Development of Convenient Immunosensors Employing Single Chain Variable Fragment (scFv)-Displayed Biogenic Magnetic Nanoparticles
Authors: Goto Ayaka , Miura Daimei , Tomoe Ryoto , Sode Koji , Tsugawa Wakako , Ikebukuro Kazunori , Yoshino Tomoko , Asano Ryutaro 
Published: December 2024

Introduction

Convenient immunosensors are desired for the early diagnosis of diseases; however, washing operation to remove unreacted sensing elements takes a long time and requires well-trained technicians in conventional immunosensors. Recently, recombinant antibody fragments such as single chain variable fragments (scFvs) have been employed to realize them. Here, we developed two wash-free immunodetection systems combining a scFv-glucose oxidase complex (scFv-GOx) (Fig.1a) and a scFv-displaying magnetic nanoparticle (scFv-MNP) (Fig. 1b). The first one is a chemiluminescent detection system based on the peroxidase activity of MNPs (Fig. 2a). When scFv-MNP and scFv-GOx bind to the target, MNP and GOx become spatially close. Substrate addition is then expected to initiate a target-dependent sequential reaction in which luminol reacts with hydrogen peroxide generated by the oxidation of GOx to produce a chemiluminescent signal. The second one is an electrochemical detection system that integrates magnetic separation (Fig. 2b). Targets are preincubated with scFv-MNP and scFv-GOx, and dropped onto an electrode. Magnetic separation allows efficient electron transfer from GOx to the electrode.

Methods

To prepare scFv-MNPs, we focused on biogenic MNPs, magnetosomes synthesized by magnetotactic bacterium, Magnetospirillum magneticum. We also integrated an efficient protein coupling module, SpyCatcher (SC)/SpyTag (ST) system, to facilitate the exchange of scFvs depending on the target. First, preparing SC fused Mms13, an endogenously existing protein in the lipid layer of magnetosome, which was utilized for protein display on magnetosomes1, then we isolated SC-displayed MNPs (SC-MNPs) (Fig. 1c) from transformed magnetic bacteria with the vector expressing SC fused Mms 13. Second, to detect C-reactive protein (CRP) with homo pentameric structure, anti-CRP scFv-MNPs were prepared by mixing SC-MNPs and ST-fused anti-CRP scFv (scFv-ST) (Fig. 1d). Then, scFv-MNPs, anti-CRP scFv-GOx, and CRP were mixed and incubated for 30 minutes. Chemiluminescence was measured after addition of glucose and luminol. For the electrochemical detection, the mixture was dropped onto a printed carbon electrode and response current was monitored after the addition of glucose and magnetic separation.

Results and discussion

CRP concentration-dependent signal increases were observed in the range of 10-250 nM and 10-100 nM, in the chemiluminescent detection and the electrochemical detection, respectively. In the chemiluminescent detection system, a more convenient operation was achieved by simply mixing scFv-MNPs and GOx-scFv, but the signal detection time took about 20 min. In contrast, the electrochemical detection time took around 5 min because the current value increases immediately after glucose is added, but the sensitivity was low under the present detection conditions. Therefore, we are working to optimize the detection conditions and detect CRP in serum with both detection systems.

Reference

Tanaka et al., Anal. Chem. 2000, 72 (15), 3518−3522.

Figure 1