propose an innovated semisynthetic biosensing molecule, the Ab tamer , 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 “ Ab tamers,” 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 Ab tamer 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 “ Ab ta-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).