Ultrasensitive detection of 2,4-dichlorophenoxyacetic acid by inhibiting alkaline phosphatase immobilized onto a highly porous gold nanocoral electrode

Angelo Tricase; Michele Catacchio; Verdiana Marchianò; Eleonora Macchia; Paolo Bollella; Luisa Torsi

doi:10.1039/d4nr04857a

Ultrasensitive detection of 2,4-dichlorophenoxyacetic acid by inhibiting alkaline phosphatase immobilized onto a highly porous gold nanocoral electrode

Angelo Tricase
, Michele Catacchio
, Verdiana Marchianò
, Eleonora Macchia
, Paolo Bollella^*
, Luisa Torsi

^*Corresponding author for this work

Physics

Research output: Contribution to journal › Article › Scientific › peer-review

Abstract

Herein, we describe the design and implementation of an ultrasensitive enzyme inhibition-based biosensor for 2,4-dichlorophenoxyacetic acid (2,4-D) detection. The biosensor utilizes alkaline phosphatase (AlP), immobilized on a photo-crosslinked polymer matrix of poly(vinyl alcohol) functionalized with N-methyl-4(4′-formylstyryl)pyridinium (PVA-SbQ), supported by electrodes coated with highly porous gold nanocorals (hPGNCs). After preliminary electrochemical and morphological characterization, the PVA-SbQ/AlP/hPGNC electrode was tested for inhibition studies employing ascorbate 2-phosphate (A2P) as the initial substrate. The biosensor preparation/sensing time from electrode preparation to final results is approximately 45 minutes, which enables the possibility to easily scale up the electrode production process on a daily basis with a reliable analytical result in only 5 minutes of amperometric measurement. Following the initial kinetic studies and evaluation of analytical performance, the PVA-SbQ/AlP/hPGNC platform demonstrated a linear detection range from 0.002 to 22 ppt, with a sensitivity of 0.121 ± 0.006 ppt⁻¹ (RSD = 4.9%, R² = 0.996, and N = 6) and a limit of detection (LoD) of 0.7 ppq. This sensitivity is 7-8 orders of magnitude below the regulatory thresholds in Europe and the USA. Furthermore, the biosensor was validated using 19 homogenized wheat leaf sample extracts, prepared in line with European Food Safety Authority (EFSA) guidelines, achieving average recoveries exceeding 96% and RSD values under 9.8%. The biosensor also exhibited robust operational and storage stability, maintaining 84% (30 hours of continuous operation) and 94% (120 days) of its initial response, respectively. These results highlight the potential of the PVA-SbQ/AlP/hPGNC biosensor for on-site 2,4-D monitoring in agricultural crops and its feasibility for integration with artificial intelligence for advanced diagnostics.

Original language	English
Pages (from-to)	9197-9204
Number of pages	8
Journal	Nanoscale
Volume	17
Issue number	15
DOIs	https://doi.org/10.1039/d4nr04857a
Publication status	Published - 25 Mar 2025
MoE publication type	A1 Journal article-refereed

Funding

The following funding agencies are acknowledged: NoOne-A binary sensor with a single-molecule digit to discriminate biofluids enclosing zero or at least one biomarker, ERC Stg2021, GA:101040383; Centro di Innovazione in Single-Molecule Digital Assay – “Digital Assay” from Regione Puglia DGR N. 218 of 21/02/2022; the Italian network of excellence for advanced diagnosis (INNOVA), Ministero della Salute -code PNC-E3-2022-23683266 PNC-HLS-DA, CUP: C43C22001630001; Complementary National Plan PNC-I.1 “Research initiatives for innovative technologies and pathways in the health and welfare sector” D.D. 931 of 06/06/2022, DARE – DigitAl lifelong pRevEntion initiative, code PNC0000002. This work was partially supported by the European Union – Next Generation EU under the Italian National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, Investment 1.3, CUP D43C22003080001, partnership on “Telecommunications of the Future” (PE00000001 – program “RESTART”), and CSGI (Centre for Colloid and Surface Science). The following funding agencies are acknowledged: NoOne-A binary sensor with a single-molecule digit to discriminate biofluids enclosing zero or at least one biomarker, ERC Stg2021, GA:101040383; Centro di Innovazione in Single-Molecule Digital Assay - “Digital Assay” from Regione Puglia DGR N. 218 of 21/02/2022; the Italian network of excellence for advanced diagnosis (INNOVA), Ministero della Salute -code PNC-E3-2022-23683266 PNC-HLS-DA, CUP: C43C22001630001; Complementary National Plan PNC-I.1 “Research initiatives for innovative technologies and pathways in the health and welfare sector” D.D. 931 of 06/06/2022, DARE - DigitAl lifelong pRevEntion initiative, code PNC0000002. This work was partially supported by the European Union - Next Generation EU under the Italian National Recovery and Resilience Plan (NRRP), Mission 4, Component 2, Investment 1.3, CUP D43C22003080001, partnership on “Telecommunications of the Future” (PE00000001 - program “RESTART”), and CSGI (Centre for Colloid and Surface Science).

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https://urn.fi/URN:NBN:fi-fe2026021914516

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title = "Ultrasensitive detection of 2,4-dichlorophenoxyacetic acid by inhibiting alkaline phosphatase immobilized onto a highly porous gold nanocoral electrode",

abstract = "Herein, we describe the design and implementation of an ultrasensitive enzyme inhibition-based biosensor for 2,4-dichlorophenoxyacetic acid (2,4-D) detection. The biosensor utilizes alkaline phosphatase (AlP), immobilized on a photo-crosslinked polymer matrix of poly(vinyl alcohol) functionalized with N-methyl-4(4′-formylstyryl)pyridinium (PVA-SbQ), supported by electrodes coated with highly porous gold nanocorals (hPGNCs). After preliminary electrochemical and morphological characterization, the PVA-SbQ/AlP/hPGNC electrode was tested for inhibition studies employing ascorbate 2-phosphate (A2P) as the initial substrate. The biosensor preparation/sensing time from electrode preparation to final results is approximately 45 minutes, which enables the possibility to easily scale up the electrode production process on a daily basis with a reliable analytical result in only 5 minutes of amperometric measurement. Following the initial kinetic studies and evaluation of analytical performance, the PVA-SbQ/AlP/hPGNC platform demonstrated a linear detection range from 0.002 to 22 ppt, with a sensitivity of 0.121 ± 0.006 ppt−1 (RSD = 4.9\%, R2 = 0.996, and N = 6) and a limit of detection (LoD) of 0.7 ppq. This sensitivity is 7-8 orders of magnitude below the regulatory thresholds in Europe and the USA. Furthermore, the biosensor was validated using 19 homogenized wheat leaf sample extracts, prepared in line with European Food Safety Authority (EFSA) guidelines, achieving average recoveries exceeding 96\% and RSD values under 9.8\%. The biosensor also exhibited robust operational and storage stability, maintaining 84\% (30 hours of continuous operation) and 94\% (120 days) of its initial response, respectively. These results highlight the potential of the PVA-SbQ/AlP/hPGNC biosensor for on-site 2,4-D monitoring in agricultural crops and its feasibility for integration with artificial intelligence for advanced diagnostics.",

author = "Angelo Tricase and Michele Catacchio and Verdiana Marchian{\`o} and Eleonora Macchia and Paolo Bollella and Luisa Torsi",

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T1 - Ultrasensitive detection of 2,4-dichlorophenoxyacetic acid by inhibiting alkaline phosphatase immobilized onto a highly porous gold nanocoral electrode

AU - Tricase, Angelo

AU - Catacchio, Michele

AU - Marchianò, Verdiana

AU - Macchia, Eleonora

AU - Bollella, Paolo

AU - Torsi, Luisa

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N2 - Herein, we describe the design and implementation of an ultrasensitive enzyme inhibition-based biosensor for 2,4-dichlorophenoxyacetic acid (2,4-D) detection. The biosensor utilizes alkaline phosphatase (AlP), immobilized on a photo-crosslinked polymer matrix of poly(vinyl alcohol) functionalized with N-methyl-4(4′-formylstyryl)pyridinium (PVA-SbQ), supported by electrodes coated with highly porous gold nanocorals (hPGNCs). After preliminary electrochemical and morphological characterization, the PVA-SbQ/AlP/hPGNC electrode was tested for inhibition studies employing ascorbate 2-phosphate (A2P) as the initial substrate. The biosensor preparation/sensing time from electrode preparation to final results is approximately 45 minutes, which enables the possibility to easily scale up the electrode production process on a daily basis with a reliable analytical result in only 5 minutes of amperometric measurement. Following the initial kinetic studies and evaluation of analytical performance, the PVA-SbQ/AlP/hPGNC platform demonstrated a linear detection range from 0.002 to 22 ppt, with a sensitivity of 0.121 ± 0.006 ppt−1 (RSD = 4.9%, R2 = 0.996, and N = 6) and a limit of detection (LoD) of 0.7 ppq. This sensitivity is 7-8 orders of magnitude below the regulatory thresholds in Europe and the USA. Furthermore, the biosensor was validated using 19 homogenized wheat leaf sample extracts, prepared in line with European Food Safety Authority (EFSA) guidelines, achieving average recoveries exceeding 96% and RSD values under 9.8%. The biosensor also exhibited robust operational and storage stability, maintaining 84% (30 hours of continuous operation) and 94% (120 days) of its initial response, respectively. These results highlight the potential of the PVA-SbQ/AlP/hPGNC biosensor for on-site 2,4-D monitoring in agricultural crops and its feasibility for integration with artificial intelligence for advanced diagnostics.

AB - Herein, we describe the design and implementation of an ultrasensitive enzyme inhibition-based biosensor for 2,4-dichlorophenoxyacetic acid (2,4-D) detection. The biosensor utilizes alkaline phosphatase (AlP), immobilized on a photo-crosslinked polymer matrix of poly(vinyl alcohol) functionalized with N-methyl-4(4′-formylstyryl)pyridinium (PVA-SbQ), supported by electrodes coated with highly porous gold nanocorals (hPGNCs). After preliminary electrochemical and morphological characterization, the PVA-SbQ/AlP/hPGNC electrode was tested for inhibition studies employing ascorbate 2-phosphate (A2P) as the initial substrate. The biosensor preparation/sensing time from electrode preparation to final results is approximately 45 minutes, which enables the possibility to easily scale up the electrode production process on a daily basis with a reliable analytical result in only 5 minutes of amperometric measurement. Following the initial kinetic studies and evaluation of analytical performance, the PVA-SbQ/AlP/hPGNC platform demonstrated a linear detection range from 0.002 to 22 ppt, with a sensitivity of 0.121 ± 0.006 ppt−1 (RSD = 4.9%, R2 = 0.996, and N = 6) and a limit of detection (LoD) of 0.7 ppq. This sensitivity is 7-8 orders of magnitude below the regulatory thresholds in Europe and the USA. Furthermore, the biosensor was validated using 19 homogenized wheat leaf sample extracts, prepared in line with European Food Safety Authority (EFSA) guidelines, achieving average recoveries exceeding 96% and RSD values under 9.8%. The biosensor also exhibited robust operational and storage stability, maintaining 84% (30 hours of continuous operation) and 94% (120 days) of its initial response, respectively. These results highlight the potential of the PVA-SbQ/AlP/hPGNC biosensor for on-site 2,4-D monitoring in agricultural crops and its feasibility for integration with artificial intelligence for advanced diagnostics.

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DO - 10.1039/d4nr04857a

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AN - SCOPUS:105003026518

SN - 2040-3364

VL - 17

SP - 9197

EP - 9204

JO - Nanoscale

JF - Nanoscale

IS - 15

ER -

Ultrasensitive detection of 2,4-dichlorophenoxyacetic acid by inhibiting alkaline phosphatase immobilized onto a highly porous gold nanocoral electrode

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