A Rapid, Homogeneous, and Universal Electrochemical Immunosensing System Using an Antibody/Split-Enzyme Complex
Authors: Miura Daimei , Tobita Yuka , Hirano Kensuke , Hatano Yuma , Tsugawa Wakako , Sode Koji , Ikebukuro Kazunori , Asano Ryutaro 
Published: December 2024

Introduction

Biosensors for point-of-care testing (POCT) must be rapid, user-friendly, and equipment-free. However, the conventional immunosensor principle employing the labelled secondary antibodies and the washing processes like enzyme-linked immunosorbent assay (ELISA) prevents the use for POCT applications. Our group has been developing antibody-enzyme complexes (AECs)1, which are composed of fragment antibodies and redox enzymes by applying a protein coupling module, SpyCatcher (SC)/SpyTag (ST). Although these AECs realized electrochemical immunosensors with rapid and sensitive detection2,3, but they still required magnetic bound/free (B/F) separation.

We propose an ideal immunosensor employing AECs for POCT, which uses a protein-fragment complementation assay. This is based on the reconstitution of enzyme of the AEC which is inactivated by split technology and its enzymatic activity recovers in response to the target recognition by the AEC. To date, split pyrroloquinoline quinone-glucose dehydrogenase (PQQ-GDH) was reported and applied to the construction of AEC by means of genetic fusion with fragment antibodies4. However, the genetic fusion of antibodies is limited to the C-terminus of the enzyme to avoid loss of binding ability because the N-terminus of antibodies is located nearby the binding site of antibody. Moreover, the recombinant preparation of each protein fragment after splitting enzymes often accompanies the formation of the protein aggregates due to the exposure of the hydrophobic core of the enzyme.

Here, we focused on a flavin adenine dinucleotide GDH (FAD-GDH) as a source for split enzyme, which is introduced to a loop of the enzyme, and employed SC and ST to construct universal split AECs. In addition, it is expected to prepare the split enzyme as soluble proteins because it is digested after the recombinant production. After the preparation of AECs, we applied the split AECs to electrochemical detection of biomarkers, C-reactive protein (CRP) and hemoglobin (Hb), and infectious virus, SARS-CoV-2.

Methods

First, we selected three split site candidates such as a loop exposed to the protein surface and a site which are not important for the protein structure. Then, tobacco etch virus (TEV) protease recognition sequence was inserted to each candidate site and SC was genetically fused to both terminals of FAD-GDH (SC-GDH-SC). ST was genetically fused to the C-terminus of single-chain variable fragments (scFv-ST). All the proteins were recombinantly produced in E. coli and purified. SC-GDH-SC was digested with TEV protease, and the preparation of split GDH was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Then, they were stoichiometrically mixed with the purified scFv-ST and incubated overnight at 4℃. The formation of split AEC was also evaluated by SDS-PAGE analysis. The binding ability and enzymatic activity caused by reconstitution upon target binding of the split AEC were evaluated. Finally, electrochemical detection system was constructed. The target was incubated with the split GDH, then mixed with the reaction solution containing a redox probe and glucose, and loaded onto a disposable electrode-printed chip. After incubation for 90 s, chronoamperometric measurement was performed.

Results and discussion

The result of SDS-PAGE analysis showed that all the split AECs were successfully prepared using the SC/ST system. Among the split site candidates, the GDH split between G407 and N408 showed the highest recovery of enzymatic activity after mixing with CRP. The enzymatic activity was not decreased even after protease digestion, which suggested that the GDH was not completely split into individual fragments in solution. This can also be confirmed by the binding ability of the split AEC, which showed enhanced affinity compared to scFv-ST due to the bivalent effect. However, the enzymatic activity of the split GDH was lowered in the absence of the target and recovered in response to the target concentration. When the different scFv-ST was used for the split AEC preparation, other targets were successfully detected, demonstrating the versatility of the system. As a result of electrochemical CRP detection, CRP concentration-dependent current increase was observed, and the linear range met the clinically required range.

Conclusion

We designed split AECs with high versatility, and applied it to the electrochemical detection of the protein biomarker. The detection can be completed within 5 min without any B/F separation, which can meet the criteria for POCT application. In addition, it was based on chronoamperometric measurement using GDH, a sensor configuration of a currently available glucose meter can be used for future applications.

Reference

1Kimura et al., Anal. Chem., 2018, 90, 14500-14506.

2Kimura et al., Biosens. Bioelectron., 2021, 175, 112885.

3Miura et al., Talanta, 2021, 234, 122638.

4Guo et al., JACS, 2016, 138, 10108-10111.