The exploration of nanomaterials with mimic enzyme activity (named nanozyme) has gained extensive attention in the fields of advanced analytical chemistry and materials science. Herein, the gold nanoparticles doped covalent organic frameworks (COFs) were prepared, which exhibited not only excellent mimic nitroreductase activity but also robust stability. By replacing the traditional natural enzyme tag in an enzyme-linked immunosorbent assay (ELISA), we employed the proposed nanozyme to label the detecting antibody. According to the catalytic properties of the nanozyme, 4-nitrothiophenol (4-NTP) was introduced as the substrate, which can be transformed to 4-aminothiophenol (4-ATP) in the presence of NaBH 4 . In a surface enhanced Raman scattering (SERS) assay, 4-ATP was capable of functioning as a powerful bridge to connect the gold nanostars (with excellent SERS performance) by both the Au−S bond and electrostatic force to further produce a Raman "hot spot". Meanwhile, the Raman signal of 4-nitrothiophenol at 1573 cm −1 was weakened, and a new signal at 1591 cm −1 generated by 4-ATP was turned on, leading to the generation of a ratiometric SERS signal. Based on this performance, a ratiometric nanozymelinked immunosorbent assay (NELISA) strategy was developed delicately, which was applied to detect β-lactoglobulin (allergenic protein) by monitoring the ratiometric signal of I 1591 /I 1573 with a limit of detection (LOD) of 0.01 ng/mL. The linear range is 25.65−6.2 × 10 4 ng/mL, covering more than 3 orders of magnitude. The developed method showed many advantages such as low-cost, higher recovery, and lower cross-reactivity, providing new insight into the application of SERS technology for trace target analysis.

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An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN) 6 4− ), which could exhibit a unique characteristic Raman peak that was located in the "biological Raman-silent region". By mixing the alkali-treated solution with the SERS substrate, Au@Ag core−shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.