ABSTRACT
The global spread of monkeypox has become a worldwide public healthcare issue. Therefore, there is an urgent need for accurate and sensitive detection methods to effectively control its spreading. Herein, we screened by phage display two peptides M4 (sequence: DPCGERICSIAL) and M6 (sequence: SCSSFLCSLKVG) with good affinity and specificity to monkeypox virus (MPXV) B21R protein. To simulate the state of the peptide in the phage and to avoid spatial obstacles of the peptide, GGGSK was added at the C terminus of M4 and named as M4a. Molecular docking shows that peptide M4a and peptide M6 are bound to different epitopes of B21R by hydrogen bonds and salt-bridge interactions, respectively. Then, peptide M4a was selected as the capture probe, phage M6 as the detection probe, and carbonized polymer dots (CPDs) as the fluorescent probe, and a colorimetric and fluorescent double-signal capture peptide/antigen/signal peptide-displayed phage sandwich ELISA triggered by horseradish peroxidase (HRP) through a simple internal filtration effect (IFE) was constructed. HRP catalyzes H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB, which can further quench the fluorescence of CPDs through IFE, enabling to detect MPXV B21R in colorimetric and fluorescent modes. The proposed simple immunoassay platform shows good sensitivity and reliability in MPXV B21R detection. The limit of detection for colorimetric and fluorescent modes was 27.8 and 9.14 pg/mL MPXV B21R, respectively. Thus, the established double-peptide sandwich-based dual-signal immunoassay provides guidance for the development of reliable and sensitive antigen detection capable of mutual confirmation, which also has great potential for exploring various analytical strategies for other respiratory virus surveillance.
Subject(s)
Enzyme-Linked Immunosorbent Assay , Peptides , Enzyme-Linked Immunosorbent Assay/methods , Peptides/chemistry , Antigens, Viral/immunology , Antigens, Viral/analysis , Antigens, Viral/chemistry , Molecular Docking Simulation , Horseradish Peroxidase/chemistry , Horseradish Peroxidase/metabolism , Limit of Detection , Fluorescent Dyes/chemistry , Peptide Library , Benzidines/chemistry , Colorimetry/methodsABSTRACT
In the current study, the effect of trivalent cerium (Ce3+ ) on the production of reactive oxygen species (ROS) was investigated in the root of Arabidopsis thaliana by an in vitro study. The roots of A. thaliana were exposed with 0, 1, and 5 µmol/L Ce3+ for 12 h in vitro. It was found that the level of H2 O2 , O2 .- , and ·OH was enhanced by 5 µmol/L Ce3+ , but reduced by 1 µmol/L Ce3+ . The activities of peroxidase (POD), catalase (CAT), and superoxidase dismutase (SOD) were enhanced by 1 µmol/L Ce3+ , but reduced by 5 µmol/L Ce3+ . Moreover, we used a laser-scanning confocal microscopy to detect the changes of ROS in the root cells of A. thaliana by using a fluorochrome 2',7'-dichlorofluorescein diacetate (H2 DCF-DA). It showed that the level of ROS was declined in the root cells treated by 1 µmol/L Ce3+ , but the oscillation of ROS was found in the root cells treated with 5 µmol/L Ce3+ . In addition, REEs affect the uptake of mineral elements, which may be related to the oxidative stress in the cells of roots. In all, the data of our study indicated that the appropriate concentration of Ce3+ exhibited an anti-oxidation property and improved the defense system in the root cells of A. thaliana.
