Electrochemiluminescence sensor based on luminous hydrogel and GO-aptamer interface and detection method thereof

Abstract

The invention discloses an electrochemical luminescence (ECL) sensor based on a luminous hydrogel and graphene oxide (GO)-aptamer interface and an acetamiprid detection method thereof. The luminescent hydrogel is formed by loading manganese dioxide nanosheets (MnO2NSs), N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and gold nanoparticles (AuNPs) grown in situ in a three-dimensional network of polyethyleneimine-glutaraldehyde (PEI-GA) together, and is used as an ECL luminescent layer with high density and stable microenvironment; a GO-aptamer composite layer is constructed on the surface of the GO-aptamer composite layer, and signal transduction of GO short-range quenching-target induced desorption-signal recovery (signal-on) is achieved. An ECL signal is collected in a buffer system containing a hydrogen peroxide co-reactant, and a quantitative relation is established by delta IECL or I / I0, so that sensitive detection of acetamiprid is realized; good linearity is realized in a range of 1.0 * 10 <-3 > to 1.0 * 10 < 3 > nM, and the detection limit is about 0.3 pM. The method is good in repeatability and stability, and can be combined with QuEChERS pretreatment to be used for sample detection of vegetables such as spinach, celery and leek.

Description

Technical Field

[0001] This invention belongs to the field of electrochemiluminescence detection technology, specifically relating to a luminescent hydrogel composite material, a method for constructing a GO-aptamer functionalized interface on the electrode surface, and an electrochemiluminescence sensor based on the interface and its detection method. Background Technology

[0002] Electrochemiluminescence (ECL) sensing has advantages such as high sensitivity, electrical controllability and low system background. However, traditional planar or nanofilm light-emitting layers generally suffer from problems such as limited emission site density, long mass transfer path, unstable microenvironment and sensitivity to quenching of complex matrices, which limit emission efficiency and quantitative stability.

[0003] While three-dimensional porous substrates combined with multi-component composites can improve ECL intensity to some extent, the dispersion and stability of luminescent components at the interface remain insufficient, and the transfer of electrons / energy / active intermediates is inadequate. The "quenching-target desorption-signal recovery" strategy based on graphene oxide (GO) and single-stranded DNA aptamers can achieve specific recognition, but it is susceptible to non-specific adsorption and interfacial contamination in complex samples, and its synergistic construction with the luminescent layer and its resistance to matrix fluctuations still need improvement.

[0004] Therefore, there is an urgent need for a sensing system that combines high ECL emission efficiency, good matrix tolerance, and stable recognition and transduction capabilities to achieve sensitive, rapid, and reliable detection of small molecule targets in complex vegetable matrices. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of insufficient luminescence efficiency, significant matrix quenching and poor signal transduction stability in existing ECL sensors, and to provide an ECL sensor and its detection method based on the interface of luminescent hydrogel and GO-aptamer.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a luminescent hydrogel comprising manganese dioxide nanosheets (MnO2NSs), a luminescent substrate ABEI, in-situ grown gold nanoparticles (AuNPs), and a three-dimensional gel network formed by crosslinking polyethyleneimine and glutaraldehyde (PEI-GA); wherein the MnO2NSs / ABEI / AuNPs are dispersed and fixed in the three-dimensional gel network as an electrochemiluminescence emission complex to form a stable ECL microenvironment.

[0007] Preferably, MnO2NSs promotes the reactive oxygen species (ROS)-related ECL pathway and shortens the reaction mass transfer path, ABEI provides high quantum efficiency luminescence, AuNPs enhance electron transfer and local surface effects, and the PEI-GA three-dimensional network provides a stable microenvironment and high specific surface area loading, so that the ECL emission efficiency is improved by about 20% compared with the nanofilm type luminescent layer under the same loading and the same potential window.

[0008] The present invention also provides an ECL sensor, comprising a conductive substrate, an electrode-modified luminescent hydrogel layer, and a GO-aptamer composite layer on its surface; wherein the aptamer is an oligonucleotide sequence capable of specifically recognizing a target small molecule pesticide, and GO and the aptamer form a reversible complex through π-π stacking and / or electrostatic interaction, and target binding induces the aptamer to desorb from the GO surface to recover the ECL signal.

[0009] The present invention also provides a detection method in which an ECL signal is recorded in a buffer system containing co-reactants, and a quantitative relationship is established with ΔI_ECL or I / I0 as the response quantity to achieve quantitative detection of target small molecules.

[0010] Compared with existing technologies, this invention has the following advantages: It introduces a three-dimensional PEI-GA hydrogel to co-load MnO2NSs / ABEI / AuNPs to construct stable and high-density luminescent sites and shorten the electron and ROS transport paths; the GO-aptamer composite layer achieves high-contrast "quenching-release-recovery" signal transduction and suppresses matrix effects; the method achieves high-contrast "quenching-release-recovery" signal transduction and suppresses matrix effects; the method achieves high-contrast "quenching-release-recovery" signal transduction and suppresses matrix effects. -3 -10 3 It exhibits good linearity and a detection limit of 0.3 pM in the nM range, with good repeatability and stability, making it suitable for rapid detection of acetamiprid in vegetable matrices. Attached Figure Description

[0011] Figure 1 This is a schematic diagram illustrating the composition and formation mechanism of luminescent hydrogels.

[0012] Figure 2 This is a schematic diagram of the ECL sensor configuration (conductive substrate / luminescent hydrogel / GO-aptamer composite layer).

[0013] Figure 3 This is a schematic diagram of the ECL signal transduction mechanism (signal recovery from GO quenching and target-induced desorption).

[0014] Figure 4 Electrochemical characterization diagrams of the sensor assembly process (CV / EIS / DPV / ECL comparison).

[0015] Figure 5 The results of parameter optimization (pH, H2O2 dosage, incubation time and aptamer concentration).

[0016] Figure 6 To calibrate the curve and regression line (the linear relationship between logarithmic concentration and ΔI_ECL).

[0017] Figure 7 The results represent the evaluation of the method's specificity, stability, and repeatability.

[0018] Figure 8 The statistical results are for the spiked recovery experiment. Detailed Implementation

[0019] The following embodiments are used to further illustrate the present invention, but do not limit the scope of protection of the present invention in any way. Equivalent substitutions and modifications made by those skilled in the art without departing from the spirit and substance of the present invention are all within the scope of protection of the present invention.

[0020] Example 1: Preparation of luminescent hydrogels (1) Preparation of MnO2NSs Using potassium permanganate as the oxidant and fatty acid salt surface modifiers (preferably sodium oleate, or sodium octanoate, etc.) as the modulating components, the mixture was reacted under pH 8.5–9.5 conditions and then reacted in a water bath at about 80°C to obtain a dispersion of manganese dioxide nanosheets. The obtained product was centrifuged and washed to remove residual ions and surface modifiers, then resuspended and stored in the dark for later use.

[0021] (2) ABEI adsorption and in-situ growth of AuNPs Add ABEI solution (e.g., millimolecular concentration) to the MnO2NSs dispersion and stir or allow it to stand for adsorption; then add chloroauric acid solution and react under weakly alkaline conditions to generate AuNPs in situ on the MnO2NSs surface. The system color gradually changes from brown to wine red, resulting in a MnO2NSs@ABEI@AuNPs composite dispersion; after the reaction, remove free ABEI and residual gold precursors by centrifugation and wash, and redisperse for later use.

[0022] (3) PEI-GA three-dimensional crosslinking The above composite dispersion was mixed with a certain mass fraction of PEI solution and adjusted to pH 7.2–8.0. Glutaraldehyde (GA) solution was slowly added for cross-linking, and the mixture was allowed to stand at room temperature for about 30–60 min to gel. After gelation, the mixture was gently washed with deionized water to remove residual GA and free small molecules, resulting in a luminescent hydrogel.

[0023] Preferred parameters: The molar ratio of MnO2NSs, ABEI to gold precursor is (0.5–3):(1–5):(0.1–1); the mass fraction of PEI is 0.1%–2.0%; and the volume fraction of GA is 1%–5%.

[0024] Example 2: Construction of GO-aptamer composite layer Aptamer sequence: 5′-CTGACACCATATTATGAAGA-3′.

[0025] Composite preparation: GO dispersion (e.g., 20 μg·mL⁻¹) –1 The GO-aptamer complex (GO-aptamer) is placed in a buffer solution and mixed with an aptamer solution (10–200 nM, preferably about 100 nM). The mixture is incubated at room temperature or 25°C for 10–60 min to allow the aptamer to adsorb onto the GO surface via π-π stacking and electrostatic interactions, forming a GO-aptamer complex. The supernatant is preferably removed by centrifugation and resuspended after washing with the same buffer to obtain a stable GO-aptamer dispersion. If necessary, Na+ can be used. + / Mg 2+ Adjusting ionic strength to stabilize the recombination.

[0026] Example 3: Electrode Modification and Sensor Assembly A conductive substrate (preferably a carbon-based screen-printed electrode, but glassy carbon or gold electrodes can also be used) is selected. A luminescent hydrogel is dropped onto its surface and immobilized into a film under room temperature and light-protected conditions (e.g., 8 μL drop volume, immobilization for 30–60 min). Free matter is removed by gentle washing with buffer solution. Subsequently, a GO-aptamer complex is added and incubated (e.g., 8 μL drop volume, incubation for about 30 min). After two gentle washes, non-covalent immobilization is achieved through the positive charge of the hydrogel surface, the negative charge of GO, and π-π interactions. A blocking agent (preferably 0.05% BSA, incubation for about 30 min) can be further added to block non-specific adsorption sites and then gently washed to obtain a layered ECL sensor consisting of "substrate / luminescent hydrogel / GO-aptamer ( / blocking layer)". During sensor assembly, interfacial charge transfer was progressively restricted and baseline ECL was quenched. After the target was added, the aptamer desorbed from the GO surface, the interfacial barrier was partially removed, and the electrochemical response and ECL signal were restored, verifying the target-triggered signal-on mechanism and successful interface construction.

[0027] Example 4: Detection Principle In a buffer system containing co-reactants such as hydrogen peroxide, the luminescent hydrogel provides a high-density luminescent site and a stable microenvironment. When GO approaches the luminescent interface, it generates short-range quenching and forms a mass transfer / charge shielding effect, which reduces the baseline ECL. When the target binds to the aptamer, the aptamer desorbs from the GO surface and moves the GO away or weakens its short-range quenching effect. The ECL signal recovers and is enhanced as the target concentration increases, thereby achieving signal-on quantitative detection.

[0028] Example 5: Optimization of Detection Method Without altering the configuration, a single-factor method was used to systematically optimize the working pH, H2O2 addition volume, target incubation time, and aptamer apparent coverage. Results showed that the signal increased with pH from 7.5 to 8.5, plateauing at 9.5; considering both background and stability, 0.1 M PBS at pH 8.5 was selected. The signal significantly increased with H2O2 volume from 5 μL to 40 μL; 40–80 μL entered a plateau region with a risk of background increase, so 40 μL was chosen. Incubation time exhibited saturation kinetics, rapidly increasing from 20–40 min, with little change from 40–60 min; 40 min was selected. Aptamer loading significantly increased the signal from 10 to 100 nM, slightly decreasing at 200 nM; considering both surface coverage and electrostatic / steric hindrance effects, 100 nM was selected, and composite layers were prepared using a GO:aptamer mass ratio window of 1:20–1:200. These selections collectively resulted in a high signal-to-noise ratio and low RSD.

[0029] Example 6: Methodological linearity and detection limit Under optimized conditions, the concentration of the target compound acetamiprid is 10. –3 -10 3 The regression equation shows good linearity with ΔI_ECL within the nM range, and the detection limit calculated based on the 3σ / slope method (n≥11 blank) is approximately 0.3 pM; the corresponding regression equation is: I_ECL = 10508.64858 + 1018.24993·LgC_ACE, R 2 = 0.99899.

[0030] Example 7: Specificity, stability, and repeatability of the detection method The responses of non-target small molecules at the same concentration were weak or close to the background, while acetamiprid triggered a significant signal-on recovery, indicating good molecular specificity. When the sealed electrode was stored at 4°C and tested at fixed intervals, the ECL intensity remained basically stable over 20 days, with a signal retention rate of ≥95%. The inter-batch RSD of 10 independently prepared electrodes was <3%, and the RSD of 10 consecutive tests of the same sample was <3%, showing excellent reproducibility and operational stability.

[0031] Example 8: Spiked Recovery Experiment Using spinach, celery and leek homogenates as the matrix, the QuEChERS process (total time approximately 30–40 min) was employed for acetonitrile extraction-MgSO4 / NaCl salting-PSA / C18 dispersion solid-phase purification. Detection was performed at different spiking levels and the recovery rate was calculated using the calibration equation. The results showed a recovery rate of 80%–115% and RSD ≤ 8%.

Claims

1. A luminescent hydrogel, characterized in that, The hydrogel comprises manganese dioxide nanosheets, a luminescent substrate ABEI, in-situ generated gold nanoparticles, and a three-dimensional gel network formed by crosslinking polyethyleneimine and glutaraldehyde. The manganese dioxide nanosheets, ABEI, and gold nanoparticles are dispersed and fixed in the three-dimensional gel network as an electrochemiluminescence emission complex to promote the generation of reactive oxygen species and enhance the electrochemiluminescence efficiency.

2. The luminescent hydrogel as described in claim 1, characterized in that, The molar ratio of manganese dioxide nanosheets, ABEI and chloroauric acid is (0.5–3):(1–5):(0.1–1), the mass fraction of polyethyleneimine is 0.1%–2.0%, and the volume fraction of glutaraldehyde is 1%–5%. The mixture is crosslinked for 30–60 min at pH 7.2–8.0 to form a gel.

3. An electrochemiluminescence sensor, characterized in that, The device comprises a conductive substrate and a luminescent hydrogel layer as described in claim 1 or 2, fixed on its surface, wherein a graphene oxide-aptamer composite layer is constructed on the hydrogel layer; the aptamer is a single-stranded oligonucleotide capable of specifically binding to a target small molecule pesticide, and the graphene oxide and the aptamer form a reversible composite through π-π stacking and / or electrostatic interaction to generate a near-interface quenching effect, and the target binding induces the aptamer to desorb from the graphene oxide surface and causes the electrochemiluminescence signal to recover.

4. The electrochemiluminescence sensor as described in claim 3, characterized in that, The target small molecule pesticide is acetamiprid, and the aptamer nucleic acid sequence is 5′-CTGACACCATATTATGAAGA-3′.

5. The electrochemiluminescence sensor as described in claim 3 or 4, characterized in that, The mass ratio of graphene oxide to aptamer is 1:20–1:200, and the apparent coverage of the aptamer corresponds to a solution concentration of 10–200 nM, preferably about 100 nM.

6. The electrochemiluminescence sensor according to any one of claims 3-5, characterized in that, The conductive substrate is one of a screen-printed electrode, a glassy carbon electrode, or a gold electrode.

7. The electrochemiluminescence sensor according to any one of claims 3-6, characterized in that, A sealing layer is further provided outside the graphene oxide-aptamer composite layer. The sealing layer is a protein sealing agent and / or a polymer sealing agent, preferably a 0.01%–0.2% (w / v) bovine serum albumin solution.

8. A method for detecting target small molecule pesticides based on an electrochemiluminescence sensor as described in any one of claims 3–7, characterized in that, include: The sensor is brought into contact with and incubated with the sample to be tested; The sensor was placed in a buffer system containing co-reactants, and electrochemiluminescence signals were collected under set electrochemical conditions. A quantitative relationship between the response quantity ΔI_ECL or I / I0 and the target concentration is established.

9. The detection method as described in claim 8, characterized in that, The buffer solution has a pH of 7.5–9.5, preferably 8.5, and the co-reactant is hydrogen peroxide. The volume of hydrogen peroxide added is 5–80 μL, preferably about 40 μL, and the incubation time is 20–60 min, preferably about 40 min.

10. The detection method as described in claim 8 or 9, characterized in that, The pretreatment of vegetable samples was carried out using the QuEChERS process, which included acetonitrile extraction, MgSO4 / NaCl salting out, and PSA / C18 dispersion solid phase purification. The overall processing time was about 30–40 min, and the spiked recoveries were 80%–115% with an RSD of no more than 8%.

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