ECL sensor based on ferrocene hollow nanospheres and method for detecting chlorpyrifos

Abstract

The present application relates to the technical field of pesticide residue analysis determination, in particular to a ferrocene hollow nanosphere-based ECL sensor and a chlorpyrifos detection method, comprising an ECL sensor preparation step and an operation method for determining chlorpyrifos by using the sensor; the sensor outputs an ECL signal, can detect the content of chlorpyrifos, and the results are mutually verified, and the accuracy is high; the determination method is simple, rapid, good in selectivity and high in sensitivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pesticide residue analysis determination, in particular to a ferrocene hollow nanosphere-based ECL sensor and chlorpyrifos detection method, including ECL sensor preparation steps and operation method for determining chlorpyrifos using the sensor; ECL signal output can detect the content of chlorpyrifos, and the results are mutually confirmed, with high accuracy; the determination method is simple, rapid, selective, and highly sensitive. BACKGROUND

[0002] Electrochemiluminescence sensor is a new type of sensor technology, which can be used for detecting pesticide residues through its high sensitivity and fast response. The advantage of this technology is that it can quickly and accurately detect pesticide residues without complex sample preparation process, and has high practicability, and is expected to be widely used in today's society which pays more and more attention to food safety;

[0003] Sulfur quantum dots have good water dispersibility, low toxicity and excellent optical properties, and have become a research hotspot in the field of electrochemistry. Shen et al. synthesized a new type of sulfur quantum dots by using sublimed sulfur as raw material after 125 hr treatment, proposed the assembly-fission mechanism in the formation process of sulfur quantum dots, and first observed the significant electrochemiluminescence of sulfur quantum dots. However, the synthesis time of the prepared sulfur quantum dots is long and the quantum efficiency is low; therefore, scientists are committed to exploring more efficient methods.

[0004] By using different nanomaterials to design effective ECL sensing strategies, the sensitivity of the sensor can be significantly improved. Porous polymers are satisfactory carriers due to their lightweight, high porosity and high specific surface area. Electrostatic self-assembly is an effective strategy for constructing functional materials of porous polymers, which is usually composed of a template and two polymers with opposite charges. Silicon spheres are often used as self-assembly templates due to their stable performance and uniform particle size, and the polymers with opposite charges are sequentially adsorbed on the surface of the template by electrostatic interaction. For self-assembly technology, the function can be changed by simply changing the adsorbed material. SUMMARY

[0005] One of the purposes of the present application is to prepare blue luminescent sulfur quantum dots with short synthesis time and high quantum efficiency, and the luminescence color is regulated by changing the particle size of sulfur dots through H2O2 etching, and the amount and concentration of H2O2 are explored.

[0006] The second purpose of the present application is to use covalent crosslinking and electrostatic interaction to adsorb PEI-Fc-PAA on the surface of silicon spheres layer by layer, and remove the silicon sphere template to prepare ferrocene hollow nanospheres Fc-HPNs. Fc can compete with SO4· and SQDs· to make sulfur quantum dots emit light. - - ​The prepared Fc-HPNs realize quenching agent enrichment in the process of quenching the luminescence of sulfur quantum dots; wherein the Fc-HPNs are prepared in a water-based medium;

[0007] The third purpose of the present application is to modify the blue luminescent sulfur quantum dots on the electrode surface, load the complementary chain of chlorpyrifos on the Fc-HPNs in a high efficiency, play the roles of catalysis and signal amplification, construct a signal-off type ECL aptamer sensor, and realize sensitive detection of chlorpyrifos.

[0008] The technical scheme of the present application is as follows:

[0009] The complementary chain cDNA of chlorpyrifos aptamer is loaded on the hollow Fc-HPNs, the cDNA is hybridized with the aptamer on the electrode surface to form a double-stranded structure, an ECL aptamer sensor is constructed with sulfur quantum dots as a luminescent body and Fc as a quencher, and the signal is read out in the ECL signal mode of cyclic voltammetry. When chlorpyrifos exists, the aptamer is combined with chlorpyrifos, the double-stranded DNA is dispiraled, and the Fc-HPNs also separate from the electrode surface. Therefore, the ECL signal will rise. The specific scheme is as follows:

[0010] 1. An ECL sensor based on ferrocene hollow nanospheres and a chlorpyrifos detection method, comprising the following steps:

[0011] (1) Preparation of Fc-HPNs

[0012] 0.3~0.4 g of ferrocene formate is weighed into 20~30 mL of ultrapure water and continuously stirred for 10~15 min, then 2.5~3.5 mL of EDC solution with a concentration of 3.8~4.2 mmol·L -1 and 0.8~1.0 mmol·L -1 of NHS solution are added and stirred overnight; 55~60 µL of PEI is added to the mixed solution and stirred on a magnetic stirrer for 2 hr, and the PEI-Fc complex is successfully prepared by cross-linking the PEI and ferrocene formate through an amide bond;

[0013] 0.02~0.03 g of SiO2 is ultrasonically dispersed in 30~35 mL of ultrapure water, then 2.5~3 mL of PEI-Fc complex is added and continuously stirred for 30~50 min, the precipitate is collected by centrifugation at 8000 rpm, and the precipitate is washed with water and ethanol to obtain SiO2@PEI-Fc, which is then dispersed in 15~20 mL of ultrapure water; then 5.0~5.5 mL of 0.05~0.1 g·mL -1PAA was added into the above solution under continuous stirring for 30-50 min, and then SiO2@PEI-Fc-PAA core-shell polymer nanomaterials were obtained after centrifugation and multiple washing; the above PEI-Fc coating process was repeated twice, and then 1.25 mL of EDC solution with a concentration of 3.8-4.2 mmol·L -1 and 1.0-1.2 mmol·L -1 of NHS was added under stirring at 4 ℃ for 2 hr to enhance the stability of the composite framework; 2.5-3.0 mL of HF solution with a concentration of 0.05-0.12 mol·L -1 was added into the SiO2@PEI-Fc-PAA core-shell polymer mixed solution, and the product was collected by centrifugation at 8000 rpm under continuous stirring at room temperature for 15-30 min and multiple washing to remove the remaining HF; finally, the product was dispersed in 5.0-10.0 mL of ultrapure water to obtain Fc-HPNs;

[0014] (2) Preparation of blue-emitting sulfur quantum dots SQDs

[0015] 1.2-1.5 g of sulfur sublimation, 3.8-4.2 g of NaOH, 2.5-3.5 mL of polyhexanediol with a molecular weight of 600, and 50-60 mL of ultrapure water were weighed into a 100 mL round-bottom flask and mixed, and the mixture was continuously stirred at 70-75 °C for 48-50 hr; as time went on, the reactants changed from the original light yellow to red and finally to orange solution, and emitted weak green fluorescence under ultraviolet light, obtaining a sulfur dot solution;

[0016] Under continuous stirring, 40-50 mL of H2O2 with a volume fraction of 1.5-2.0% was added to 30-35 mL of the sulfur dot solution, and the solution changed from light green to bright blue under ultraviolet light, and the preparation of sulfur quantum dots was completed; the above solution was placed in a dialysis bag with a molecular weight cut-off of 1000 Da for dialysis for 24-30 hr to remove unreacted small molecules, and then freeze-dried to obtain a yellow powder; 2-2.5 mg of sulfur quantum dot powder was dissolved in 0.8-1.5 mL of ultrapure water to obtain a sulfur quantum dot solution;

[0017] (3) Preparation of ECL sensor

[0018] 1) 20-30 µL of chlorpyrifos cDNA with a concentration of 1.0-1.2 µmol·L -1 and 180-200 µL of Fc-HPNs and 30-40 µL of glutaraldehyde solution with a mass fraction of 2.5-3.0 wt% were mixed in a centrifuge tube, continuously oscillated on a shaker for 1.5-2.0 hr to obtain Fc-HPNs-cDNA, and stored in a 4 ℃ refrigerator for standby use;

[0019] 2) The treated glassy carbon electrode GCE is placed into a 1% by mass chloroauric acid solution, gold is electrodeposited at a potential of -0.2 V for 1 min using an i-t curve method, and an AuNPs / GCE modified electrode is obtained;

[0020] 3) 8.0~10.0 μL of a sulfur quantum dot solution is taken and drop-coated on the electrode, and after being dried at room temperature, an SQDs / AuNPs / GCE is obtained;

[0021] 4) The same method is used to plate gold again for 1 min, and then the electrode is washed with a PBS buffer solution with a pH of 7.4; 8.0~10 μL of a cDNA solution with a concentration of 1.0 μmol·L -1 is added dropwise on the electrode, and after being incubated at 4 °C for 12~20 hr, an Apt / AuNPs / SQDs / AuNPs / GCE is obtained;

[0022] 5) 4~6 μL of MCH with a concentration of 8~12 μmol·L -1 is added dropwise on the electrode, and after being incubated at 37 °C for 1 hr, the electrode is washed with a PBS buffer solution with a pH of 7.4, so as to block non-specific sites of the electrode;

[0023] 6) 8.0~10.0 μL of Fc-HPNs-cDNA is drop-coated on the surface of the modified electrode, and after being incubated at 35~37 °C for 1 hr, the electrode is washed with a PBS buffer solution with a pH of 7.4, so as to obtain a Fc-HPNs-cDNA / MCH / Apt / AuNPs / SQDs / AuNPs / GCE.

[0024] 2. The method for detecting chlorpyrifos by using an ECL aptamer sensor is as follows:

[0025] (1) Different concentrations of chlorpyrifos are added dropwise on the surface of the constructed sensor, and the sensor is incubated at 35~37 °C for 1 hr, and then the sensor is washed with a PBS buffer solution with a pH of 7.4~7.8;

[0026] (2) The aptamer sensor is used as a working electrode, a saturated KCl-filled Ag / AgCl electrode is used as a reference electrode, and a platinum electrode is used as an auxiliary electrode, cyclic voltammetry scanning is performed in a potential range of -2.0-0.0 V, a photomultiplier voltage is set to 700 V, and the obtained light intensity results are recorded.

[0027] The present application has the following beneficial effects:

[0028] 1. The mode of the present application is simple, quick, and fast. The same instrument and the same determination solution system are used for the sensor, and there is no need to change the instrument and the solution system;

[0029] 2.The sensing signal is sensitive and the transmission speed is fast; Fc-HPNs are prepared in a water-based medium for the first time, Fc-HPNs are used as a quencher, the silicon ball template can be removed by using hydrofluoric acid to improve the conductivity, and the low quenching efficiency of Fc is improved, and the detection sensitivity is improved; the hollow Fc-HPNs have the advantages of large specific surface area, low density, high loading capacity and short mass-to-charge transfer distance, which can improve the conductivity of the sensor and amplify the signal;

[0030] 3.The size and color of the SQDs are successfully controlled by the H2O2 post-etching method, and the luminescence performance of the SQDs is further improved, and the SQDs are successfully used for the ECL aptamer sensor to detect pesticide residues;

[0031] 4.It is proved that the signal-based ECL aptamer sensor based on Fc-HPNs has good analytical performance, good reproducibility, stability and selectivity, and shows good application prospect in actual pesticide residue detection, and provides a new method for pesticide residue detection;

[0032] 5.The sample determination procedure is simple, and the operation is simple and fast. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The ECL time-intensity graph (A) and the linear relationship graph (B) of different concentrations of chlorpyrifos pesticide are shown

[0034] Wherein, a--0, b--10 -14 , c--10 -13 , d--10 -12 , e--10 -11 , f--10 -10 , g--10 -9 , h--10 -8 , i--10 -7 mol·L -1 . DETAILED DESCRIPTION

[0035] In order to better understand the present application, the technical solutions of the present application will be described in detail below with specific examples, but the present application is not limited thereto.

[0036] Example 1 Preparation of an ECL sensor based on Fc-HPNs quenched sulfur quantum dots:

[0037] (1) Preparation of Fc-HPNs

[0038] 0.36 g of ferrocene formic acid was weighed into 25 mL of ultrapure water and continuously stirred for 10 min, then 2.5 mL of EDC with a concentration of 3.8 mmol·L -1 and 1.0 mmol·L-1 NHS solution was stirred overnight; 60 µL PEI was added to the mixed solution and stirred on a magnetic stirrer for 2 hr, and PEI was successfully crosslinked with ferrocene formate by an amide bond to prepare a PEI-Fc complex;

[0039] 0.03 g SiO2 was ultrasonically dispersed in 30 mL ultrapure water, followed by the addition of 3 mL PEI-Fc complex under continuous stirring for 40 min, and the precipitate was collected by centrifugation at 8000 rpm and washed with water and ethanol to obtain SiO2@PEI-Fc, which was then dispersed in 20 mL ultrapure water; then, 5.2 mL PAA with a concentration of 0.05 g·mL -1 was added to the above solution under continuous stirring for 50 min, followed by centrifugation and multiple washing to obtain a SiO2@PEI-Fc-PAA core-shell polymer nanomaterial; the above PEI-Fc coating process was repeated twice, followed by the addition of 1.25 mL EDC with a concentration of 4.0 mmol·L -1 and NHS solution with a concentration of 1.0 mmol·L -1 was stirred at 4 °C for 2 hr to enhance the stability of the composite framework; 2.8 mL HF with a concentration of 0.1 mol·L -1 was added dropwise to the SiO2@PEI-Fc-PAA core-shell polymer mixed solution, and the product was collected by centrifugation at 8000 rpm and washed multiple times to remove the remaining HF, and finally the product was dispersed in 8 mL ultrapure water to obtain Fc-HPNs;

[0040] (2) Preparation of blue-emitting sulfur quantum dots SQDs

[0041] 1.5 g of sulfur sublimation, 4.0 g of NaOH, 2.5 mL of polyhexanediol with a molecular weight of 600, and 60 mL of ultrapure water were weighed into a 100 mL round-bottom flask and mixed, and the mixture was continuously stirred at 70 °C for 48 hr; as time went on, the reactants changed from the original light yellow to red and finally to an orange solution, and emitted a weak green fluorescence under a UV lamp, obtaining a sulfur dot solution;

[0042] Under continuous stirring, 40 mL of 1.5% H2O2 was added to 30 mL of the sulfur dot solution, and the solution changed from light green to bright blue under a UV lamp, and the preparation of the sulfur quantum dots was completed; the above solution was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed for 24 hr to remove unreacted small molecules, and then freeze-dried to obtain a yellow powder; 2 mg of the sulfur quantum dot powder was dissolved in 0.8 mL of ultrapure water to obtain a sulfur quantum dot solution;

[0043] (3) Preparation of an ECL sensor

[0044] 1) Take 20 μL of chlorpyrifos cDNA with a concentration of 1.0 μmol·L -1 and 180 μL of Fc-HPNs and 30 μL of glutaraldehyde solution with a mass fraction of 2.5 wt% into a centrifuge tube, mix and continuously shake on a shaker for 1.5 hr to obtain Fc-HPNs-cDNA, and store in a 4 ℃ refrigerator for standby;

[0045] 2) Place the treated glassy carbon electrode GCE into a chloroauric acid solution with a mass fraction of 1%, and use the i-t curve method to deposit gold at a potential of -0.2 V for 1 min to obtain an AuNPs / GCE modified electrode;

[0046] 3) Take 8.0 μL of sulfur quantum dot solution and drop coat it on the electrode, and after air drying at room temperature, SQDs / AuNPs / GCE is obtained;

[0047] 4) Use the same method to plate gold again for 1 min, and then wash the electrode with PBS buffer solution with a pH of 7.4; add 8.0 μL of chlorpyrifos Apt with a concentration of 1.0 μmol·L -1 on the electrode, and incubate at 4 °C for 12 hr to obtain Apt / AuNPs / SQDs / AuNPs / GCE;

[0048] 5) Add 4 μL of MCH with a concentration of 8 μmol·L -1 on the electrode, and incubate at 37 °C for 1 hr to block the non-specific sites of the electrode, and then wash with PBS buffer solution with a pH of 7.4;

[0049] 6) Drop coat 8.0 μL of Fc-HPNs-cDNA on the surface of the modified electrode, incubate at 35 °C for 1 hr, and then wash with PBS buffer solution with a pH of 7.4 to obtain Fc-HPNs-cDNA / MCH / Apt / AuNPs / SQDs / AuNPs / GCE.

[0050] Example 2 Preparation of an ECL sensor based on Fc-HPNs quenching sulfur quantum dots:

[0051] (1) Preparation of ferrocene hollow nanomicrospheres

[0052] Weigh 0.4 g of ferrocene formate into 30 mL of ultrapure water and continuously stir for 15 min, then add 3.5 mL of EDC with a concentration of 4.2 mmol·L -1 and 1.0 mmol·L -1NHS solution was stirred overnight; 60 μL PEI was added to the mixed solution and stirred on a magnetic stirrer for 2 hr, and PEI was successfully crosslinked with ferrocene formate by an amide bond to prepare a PEI-Fc complex;

[0053] 0.03 g SiO2 was ultrasonically dispersed in 35 mL ultrapure water, followed by the addition of 3 mL PEI-Fc complex under continuous stirring for 50 min, and the precipitate was collected by centrifugation at 8000 rpm and washed with water and ethanol to obtain SiO2@PEI-Fc, which was then dispersed in 20 mL ultrapure water; then, 5.5 mL PAA with a concentration of 0.1 g·mL -1 was added to the above solution under continuous stirring for 50 min, followed by centrifugation and multiple washing to obtain a SiO2@PEI-Fc-PAA core-shell polymer nanomaterial; the above PEI-Fc coating process was repeated twice, followed by the addition of 1.25 mL EDC with a concentration of 4.2 mmol·L -1 and NHS solution with a concentration of 1.2 mmol·L -1 at 4 °C for 2 hr to enhance the stability of the composite framework; 3.0 mL HF with a concentration of 0.12 mol·L -1 was added dropwise to the SiO2@PEI-Fc-PAA core-shell polymer mixed solution, and the mixture was continuously stirred at room temperature for 30 min, and the product was collected by centrifugation at 8000 rpm and washed multiple times to remove the remaining HF, and finally the product was dispersed in 10.0 mL ultrapure water to obtain Fc-HPNs;

[0054] (2) Preparation of blue-emitting sulfur quantum dots SQDs

[0055] 1.5 g of sulfur sublimation, 4.2 g of NaOH, 3.5 mL of polyhexanediol with a molecular weight of 600, and 60 mL of ultrapure water were weighed into a 100 mL round-bottom flask and mixed, and the mixture was continuously stirred at 75 °C for 50 hr; as time went on, the reactants changed from the original light yellow to red and finally to an orange solution, and emitted a weak green fluorescence under a UV lamp, obtaining a sulfur dot solution;

[0056] Under continuous stirring, 50 mL of 2.0% H2O2 was added to 35 mL of the sulfur dot solution, and the solution changed from light green to bright blue under a UV lamp, and the preparation of the sulfur quantum dots was completed; the above solution was placed in a dialysis bag with a molecular weight cut-off of 1000 Da and dialyzed for 30 hr to remove unreacted small molecules, and then freeze-dried to obtain a yellow powder; 2.5 mg of the sulfur quantum dot powder was dissolved in 1.5 mL of ultrapure water to obtain a sulfur quantum dot solution;

[0057] (3) Preparation of an ECL sensor

[0058] 1) Take 30 μL of 1.2 μmol·L -1 concentration of chlorpyrifos cDNA and 200 μL of Fc-HPNs and 40 μL of 3.0 wt% glutaraldehyde solution into a centrifuge tube, mix and continuously shake on a shaker for 2.0 hr to obtain Fc-HPNs-cDNA, and store in a 4 ℃ refrigerator for standby;

[0059] 2) Put the treated GCE into a 1% chloroauric acid solution, use the i-t curve method to deposit gold at a potential of -0.2 V for 1 min to obtain an AuNPs / GCE modified electrode;

[0060] 3) Take 10.0 μL of SQDs solution and drop coat on the electrode, and after air drying at room temperature, SQDs / AuNPs / GCE is obtained;

[0061] 4) Use the same method to plate gold again for 1 min, and then wash the electrode with a PBS buffer solution with a pH of 7.4; add 10 μL of 1.0 μmol·L -1 concentration of chlorpyrifos Apt on the electrode, and incubate at 4 ℃ for 20 hr to obtain Apt / AuNPs / SQDs / AuNPs / GCE;

[0062] 5) Add 6 μL of 12 μmol·L -1 concentration of MCH on the electrode, and incubate at 37 ℃ for 1 hr to block the non-specific sites of the electrode, and then wash with a PBS buffer solution with a pH of 7.4;

[0063] 6) Drop coat 10.0 μL of Fc-HPNs-cDNA on the modified electrode surface, incubate at 35-37 ℃ for 1 hr, and then wash with a PBS buffer solution with a pH of 7.4 to obtain Fc-HPNs-cDNA / MCH / Apt / AuNPs / SQDs / AuNPs / GCE.

[0064] Example 3: Detection of chlorpyrifos by ECL aptamer sensor:

[0065] (1) Add different concentrations of chlorpyrifos pesticide on the surface of the constructed sensor, incubate at 35 ℃ for 1 hr, and then wash with a PBS buffer solution with a pH of 7.4;

[0066] (2) Take the aptamer sensor as the working electrode, the saturated KCl-filled Ag / AgCl electrode as the reference electrode, and the platinum electrode as the auxiliary electrode, and perform cyclic voltammetry scanning in a potential range of -2.0-0.0 V, set the photomultiplier voltage to 700 V, and record the obtained light intensity results.

[0067] Example 4 Method for detecting chlorpyrifos by ECL aptamer sensor

[0068] (1) Different concentrations of chlorpyrifos pesticide were added to the surface of the constructed sensor, and incubated at 37 ℃ for 1 hr, and then washed with PBS buffer solution with pH 7.8;

[0069] (2) The aptamer sensor was used as the working electrode, the saturated KCl- filled Ag / AgCl electrode was used as the reference electrode, and the platinum electrode was used as the auxiliary electrode. The cyclic voltammetry scan was carried out in the potential range of -2.0-0.0 V, the photomultiplier voltage was set to 700 V, and the recorded light intensity results were recorded.

[0070] Example 5 Detection of actual samples

[0071] The feasibility of the sensor in the detection of actual samples was verified by testing the vegetable and fruit samples purchased from the nearby market. First, 100 g of celery and 100 g of tomatoes were accurately weighed and ground into juice in a juicer; the two samples were respectively dispersed in 100 mL of 0.1 mol·L -1 of PBS buffer solution with pH 7.4, and ultrasonic treatment was carried out for 5 min. The samples were centrifuged to obtain the supernatant; compared with the results of liquid chromatography-mass spectrometry, the constructed sensor was consistent with the results; the recovery rate of chlorpyrifos was between 92.1~105%, and the relative standard deviation was less than 5.1%.

Claims

1. An ECL sensor based on ferrocene hollow nanospheres, characterized in that... It is prepared by the following method: (1) Preparation of Fc-HPNs Weigh 0.3–0.4 g of ferrocene formate and add it to 20–30 mL of ultrapure water. Stir continuously for 10–15 min, then add 2.5–3.5 mL of a solution with a concentration of 3.8–4.2 mmol·L⁻¹. -1 1-Ethyl-(3-dimethylaminopropyl)carbodiimide EDC and 0.8–1.0 mmol·L -1 The N-hydroxythiosuccinimide NHS solution was stirred overnight; 55-60 μL of polyethyleneimine (PEI) was added to the mixed solution and stirred on a magnetic stirrer for 2 hours. The polyethyleneimine-ferrocene composite PEI-Fc was successfully prepared by crosslinking polyethyleneimine and ferrocene formate through amide bonds. 0.02–0.03 g SiO2 was ultrasonically dispersed in 30–35 mL of ultrapure water. Then, 2.5–3 mL of the PEI-Fc complex was added and the mixture was stirred continuously for 30–50 min. The precipitate was collected by centrifugation at 8000 rpm and thoroughly washed with water and ethanol to obtain SiO2@PEI-Fc. This SiO2 was then dispersed in 15–20 mL of ultrapure water. Finally, 5–5.5 mL of a 0.05–0.1 g·mL⁻¹ solution was added. -1 After adding polyacrylic acid (PAA) to the above solution and stirring continuously for 30–50 min, the mixture was centrifuged and washed multiple times to obtain SiO2@PEI-Fc-PAA core-shell polymer nanomaterials. The above process of coating PEI-Fc was repeated twice, and then 1.25 mL of EDC with a concentration of 3.8–4.2 mmol·L⁻¹ was added. -1 and concentrations of 1.0–1.2 mmol·L -1 The NHS solution was stirred at 4°C for 2 hours to enhance the stability of the composite skeleton; 2.5–3.0 mL of solution with a concentration of 0.05–0.12 mol·L⁻¹ -1 HF was added dropwise to the SiO2@PEI-Fc-PAA core-shell polymer mixed solution, and the mixture was stirred continuously at room temperature for 15-30 min. The product was collected by centrifugation at 8000 rpm and washed several times to remove the remaining HF. Finally, the product was dispersed in 5-10 mL of ultrapure water to obtain Fc-HPNs. (2) Preparation of blue luminescent sulfur quantum dots (SQDs) Weigh 1.2–1.5 g of sublimed sulfur, 3.8–4.2 g of NaOH, 2.5–3.5 mL of polyethylene glycol with a molecular weight of 600, and 50–60 mL of ultrapure water and place them in a 100 mL round-bottom flask. Mix the mixture and stir continuously at 70–75 °C for 48–50 hours. As time goes on, the reactants change from the original light yellow to red and finally to an orange solution, emitting a weak green fluorescence under ultraviolet light, thus obtaining the sulfur point solution. Under continuous stirring, 40–50 mL of 1.5–2.0% (v / v) H₂O₂ was added to 30–35 mL of sulfur quantum dot solution. Under ultraviolet light, the solution changed from light green to bright blue, indicating that the sulfur quantum dots were prepared. The solution was then dialyzed for 24–30 hours in a dialysis bag with a molecular weight cutoff of 1000 Da to remove unreacted small molecules, and then freeze-dried to obtain a yellow powder. 2–2.5 mg of the sulfur quantum dot powder was dissolved in 0.8–1.5 mL of ultrapure water to obtain a sulfur quantum dot solution. (3) Fabrication of ECL sensor 1) Take 20–30 μL of a solution with a concentration of 1.0–1.2 μmol·L⁻¹. -1 The complementary strand cDNA of the chlorpyrifos aptamer, 180-200 μL of Fc-HPNs and 30-40 μL of 2.5-3.0 wt% glutaraldehyde solution were placed in a centrifuge tube and mixed. The mixture was shaken continuously on a shaker for 1.5-2.0 hr to obtain Fc-HPNs-cDNA, which was then stored in a 4°C refrigerator for later use. 2) The treated glassy carbon electrode GCE was placed in a 1% (w / w) chloroauric acid solution, and gold was electrodeposited at a potential of -0.2V for 1 min using the it curve method to obtain an AuNPs / GCE modified electrode. 3) Transfer 8.0–10.0 μL of the sulfur quantum dot solution and drop it onto the electrode. After drying at room temperature, SQDs / AuNPs / GCE are obtained. 4) After re-plating gold for 1 min using the same method, wash the electrode with PBS buffer at pH 7.4; add 8.0–10 μL of 1.0 μmol·L⁻¹ solution to the electrode. -1 Chlorpyrifos aptamer Apt, incubated at 4°C for 12-20 hours, yields Apt / AuNPs / SQDs / AuNPs / GCE; 5) Add 4–6 μL of a solution with a concentration of 8–12 μmol·L⁻¹ to the electrode. -1 The 6-mercaptohexanol MCH was incubated at 37°C for 1 hour to block non-specific sites on the electrode, and then washed with PBS buffer at pH 7.

4. 6) Drop 8.0–10.0 μL of Fc-HPNs-cDNA onto the modified electrode surface, incubate at 35–37 °C for 1 hour, and wash with PBS buffer at pH 7.4 to obtain Fc-HPNs-cDNA / MCH / Apt / AuNPs / SQDs / AuNPs / GCE.

2. The detection method for chlorpyrifos using the sensor according to claim 1, characterized in that, The detection method is as follows: (1) Different concentrations of chlorpyrifos pesticide were dropped onto the surface of the constructed sensor, incubated at 35-37°C for 1 hour, and then rinsed with PBS buffer at pH 7.4-7.

8. (2) Using the sensor as the working electrode, an Ag / AgCl electrode filled with KCl as the reference electrode, and a platinum electrode as the auxiliary electrode, a cyclic voltammetric scan was performed in the potential range of -2.0 to 0.0V. The photomultiplier tube voltage was set to 700V, and the light intensity results were recorded.

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