Detection of Hg2+ Using a Dual-Mode Biosensing Probe Constructed Using Ratiometric Fluorescent Copper Nanoclusters@Zirconia Metal-Organic Framework/ N-Methyl Mesoporphyrin IX and Colorimetry G-Quadruplex/Hemin Peroxidase-Mimicking G-Quadruplex DNAzyme

Abstract

Mercury (Hg²⁺) has been recognized as a global pollutant with a toxic, mobile, and persistent nature. It adversely affects the ecosystem and human health. Already developed biosensors for Hg²⁺ detection majorly suffer from poor sensitivity and specificity. Herein, a colorimetric/fluorimetric dual-mode sensing approach is designed for the quantitative detection of Hg²⁺. This novel sensing approach utilizes nanofluorophores, i.e., fluorescent copper nanoclusters-doped zirconia metal-organic framework (CuNCs@Zr-MOF) nanoconjugate (blue color) and N-methyl mesoporphyrin IX (NMM) (red color) in combination with peroxidase-mimicking G-quadruplex DNAzyme (PMDNAzyme). In the presence of Hg²⁺, dabcyl conjugated complementary DNA with T-T mismatches form the stable duplex with the CuNCs@Zr-MOF@G-quadruplex structure through T-Hg²⁺-T base pairing. It causes the quenching of fluorescence of CuNCs@Zr-MOF (463 nm) due to the Förster resonance energy transfer (FRET) system. Moreover, the G-quadruplex (G4) structure of the aptamer enhances the fluorescence emission of NMM (610 nm). Besides this, the peroxidase-like activity of G4/hemin DNAzyme offers the colorimetric detection of Hg²⁺. The formation of duplex with PMDNAzyme increases the catalytic activity. This novel biosensing probe quantitatively detected Hg²⁺ using both fluorimetry and colorimetry approaches with a low detection limit of 0.59 and 36.3 nM, respectively. It was also observed that the presence of interfering metal ions in case of real aqueous samples does not affect the performance of this novel biosensing probe. These findings confirm the considerable potential of the proposed biosensing probe to screen the concentration of Hg²⁺ in aquatic products.

Fig. 1.

(A) Fabrication of the PMDNAzyme sensor. (B) Dual-detection mechanism in the presence of Hg²⁺ ion.

Fig. 2.

Structural and morphological characterization of CuNCs, Zr-MOF, and CuNCs@Zr-MOF nanoconjugate. (A) Schematic representation of fabrication of the highly fluorescent CuNCs@Zr-MOF nanoprobe. HRTEM images of (B) CuNCs and (C) SAED pattern of CuNCs. HRTEM images of (D) Zr-MOF and (E) CuNCs@Zr-MOF. SEM images of (F) Zr-MOF, (G) EDS line spectrum, and (H) elemental mapping of a Zr-MOF. SEM image of (I) CuNCs@Zr-MOF nanoconjugate, (J) EDS line spectrum, and (K) elemental mapping of CuNCs@Zr-MOF. DLS distribution of (L) CuNCs, (M) Zr-MOF, and (N) CuNCs@Zr-MOF. (O) Zeta potentials of all synthesized nanostructures. (P and Q) XRD and FTIR patterns, respectively, of CuNCs, Zr-MOF, and CuNCs@Zr-MOF. (R) XPS spectra of CuNCs@Zr-MOF.

Fig. 3.

Optical characteristics. (A) UV-Vis absorption spectra. (B) CuNCs photoluminescence spectra. (C) Zr-MOF photoluminescence spectra. (D) CuNCs@Zr-MOF photoluminescence spectra. (E) Excitation-dependent emission spectra of CuNCs@Zr-MOF. (F) Photoluminescence stability of CuNCs@Zr-MOF (error bars represent 95% confidence intervals).

Fig. 4.

Characterization of CuNCs@Zr-MOF/PMDNAzyme sensing nanoprobe. (A) Particle size analysis. (B) Zeta potential. (C) FTIR. (D) EDS spectrum.

Fig. 5.

Colorimetric responses. (A) Absorption spectra of TMB, TMB/H₂O₂, probe/TMB/H₂O₂, probe-cDNA/TMB/H₂O₂, and probe-cDNA/Hg/TMB/ H₂O₂. (B) Absorption at 652 nm for the different concentrations of Hg²⁺. (C) Linear fitting curve.

Fig. 6.

Fluorescence response of CuNCs@Zr-MOF/PMDNAzyme-cDNA sensor. (A) Illustration of the ratiometric fluorescence. (B) Effects of response time. (C) Fluorescence intensity at 463 and 610 nm for the different concentrations of Hg²⁺. (D) Linear calibration curve.

Fig. 7.

Specificity of CuNCs@Zr-MOF/PMDNAzyme-cDNA system toward Hg²⁺. (A) Fluorescent mode [inset: relative fluorescent intensity (I₆₁₀/I₄₆₃*100) against different metal ions]. (B) Colorimetric mode (inset: absorbance against different metal ions).