Triple signal amplification electrochemical sensing platform for Hg<sup>2+</sup> in water without direct modification of the working electrode

doi:10.1007/s11783-024-1850-1

2024, Vol. 18

Issue (7) : 90 https://doi.org/10.1007/s11783-024-1850-1

Triple signal amplification electrochemical sensing platform for Hg²⁺ in water without direct modification of the working electrode

Liuyin Hu¹, Jiahua Cui², Tao Lu^1,³, Yalin Wang¹, Jinping Jia^1,²(

)

¹. School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
². School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China
³. International Copper Association, Ltd., Shanghai 200020, China

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Abstract

● MrGO-AuNPs, Exo III-ATC and HCR co-amplify the signal.

● MrGO-AuNPs increases the cDNA loading to improve the Hg²⁺ capture efficiency.

● The utilization of magnetism improves the mass transfer efficiency.

● The working electrode doesn’t require direct modification, simplifying operation.

● Ultrasensitive to Hg²⁺ with a LOD of 3.14 pmol/L.

An ultrasensitive electrochemical biosensor to detect trace Hg²⁺ in environmental samples was developed utilizing nanogold-decorated magnetic reduced graphene oxide (MrGO-AuNPs), exonuclease III-assisted target cycle (Exo III-ATC) and hybridization chain reaction (HCR) synergistic triple signal amplification. The MrGO-AuNPs is a superior carrier for capture DNA (cDNA) and acts as magnetic media for automatic separation and adsorption. This innovative utilization of the magnetism and improved sensing efficiency obviates the need for direct modification and repeated polishing of the working electrode. Additionally, the three DNA hairpins (cDNA, methylene blue (MB) labeled HP1 and HP2) further contribute to biosensor specificity and selectivity. When cDNA captures Hg²⁺, it activates Exo III-ATC due to the formation of a sticky end in the cDNA stem via thymine-Hg²⁺-thymidine (T-Hg²⁺-T), this leads to the hydrolysis of self-folded cDNA by Exo III-ATC to form “key” DNA (kDNA). The kDNA subsequently initiates HCR, resulting in massive super-sandwich structures (kDNA-[HP1/HP2]_n) carrying signaling molecules on MrGO-AuNPs, and this overall structure serves as a signal probe (SP). Leveraging magnetic adsorption, the SP was automatically adsorbed onto the magneto-glass carbon electrode (MGCE), generating an amplified signal. This biosensor’s detection limit (LOD) was 3.14 pmol/L, far below the limit of 10 nmol/L for mercury in drinking water set by the US EPA. The biosensor also showed excellent selectivity when challenged by interfering ions, and the results of its application in actual samples indicate that it has good potential for practical applications in environmental monitoring.

Keywords Electrochemical sensing Hg²⁺ Amplification Graphene Hybridization chain reaction Exonuclease III

Corresponding Author(s): Jinping Jia

Issue Date: 13 May 2024

Cite this article:

Liuyin Hu,Jiahua Cui,Tao Lu, et al. Triple signal amplification electrochemical sensing platform for Hg²⁺ in water without direct modification of the working electrode[J]. Front. Environ. Sci. Eng., 2024, 18(7): 90.

URL:

https://academic.hep.com.cn/fese/EN/10.1007/s11783-024-1850-1
https://academic.hep.com.cn/fese/EN/Y2024/V18/I7/90

Scheme1 (1) The microscopic schematic diagram of the proposed sensing system, containing the process of preparation of CP and the process of formation of SP after a two-step signal amplification process of Hg²⁺-induced Exo III-ATC and HCR, (2) the macroscopic flowchart of transferring SP to magnetic glassy carbon electrode (MGCE) for magnetic adsorption and subsequent electrochemical testing.

Fig.1 Characterization of the materials. (A) and (B) TEM and EDS mapping of MrGO, (C) TEM of MrGO-AuNPs, (D) comparison of GO with MrGO-AuNPs colloid before and after magnetic separation.

Fig.2 Patterns of XRD characterization, with black, red and green lines denoting GO, MrGO and MrGO-AuNPs, respectively.

Fig.3 EIS (A) and CV (B) testing of step-modified MGCE, (a) bare MGCE, (b) MGCE with MrGO-AuNPs, (c) MGCE with MCH/cDNA-MrGO-AuNPs (CP), (d) MGCE with Hg²⁺ + CP, (e) MGCE with Exo III + Hg²⁺ + CP, (f) MGCE with HP1 + Exo III + Hg²⁺ + CP, (g) MGCE with HP2 + HP1 + Exo III + Hg²⁺ + CP.

Fig.4 Feasibility and signal amplification of this biosensor, (A) square wave voltammograms of different modified electrodes, (B) histogram of peak current per unit area for different modified electrodes. a. MCH/cDNA-MrGO-AuNPs (CP) + Exo III + HP1 + HP2 + MGCE, b. CP + Hg²⁺ + Exo III + HP1 + MGCE, c. CP + Hg²⁺ + Exo III + HP1 + HP2 + MGCE, d. MCH/cDNA + Hg²⁺ + Exo III + HP1 + HP2 + AuE.

Fig.5 Optimization of detection conditions, (A) Optimization of cDNA concentration, the MrGO-AuNPs was 1 mg/mL, (B) the capture time with Hg²⁺ (100 nmol/L), CP was 5 μL, (C) the Exo III catalytic hydrolysis time, Exo III concentration was 10 U/µL, (D) optimization of HP1/HP2 concentration (HP1:HP2 = 1:1), (E) optimization of HCR time, HP1/HP2 concentration was 3.5 μmol/L, (F) pH optimization for Hg²⁺ capture, Hg²⁺ was 100 nmol/L, CP was 5 μL.

Fig.6 The shelf life of this biosensor, for the experiments, CP was 5 μL, capture pH was 7.5, capture time was 20 min, Exo III-ATC time was 25 min (Exo III concentration was 10 U/μL), HP1/HP2 (1:1) concentration was 3.5 μmol/L, HCR time was 30 min.

Fig.7 (A) SWVs at different Hg²⁺ concentrations (0 to 5 × 10⁶ pmol/L), (B) the plot of logarithms of Hg²⁺ concentrations (0.01 to 5 × 10⁶ pmol/L) corresponding to peak current differences, (C) the linear curve between logarithms of Hg²⁺ concentrations (5 to 1 × 10⁵ pmol/L) and peak current differences, and all detections were performed under optimal testing conditions.

Fig.8 The selectivity of the assay, Hg²⁺ and interfering ions were 100 and 1000 nmol/L, respectively, the capture pH was 7.5, the CP was 5 μL, the concentration of HP1/HP2 (1:1) was 3.5 μmol/L, and the Exo III concentration was 10 U/μL, and the reaction times of each step were optimized times.

Tab.1 The spiked recovery experiments for actual water samples

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