Magnetic nanobead assisted the dual targets driven fluorescent biosensor based on SPEXPAR and MNAzyme for the olfactory marker protein detection

Abstract

Fig. 1: Schematic illustration of the principle of the Dual targets driven SPEXPAR assisted MNAzyme (D-S-M) fluorescent biosensor for olfactory marker protein (OMP) detection. A. Workflow for detection of OMP in nasal swab. B. Isothermal Self-Primer EXPonential Amplification Reaction (SPEXPAR) amplification. C. The production of fluorescent signal by Multicomponent Nuclear Acid Enzyme (MNAzyme). The signal of OMP was amplified and changed into the detectable fluorescence signal based on the reactions of SPEXPAR and MNAzyme in the D-S-M fluorescence biosensor. The qualitative or quantitative analysis of OMP can be measured by the analysis of the fluorescence intensity.Image 1.

Graphical abstract

Fig. 1

Schematic illustration of the principle of the D-S-M fluorescent biosensor for OMP detection. A. Workflow for detection of OMP in nasal swab; B. Isothermal Self-Primer EXPonential Amplification Reaction (SPEXPAR) amplification; C. The production of fluorescent signal by Multicomponent Nuclear Acid Enzyme (MNAzyme).

Fig. 2

Characterization analyses of D-MH@MBs. A. The particle size and TEM image of SA-MBs; B. The particle size and TEM image of D-MH@MBs; C. The AFM images of SA-MBs and D-MH@MBs; D. The zeta potential analyses of SA-MBs and D-MH@MBs; E. The UV–vis spectroscopy analysis of SA-MBs and D-MH@MBs; F. The FT-IR spectrometer analysis of SA-MBs and D-MH@MBs.

Fig. 3

Feasibility of the D-S-M fluorescent biosensor. A. Gel electrophoresis image of the amplification feasibility of OMP by SPEXPAR. Lane 1: OMP 1; Lane 2: OMP 2; Lane 3: D-MH; Lane 4: OMP 1, OMP 2 and D-MH were incubated with other necessary components of SPEXPAR at 37 °C 1 h; Lane 5: D-MH were incubated with other necessary components of SPEXPAR at 37 °C 1 h; Lane 6: OMP 1 and OMP 2 were incubated with other necessary components of SPEXPAR at 37 °C 1 h; B. Gel electrophoresis image of the amplification feasibility of SPEXPAR triggered by OMP 1 or OMP 2 alone. Lane 1: The SPEXPAR reaction triggered with OMP 1 and OMP 2; Lane 2: The SPEXPAR reaction triggered with OMP 1; Lane 3: The SPEXPAR reaction triggered with OMP 2; C. The self-assembling and catalytic cleavage feasibility of MNAzyme; D. The feasibility of MNAzyme triggered by SPEXPAR.

Fig. 4

Performance evaluation of the D-S-M fluorescent biosensor. A. Fluorescence spectra responding to 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM and 10 μM of OMP 1 and OMP 2; B. Relationship between the fluorescence intensity at 522 nm and the logarithm of the concentration of the target (lg C). Inset: the linear relationship between the fluorescence intensity at 522 nm and lg C in the range of 1 pM–100 nM. The equation is Y = 302.08X + 1467.6 (R² = 0.9975); C. Fluorescence spectra responding to 100 nM OMP, 10 μM mismatch sequence 1–3, and 5 μM OMP; D. The fluorescence intensity of OMP detected by the D-S-M fluorescent biosensor in 11 nasal swab samples and 12 serum samples; E. The concentrations of OMP in 11 nasal swab samples and 12 serum samples; F. The fluorescence intensity of OMP detected by the D-S-M fluorescent biosensor in the nasal swab samples of 12 OD patients and 13 healthy controls; G. The concentrations of OMP in the nasal swab samples of 12 OD patients and 13 healthy controls.