A novel tri-mode detection platform for ampicillin and drug resistance genes by CRISPR-driven luminescent nanozymes

Abstract

The antibiotic residues pose significant risks for bacterial resistance. To address the practical requirements for rapid, accurate, and on-site detection of antibiotic residues and monitoring the abundance of associated resistance genes, we report a smartphone-integrated multi-mode platform. The platform is aimed to simultaneous, accurate, and visual quantitative detection of ampicillin (AMP) and β-lactam antibiotic resistance genes (blaTEM). Specifically, we developed a magnetically controlled fluorescence, colorimetric, and photothermal biosensor based on a magnetic separation unit (aminated modified complementary DNA chain (NH₂-cDNA) loading on the surface of Ferrosoferric Oxide@polydopamine (Fe₃O₄@PDA, FP), FP@cDNA) and a signal unit (the aptamer nucleic acid chain modified by phosphate group linked to Prussian blue@UiO-66@manganese dioxide (PB@UiO-66@MnO₂, PUM) through Zr-O-P bond, PUM@Apt), for the integrated detection of AMP and blaTEM. By utilizing complementary base pairing between FP@cDNA and PUM@Apt, along with precise aptamer recognition the AMP, we achieved the fluorescence quantitative detection of AMP by measuring the signal unit in the supernatant. Subsequently, the difference of signal units in colorimetric process leads to a varying conversion rate of oxidized 3,3',5,5'-Tetramethylbenzidine (oxTMB), enabling the output of colorimetric and photothermal signals. The competitive binding of aptamers permitting the determination of AMP in the range of 0-160 pM with a low detection limit (0.34 pM). Additionally, in the presence of blaTEM, the activated CRISPR/Cas12a indiscriminately cleaves the single-stranded portion of the FP@DNA@PUM complex obtained by magnetic separation. A PUM-based three-signal detection scheme was established for the sensitive determination of blaTEM with the limit of detection (LOD) of 1.03 pM. The integration of smartphone-assisted analysis broadens the potential of the platform for visual detection. Notably, the innovative platform, with its excellent stability, exhibits great potential as a simple yet robust approach for the simultaneously visually monitoring antibiotics and drug resistance genes, and holds promise in the field of kit development.

Keywords: BlaTEM; Ampicillin; Aptamer; CRISPR/Cas12a; Tri-mode detection; Visual detection.

Scheme 1

(A) Schematic representation of the preparation of FP@cDNA. (B) Schematic representation of the preparateion of PUM@Apt. (C) Schematic of tri-mode detection of AMP and blaTEM

Fig. 1

(A) TEM images of Fe₃O₄. (B) TEM images of FP. (C) Zeta potential of Fe₃O₄ and FP. (D) VSM of Fe₃O₄ and FP. (E) TEM image of PB. (F) TEM image of PU. (G) TEM image of PUM. (H) Fe, Zr and Mn element mapping for PUM. (I) Fe 2p XPS spectrum of PUM. (J) Zr 3d XPS spectrum of PUM. (K) Mn 2p XPS spectrum of PUM. (L) XRD spectrum of PUM

Fig. 2

(A) 12% native PAGE analysis. Lane M, marker; lane 1, cDNA; lane 2, aptamer; lane 3, cDNA + aptamer; lane 4, cDNA + aptamer + AMP. (B) Fluorescence spectra of Apt-FAM, Apt-FAM + FP and Apt-FAM after incubated with the FP-cDNA. (C) The change of UV–vis absorbance of P-Apt at 260 nm in the supernatant before and after the interaction of P-Apt with PUM. (D) Zeta potential values of FP, FP@cDNA, PUM, PUM@Apt. (E) PUM fluorescence emission spectra at different excitation wavelengths (280–360 nm). (F) UV–vis spectra of different reaction systems, PUM + H₂O₂ + TMB, PUM + TMB, PUM + H₂O₂, H₂O₂ + TMB, TMB and H₂O₂. (G) EPR spectra of PUM + H₂O₂ with DMPO/TEMP as spin trapping agent. (H) Photothermal images of the solutions under the irradiation of 808 nm of NIR light with different concentration of oxTMB (0, 10, 20, 40 µM). (I) Macroscopic characterization of magnetic properties of FP@DDA@PUM. (J) The feasibility of AMP fluorescence detection. (K) The feasibility of AMP colorimetric detection. (L) The feasibility of AMP photothermal detection. (M) 12% native PAGE analysis of the ssDNA trans-cleavage ability of Cas12a. (Cas12a:10 nM, crRNA: 10 nM, blaTEM gene 1 nM, ssDNA: 1 µM, cleavage time: 30 min). (N) The feasibility of blaTEM fluorescence detection. (O) The feasibility of blaTEM colorimetric detection. (P) The feasibility of blaTEM photothermal detection

Fig. 3

(A–C) Fluorescence spectra, linear relationship, and color heatmap visualization standardized fluorescence brightness under the ultraviolet lamp with increasing AMP. (D) Selectivity of AMP fluorescence detection. (E–G) UV–vis spectra, linear relationship, and corresponding color heatmap of colorimetric with increasing AMP. (H) Selectivity of AMP colorimetric detection. (I–K) Photothermal curves, linear relationship, and corresponding color heatmap of photothermal with increasing AMP. (L) Selectivity of AMP Photothermal detection

Fig. 4

(A–C) Fluorescence spectra, linear relationship, and color heatmap visualization standardized fluorescence brightness under the ultraviolet lamp with increasing blaTEM. (D) Selectivity of blaTEM fluorescence detection. (E–G) UV–vis spectra, linear relationship, and corresponding color heatmap of colorimetric with increasing blaTEM. (H) Selectivity of blaTEM colorimetric detection. (I–K) Photothermal curves, linear relationship, and corresponding color heatmap of photothermal with increasing blaTEM. (L) Selectivity of blaTEM photothermal detection

Fig. 5

(A) Schematic illustration of the programmable AMP and blaTEM sensing platform for tri-mode visual quantitative detection. Relationship between G/B, G/R, ΔT and AMP (B–D) or blaTEM (E–G). The inset shows the corresponding optical and thermal images