Function of Graphene Oxide as the "Nanoquencher" for Hg2+ Detection Using an Exonuclease I-Assisted Biosensor

Abstract

Graphene oxide is well known for its excellent fluorescence quenching ability. In this study, positively charged graphene oxide (pGO25000) was developed as a fluorescence quencher that is water-soluble and synthesized by grafting polyetherimide onto graphene oxide nanosheets by a carbodiimide reaction. Compared to graphene oxide, the fluorescence quenching ability of pGO25000 is significantly improved by the increase in the affinity between pGO25000 and the DNA strand, which is introduced by the additional electrostatic interaction. The FAM-labeled single-stranded DNA probe can be almost completely quenched at concentrations of pGO25000 as low as 0.1 μg/mL. A simple and novel FAM-labeled single-stranded DNA sensor was designed for Hg²⁺ detection to take advantage of exonuclease I-triggered single-stranded DNA hydrolysis, and pGO25000 acted as a fluorescence quencher. The FAM-labeled single-stranded DNA probe is present as a hairpin structure by the formation of T-Hg²⁺-T when Hg²⁺ is present, and no fluorescence is observed. It is digested by exonuclease I without Hg²⁺, and fluorescence is recovered. The fluorescence intensity of the proposed biosensor was positively correlated with the Hg²⁺ concentration in the range of 0-250 nM (R² = 0.9955), with a seasonable limit of detection (3σ) cal. 3.93 nM. It was successfully applied to real samples of pond water for Hg²⁺ detection, obtaining a recovery rate from 99.6% to 101.1%.

Keywords: T–Hg2+–T; exonuclease I; fluorescence quencher; hairpin structure; positively charged graphene oxide (pGO).

Scheme 1

Schematic diagram of the designed fluorescence “turn-off” strategy for Hg²⁺ detection with the assistance of Exo I nuclease using a FAM–ssDNA probe.

Figure 1

Charge analysis of GO and pGO25000.

Figure 2

Characterization of GO and pGO25000 by (A) Raman spectroscopy and (B) FT-IR.

Figure 3

High-resolution XPS spectra of GO and pGO25000: (A) wide scan, (B) C1s spectrum of pGO25000, (C) N1s spectrum of pGO25000, and (D) O1s spectrum of pGO25000.

Figure 4

Effect of pGO25000 concentrations from 0 to 0.1 μg/mL on fluorescence quenching of FAM–ssDNA.

Figure 5

(A) Fluorescence emission spectra upon addition of various Hg²⁺ concentrations ranging from 0 to 800 nM in Tris–HNO₃ buffer (10 mM, pH 8.5) containing 40 mM NaNO₃. The Hg²⁺ concentrations were as follows: 0, 2, 5, 10, 30, 50, 120, 150, 200, 250, 300, 400, 600, and 800 nM. (B) Linear response of the relative fluorescence intensity (F/F₀, F: fluorescence-detected, with various Hg²⁺ concentrations from 0 to 800 nM, F₀: fluorescence-initial, without Hg²⁺). An amount of 50 nM FAM–ssDNA was used for each reaction, and the reactions were performed in Tris–HNO₃ buffer (10 mM, 40 mM NaNO₃, pH 8.5). Ex = 495 nm, Em = 520 nm.

Figure 6

Selectivity of Hg²⁺ detection by the FAM–ssDNA probe. The fluorescence intensities of the Exo I (2 U)/pGO25000 (0.1 μg/mL)-assisted FAM–ssDNA probes were recorded without or with Hg²⁺ (600 nM) and mixed with other possible interfering metal ions (K⁺, Fe²⁺, Sn²⁺, Al³⁺, Ni²⁺, Mn²⁺, Mg²⁺, Cu²⁺, and Co²⁺, 6 mM). Ex = 495 nm, Em = 520 nm. Each sample was repeated three times.