Optical biosensing of monkeypox virus using novel recombinant silica-binding proteins for site-directed antibody immobilization

Abstract

The efficient immobilization of capture antibodies is crucial for timely pathogen detection during global pandemic outbreaks. Therefore, we proposed a silica-binding protein featuring core functional domains (cSP). It comprises a peptide with a silica-binding tag designed to adhere to silica surfaces and tandem protein G fragments (2C2) for effective antibody capture. This innovation facilitates precise site-directed immobilization of antibodies onto silica surfaces. We applied cSP to silica-coated optical fibers, creating a fiber-optic biolayer interferometer (FO-BLI) biosensor capable of monitoring the monkeypox virus (MPXV) protein A29L in spiked clinical samples to rapidly detect the MPXV. The cSP-based FO-BLI biosensor for MPXV demonstrated a limit of detection (LOD) of 0.62 ng/mL in buffer, comparable to the 0.52 ng/mL LOD achieved using a conventional streptavidin (SA)-based FO-BLI biosensor. Furthermore, it achieved LODs of 0.77 ng/mL in spiked serum and 0.80 ng/mL in spiked saliva, exhibiting no cross-reactivity with other viral antigens. The MPXV detection process was completed within 14 min. We further proposed a cSP-based multi-virus biosensor strategy capable of detecting various pandemic strains, such as MPXV, the latest coronavirus disease (COVID) variants, and influenza A protein, to extend its versatility. The proposed cSP-modified FO-BLI biosensor has a high potential for rapidly and accurately detecting MPXV antigens, making valuable contributions to epidemiological studies.

Keywords: Monkeypox virus; Multi-virus biosensor; Optical biosensing; Silica-binding proteins; Site-directed immobilization; Spiked clinical samples.

Graphical abstract

Fig. 1

Scheme for designing and purifying recombinant silica-binding protein featuring core-functional domains (cSP). (A) Schematic of the three-dimensional (3D) structure of cSP projected through Modeller10.4. (B) The structural diagram of the implemented cSP fusion gene in the pET26b plasmid. The cSP expression was regulated by the T7 promoter with a lac operator. (C) Validation of cSP expression and purification via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). 2C2: tandem protein G fragments; Si-tag: silica-binding peptide. MW: molecular weight marker; CL: whole-cell lysate; P1 and P2: purified cSP proteins.

Fig. 2

Construction and characterization of the bare-silica fibers in silica-binding protein featuring core-functional domains (cSP)-based monoclonal antibody (mAb) loading on silica surface. (A) Evaluation of the optimized oxidizing treatment time on bare-silica fiber conversion. (B) Representative result of the cSP-driven mAbs loading on the fiber-optic biolayer interferometer (FO-BLI) biosensor using silica-coated fibers. (C) Validation of the cSP capacity in capturing mAbs generated from different species.

Fig. 3

Flowchart of the silica-binding protein featuring core-functional domains (cSP)-based monkeypox virus (MPXV) detection in buffer and spiked clinical samples. mAb: monoclonal antibody; HRP: horseradish peroxidase; AMEC: 3-amino-9-ethylcarbazole.

Fig. 4

The silica-binding protein featuring core-functional domains (cSP)-based fiber-optic biolayer interferometer (FO-BLI) biosensor for monkeypox virus (MPXV) detection in buffer and spiked clinical samples. (A) The MPXV antibody screening of cSP-based assay. (B) The cross-reactivity assay towards MPXV, vaccinia virus (VACV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) XBB.1.5 (XBB.1.5), and influenza A H3N2 (H3N2). (C) A comparison of the standard curves generated using the cSP-based vs. streptavidin (SA)-based biosensing strategy for MPXV detection in buffer. (D) The cSP-based biosensing performance for MPXV detection in spiked saliva and serum. MPXV-Ab: MPXV antibody; LOD: limit of detection.

Fig. 5

Future perspective for multi-virus detection using the silica-binding protein featuring core-functional domains (cSP)-based optical biosensor. The cSP-based biosensing supports the loading of antibodies through a uniform and straightforward process. This capability enables the simultaneous detection of multiple viruses in one single sample, streamlining the analytical procedure. (A) The illustration depicts the sequential steps of loading antibodies, samples, and standards on a fiber-optic biolayer interferometer (FO-BLI) system. The entire assay supports the simultaneous detection of eight pathogens in four samples in less than 15 min. (B) The illustration shows high-throughput immobilization of antibodies using recombinant cSP on a biosensing platform with silica surface for multiplexed virus detection. SD buffer: sample diluent buffer; HRP: horseradish peroxidase; AMEC: 3-amino-9-ethylcarbazole. The figure was created with BioRender.com.