Electrochemiluminescence aptamer sensor based on nh2-luminol / Ag@siO2ns and preparation method and application thereof

Abstract

A novel electrochemiluminescence (ECL) aptamer biosensor based on functional nanomaterials was developed for the detection of malathion in tea. Firstly, multi-walled carbon nanotubes (MWCNTs) were used to immobilize gold nanoparticles (AuNPs) on the surface of the electrode by potentiostatic electrodeposition. Secondly, amino-luminol / silver nanoparticles@silica nanospheres (NH2-Luminol / Ag@SiO2NSs) were synthesized by one-pot method. Finally, the signal probes (SPs) were prepared by combining NH2-Luminol / Ag@SiO2NSs with malathion aptamer. Under the optimized conditions, the biosensor showed a wide linear range (1´10 ‑3 3 ng / mL) and a low detection limit (0.3×10 ‑3 ng / mL). The aptamer biosensor was sensitive, stable and specific, and could be used for the detection of malathion in tea. This study provided an effective method for the construction of aptamer biosensor with rapid, high sensitivity and low cost.​

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety rapid detection, and particularly relates to an electrochemiluminescence aptamer sensor based on NH2-Luminol / Ag@SiO2NSs, a preparation method and application thereof. BACKGROUND

[0002] Malathion is an organophosphorus insecticide, which is mainly used for preventing and treating various pests and diseases, including aphids, beetles, planthoppers, spider mites, ladybugs, whiteflies, borer, etc., and can also be used for soil disinfection and seed treatment. In addition, malathion is also used as an insecticide for home and public places to prevent and control pests such as mosquitoes, flies, and cockroaches. Malathion is an inhibitor of acetylcholinesterase (AChE). Acetylcholinesterase is an enzyme that degrades the neurotransmitter acetylcholine in the synaptic cleft, and malathion hinders the activity of the enzyme, causing the accumulation of neurotransmitters in the synaptic cleft, leading to nerve conduction disorders, and ultimately causing paralysis and death of pests. And there is no cross-resistance with other pesticides. It is widely used for pest control in vegetables, tea trees and tea leaves. Malathion has the characteristics of high efficiency, spectrum, and persistence, but the pesticide residues in tea leaves are harmful to human health through long-term enrichment. Therefore, it is of great significance to develop a high-sensitivity and high-specificity detection method for detecting malathion residues in tea leaves to protect human health.

[0003] Current rapid detection methods cannot meet the needs of high sensitivity and on-site detection. Therefore, it has become a development trend to establish a rapid and simple detection method. SUMMARY

[0004] To solve the above technical problems, the application provides an electrochemiluminescence aptamer sensor based on NH2-Luminol / Ag@SiO2NSs and a preparation method and application thereof, which has good stability, repeatability, specificity and sensitivity.

[0005] The technical scheme provided by the application is as follows.

[0006] The electrochemiluminescence aptamer sensor based on NH2-Luminol / Ag@SiO2NSs is characterized in that AuNPs / MWCNTs are used as a substrate, and the complementary chain (cDNA) of the aptamer is fixed by gold-sulfur bond. NH2-Luminol / Ag@SiO2NSs is used as an ECL luminophore, and the aptamer (Apt) is used as a recognition element; the NH2-Luminol / Ag@SiO2NSs is Luminol and AgNPs functionalized SiO2NSs.

[0007] Further, the preparation method of the AuNPs / MWCNTs is as follows: MWCNTs are added dropwise to the surface of a glassy carbon electrode (GCE), dried at room temperature, and then subjected to constant potential electrodeposition in a HAuCl4·3H2O solution by using a current i-t curve to obtain AuNPs / MWCNTs.

[0008] Further, the preparation method of the NH2-Luminol / Ag@SiO2NSs is as follows: under light shielding conditions, anhydrous ethanol, a Luminol solution, PVP-AgNPs, ultrapure water are added to a single-neck flask, and stirred; NH3·H2O is added, and ultrasonic treatment is performed; 1 mL of C8H 20 O4Si is added to the mixed solution, and after stirring, 1 mL of C8H 20 O4Si is added, and stirring is continued; finally, (3-Aminopropyl) triethoxysilane is added, and stirring is performed at 1500 rpm; the obtained product is centrifuged and washed three times to obtain NH2-Luminol / Ag@SiO2NSs, which is stored at 4℃ under light shielding conditions.

[0009] Further, the aptamer of the signal probe (Apt-Luminol / Ag@SiO2NSs) is a carboxyl-modified aptamer.

[0010] Further, the sequences of the thiol-modified malathion aptamer complementary strand and the carboxyl-modified malathion aptamer are as follows:

[0011] 5'-SH-(CH2)6-GGGAGCCAACACCAG-3';

[0012] 5'-COOH-ATCCGTCACACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'.

[0013] The application provides application of the above-mentioned electrochemiluminescence aptamer sensor based on NH2-Luminol / Ag@SiO2NSs in malathion detection.

[0014] Further, a glassy carbon electrode is used as a sensing platform, NH2-Luminol / Ag@SiO2NSs is used as an ECL luminophore, an aptamer is used as a recognition element, bovine serum albumin is added to block specific sites, and then reacted with a sample to be detected for detection.

[0015] Further, the electrochemiluminescence detection was performed using a Luminol-hydrogen peroxide ECL light-emitting system, the pH value of PBS was 9.0, the volume ratio of PBS to H2O2 was 1:500, the concentration of aptamer was 200 nM, and the incubation time of the sample to be tested was 40 min. Advantages

[0016] In the experiment, Luminol and AgNPs functionalized SiO2NSs were used as light-emitting bodies, NH2-Luminol / Ag@SiO2NSs and carboxyl-modified aptamer were connected through amide bond to form Apt-Luminol / Ag@SiO2NSs as a signal probe. When there is no malathion, the connection of the signal probe and the cDNA fixes the signal probe on the electrode surface through the action of base complementary pairing and hydrogen bond, and the ECL signal is strong. When malathion exists, the Apt on the surface of the signal probe specifically binds to it and is stripped from the electrode surface, and the ECL signal decreases, because the force between Apt and Malathion is stronger than that between complementary double strands. Through the monitoring of the signal change, the quantitative detection of malathion is realized.

[0017] Luminol is a highly efficient light-emitting chemical reagent with excellent chemical stability and high sensitivity. It can specifically bind to biological molecules such as proteins and nucleic acids, and is widely used in the research of biological molecule labeling and molecular probes, especially showing significant advantages in the detection and imaging of low concentration targets.

[0018] SiO2NSs is an inorganic compound material with high specific surface area, controllable size, good biocompatibility and adjustable surface properties. It can be doped with a large amount of Luminol and AgNPs to provide a stable reaction environment for the ECL light-emitting system. SiO2NSs can be loaded with Luminol and AgNPs to enhance the light-emitting effect and improve the sensitivity and efficiency of the ECL reaction.

[0019] AgNPs can catalyze the decomposition of H2O2 to enhance the light-emitting efficiency and stability of the ECL light-emitting system, and improve the sensitivity of the ECL system.

[0020] AuNPs can fix cDNA on the electrode surface through gold-sulfur bond, and can catalyze the decomposition of H2O2 to enhance the light-emitting efficiency and stability of the ECL light-emitting system, and improve the sensitivity and efficiency of the ECL reaction.

[0021] The application takes NH2-Luminol / Ag@SiO2NSs as an ECL luminophore, uses an aptamer complementary chain and an aptamer as a recognition element, and when malathion exists, the force between Apt and malathion is stronger than the strength between the complementary double strands, so that the Apt on the surface of the signal probe specifically binds and peels off from the electrode surface, and the ECL signal is reduced. The quantitative detection of malathion is realized by monitoring the change of the quenching signal. The detection range of the ECL aptamer sensor is 1´10 -3 -1´10 3 ng / mL, the detection limit is 0.3´10 -3 ng / mL, and there is no cross reaction with profenofos, phoxim, rogor and phorate. The ECL aptamer sensor can be used for the detection of malathion residues in green tea, black tea and puer tea samples, and has good stability, specificity and repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Schematic diagram of the synthesis and assembly steps of the sensing material.

[0023] Figure 2 TEM images (a, b, c) and STEM-EDS element mapping diagrams (d, e, f, g) of NH2-Luminol / Ag@SiO2NSs.

[0024] Figure 3 SEM images (a, b) of NH2-Luminol / Ag@SiO2NSs and EDS spectra (c) of NH2-Luminol / Ag@SiO2NSs.

[0025] Figure 4 Infrared spectrograms (A) of NH2-Luminol / Ag@SiO2NSs and Luminol / Ag@SiO2NSs; ultraviolet-visible spectrograms (B) of AgNPs, SiO2NSs and NH2-Luminol / Ag@SiO2NSs.

[0026] Figure 5 XPS full spectrum (a) and fine spectrum (b, c) of NH2-Luminol / Ag@SiO2NSs before and after modification of Apt.

[0027] Figure 6The images show the assembly diagrams of the aptamer sensor, including CV characterization (A) and EIS characterization (B): a: AuNPs / MWCNTs / GCE; b: MWCNTs / GCE; c: naked GCE; d: cDNA / AuNPs / MWCNTs / GCE; e: BSA / cDNA / AuNPs / MWCNTs / GCE; f: SPs / BSA / cDNA / AuNPs / MWCNTs / GCE; g: malathion / SPs / BSA / cDNA / AuNPs / MWCNTs / GCE; and the assembly diagram of the ECL aptamer sensor (C): a: NH2-Luminol / Ag@SiO2NSs / AuNPs / MWCNTs / GCE; b: NH2-Luminol / Ag@SiO2NSs / GCE; c: SPs / BSA / cDNA / AuNPs / MWCNTs / GCE; d: Comparison of ECL intensity of electrodes modified with different materials (D): a: NH2-Luminol / Ag@SiO2NSs / GCE; b: Luminol / AgNPs / GCE; c: Luminol@SiO2NSs / GCE; d: Luminol / GCE.

[0028] Figure 7 The experimental parameters to be optimized include: chloroauric acid concentration (A); aptamer concentration (B); pH of the test substrate (C); and incubation time (D).

[0029] Figure 8 The response of the aptamer sensor to different concentrations of malathion (A); the calibration curve of the aptamer sensor for detecting different concentrations of malathion (B).

[0030] Figure 9 For aptamer sensor stability assessment (A); reproducibility assessment (B); specificity assessment (C): (a) profenofos; (b) phoxim; (c) chlorpyrifos; (d) phorate; (e) malathion; (f) profenofos, phoxim, chlorpyrifos, phorate; (g) profenofos, phoxim, chlorpyrifos, phorate, malathion. Detailed Implementation

[0031] The reagent and culture medium formulations involved in the examples.

[0032] (1) Add 5 mL of luminol solution (0.01 M, which needs to be stored at 4°C in the dark for one week before use), 5 mL of PVP-AgNPs and 5 mL of ultrapure water to 40 mL of anhydrous ethanol. Then add 1 mL of NH3·H2O dropwise to the mixed solution, followed by adding 1 mL of C8H2O twice.20 O4Si, and finally 500 μL (3-Aminopropyl) triethoxysilane was added.

[0033] (2) HAuCl4·3H2O: 2 mL HAuCl4·3H2O (1 mM, 2 mM, 3 mM, 4 mM, 5 mM).

[0034] (3) PBS: 5.3 nM NaH2PO4·2H2O, 94.7 nM Na2HPO4·12H2O, 99.9 nM KCl.

[0035] (4) 30% H2O2: The volume ratio of PBS to H2O2 was 1:500.

[0036] (5) Malathion standard (1 ng / mL): 0.5 mg malathion was dissolved in 500 mL 5% methanol, and stored at 4°C.

[0037] Sources of materials involved in the examples.

[0038] BSA, KCl, NaH2PO4·2H2O, Na2HPO4·12H2O, ethanolum absolute, methanol, acetone were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). HAuCl4·3H2O, (3-Aminopropyl) triethoxysilane (98%) were purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd. H2O2 (30%) was purchased from Xi'an Chemical Reagent Factory. MWCNTs, AgNO3, Na3C6H5O7·2H2O, Polyvinyl pyrrolidone (PVP, High Purity Grade, K30), Luminol (98%), NaOH, C8H 20 O4Si, NH3·H2O were purchased from Shanghai Aldrin Biotechnology Co., Ltd.; Pesticide standard (analytical pure) was purchased from Beijing Putian Tongchuang Biological Technology Co., Ltd., and aptamer was purchased from Shengong Biological Engineering (Shanghai) Co., Ltd.

[0039] The complementary strand sequence of the thiol-modified malathion aptamer is as follows.

[0040] 5'-SH-(CH2)6-GGGAGCCAACACCAG-3'.

[0041] The sequence of the carboxyl-modified malathion aptamer is as follows.

[0042] 5'-COOH-ATCCGTCACACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'.

[0043] Example 1 Preparation of AuNPs / MWCNTs.

[0044] Add 10 mg MWCNTs in 5 mL ultrapure water, put on the shaker for 1 h, ultrasonic for 24 h to make it completely dissolved, get 2 mg / mL MWCNTs solution, 4℃ preservation for standby. Bare GCE is polished with 0.3 μm and 0.05 μm alumina polishing powder on the napped skin in turn before constant potential electrodeposition, then ultrasonic cleaning with anhydrous ethanol and ultrapure water, nitrogen blowing dry, then add 5 μL prepared 2 mg / mL MWCNTs solution on the electrode surface, dry at room temperature. In HAuCl4·3H2O solution (2 mM), electrodeposition is carried out by using current i-t curve (initial potential: -0.2 V, sampling interval: 0.1 s, running time: 300 s), AuNPs are assembled on MWCNTs, and AuNPs / MWCNTs composite material is prepared.

[0045] Example 2 Preparation of NH2-Luminol / Ag@SiO2NSs.

[0046] Under the condition of light shielding, add 40 mL anhydrous ethanol, 5 mL luminol solution (0.01 M, need to be placed at 4℃ for a week before use), 5 mL PVP-AgNPs, 5 mL ultrapure water in a single neck flask, stir at 400 rpm for 10 min. Add 1 mL NH3·H2O, ultrasonic for 5 min to make it fully mixed. Slowly add 1 mL C8H 20 O4Si drop by drop to the mixed solution, stir for 4 h, then add 1 mL C8H 20 O4Si drop by drop, continue to stir for 4 h. Finally add 500 μL (3-Aminopropyl)triethoxysilane, stir at 1500 rpm for 12 h. Centrifuge the obtained product and wash it with ultrapure water-anhydrous ethanol-ultrapure water in turn for three times to get NH2-Luminol / Ag@SiO2NSs composite nanomaterial.

[0047] Example 3 Preparation of electrochemiluminescence aptamer sensor based on NH2-Luminol / Ag@SiO2NSs.

[0048] Firstly, the glassy carbon electrode (GCE) was polished in alumina powder, then it was put into ethanol solution for ultrasonic treatment, and finally it was washed by ultrapure water and dried by nitrogen to obtain a mirror surface. 5 μΐ^of MWCNTs was dropped onto the surface of GCE, and after air-drying at room temperature, constant potential electrodeposition was carried out in 2 mM HAuCl4·3H2O by using an electrochemical workstation, so as to electrodeposited AuNPs on MWCNTs, and then the electrode was gently washed by ultrapure water and dried. 5 μΐ^of thiol-modified cDNA with a concentration of 100 nM was dropped onto the surface of the electrode, and the cDNA was fixed by using the Au-S bond formed between the cDNA and AuNPs. The non-specific binding sites exposed on the surface of the electrode were blocked by dropping 5 μΐ^of BSA (0.05%) solution, and then 5 μΐ^of signal probe was dropped onto the electrode, and reacted for 30 min, so as to fix the signal probe by hydrogen bond between complementary double strands. The GCE was gently washed by ultrapure water to remove the unreacted and combined signal probe. In order to evaluate the electrochemiluminescence effect of the aptamer sensor, 5 μΐ^of malathion pesticide standard with a concentration of 1 ng / mL was dropped onto the surface of the modified GCE and incubated for 40 min.

[0049] Principle of detection of Example 4.

[0050] When malathion does not exist, the connection between the signal probe and the cDNA is fixed on the surface of the electrode by base complementary pairing and hydrogen bond, and the ECL signal is strong. When malathion exists, the Apt on the surface of the signal probe specifically binds with it and is stripped from the surface of the electrode, and the ECL signal is reduced, because the force between Apt and Malathion is stronger than the strength between complementary double strands.

[0051] Optimization of detection method of Example 5.

[0052] In order to verify the performance of the aptamer sensor, optimization experiments were carried out on the concentration of chloroauric acid, the concentration of aptamer, the pH value of the test base solution and the incubation time. In the optimization experiment, 1 ng / mL of malathion was used as the experimental condition. The concentration of chloroauric acid is the main condition affecting the conductivity of the sensor, and the best concentration of chloroauric acid for malathion detection is verified in the work, as shown in Figure 7 A, and the best choice is 3 mM. The concentration of aptamer is an important condition affecting the performance of the aptamer sensor. The best concentration of aptamer for malathion detection is verified in the work, as shown in Figure 7 B, and the best choice is 200 nM. As shown in Figure 7 C, the ECL signal intensity in the range of pH 8.0-10.0 was investigated, and the best effect was obtained when the pH value was 9. As shown in Figure 7The effect of incubation time (20 min, 30 min, 40 min, 50 min, 60 min) on the performance of the aptamer sensor was studied, as shown in D. 40 min was selected as the optimal incubation time.

[0053] Example 6 Establishment of standard curve.

[0054] Under the optimal conditions, different concentrations of malathion were detected. As shown in Figure 8 B, the concentration of malathion in the range of 1 x 10 -3 ~1 x 10 3 ng / mL showed a good linearity with the ECL signal intensity, realizing the quantitative detection of malathion. The regression equation was I ECL = 5065.24-996.66LgC Malathion , R 2 = 0.9987. The detection limit of the aptamer sensor was 0.3 x 10 -3 ng / mL.

[0055] Example 7 Stability, specificity and repeatability of the detection method.

[0056] As shown in Figure 9 A, 12 aptamer sensors were prepared under the same conditions and stored at 4℃. After 0 days, 1 day, 7 days and 14 days, 1 ng / mL of malathion was detected, and the aptamer sensor had good stability. As shown in Figure 9 B, 7 aptamer sensors were developed under the same conditions to detect 1 ng / mL of malathion, and the RSD of the 7 electrodes was 1.69%, indicating that the sensor had good repeatability. As shown in Figure 7 C, using profenofos, phoxim, rogor and phorate as interference sources, the specificity of the aptamer sensor was studied, which showed outstanding specificity.

[0057] Example 8 Recovery test.

[0058] In order to verify the efficiency of the aptamer sensor in actual scenarios, green tea, black tea and pu'er tea were used as tea samples to detect malathion. The standard concentration of malathion (0, 1, 10 and 100 ng / mL) was added to the tea samples for detection. After extraction, the aptamer sensor was used for detection, and the detection results were calculated by the regression equation. The calculation results are shown in Table 1, and the aptamer sensor showed good performance in actual application.

[0059] Table 1 Recovery results of different concentrations of malathion added to tea samples by ECL evaluation.

[0060] Sample Added concentration (ng / mL) Measured concentration (ng / mL) Relative standard deviation (%) Recovery (%) Green tea 0 0 - - 1 1.09 2.06 108.82 10 10.72 1.98 107.15 100 97.72 2.30 97.72 Black tea 0 0 - - 1 1.07 3.46 107.15 10 10.96 1.60 109.65 100 112.20 1.67 112.20 Pu-erh tea 0 0 - - 1 1.23 2.14 123.03 10 10.42 1.24 104.23 100 104.71 3.69 104.71

Claims

1. An electrochemiluminescence aptamer sensor based on NH2-Luminol / Ag@SiO2 NSs, characterized in that, The AuNPs / MWCNTs are used as a substrate to fix the complementary strand of aptamer through gold-sulfur bond; the NH2-Luminol / Ag@SiO2NSs are used as an ECL luminophore, and the complementary strand of aptamer and aptamer are used as recognition elements; the NH2-Luminol / Ag@SiO2NSs are luminol and AgNPs functionalized SiO2NSs.

2. The method for preparing NH2-Luminol / Ag@SiO2 NSs-based electrochemiluminescence aptamer sensor according to claim 1, characterized in that, The AuNPs / MWCNTs are prepared by adding MWCNTs to the surface of a glassy carbon electrode, drying at room temperature, and then performing constant potential electrodeposition in a HAuCl4·3H2O solution by using current i-t curve; The preparation method of the NH2-Luminol / Ag@SiO2NSs is as follows: under the light-proof condition, anhydrous ethanol, a luminol solution, PVP-AgNPs, ultrapure water are added into a single-neck flask, and stirring is performed; NH3·H2O is added, and ultrasonic is performed; 1 mL of C8H 20 O4Si is added into the mixed solution, stirring is performed, and then 1 mL of C8H 20 O4Si is added, and stirring is continuously performed; finally, (3-Aminopropyl)triethoxysilane is added, and stirring is performed at 1500 rpm; the obtained product is centrifuged and cleaned three times to obtain the NH2-Luminol / Ag@SiO2NSs, which is stored at 4°C under the light-proof condition.

3. The method for preparing NH2-Luminol / Ag@SiO2 NSs-based electrochemiluminescence aptamer sensor according to claim 2, characterized in that, The AuNPs / MWCNTs are combined with the complementary strand of aptamer modified by thiol through gold-sulfur bond; the NH2-Luminol / Ag@SiO2NSs are combined with the aptamer modified by carboxyl through amide bond.

4. The method for preparing the NH2-Luminol / Ag@SiO2 NSs-based electrochemiluminescence aptamer sensor according to claim 3, characterized in that, The sequence of the complementary strand of malathion aptamer modified by thiol and the sequence of malathion aptamer modified by carboxyl are as follows: 5'-SH-(CH2)6-GGGAGCCAACACCAG-3'; 5'-COOH-ATCCGTCACACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'.

5. Application of the NH2-Luminol / Ag@SiO2NSs-based electrochemiluminescence aptamer sensor in malathion detection according to claim 1.

6. Use according to claim 5, characterized in that, A platinum electrode is used as a sensing platform, NH2-Luminol / Ag@SiO2NSs are used as an ECL luminophore, aptamer and its complementary strand are used as recognition elements, and after adding bovine serum albumin to block specific sites, the sensor is reacted with a sample to be detected.

7. Use according to claim 5, characterized in that, Electrochemiluminescence detection is performed by using a luminol-hydrogen peroxide ECL light-emitting system, the pH value of PBS is 9, the volume ratio of PBS to H2O2 is 1:500, the concentration of aptamer is 200 nM, and the incubation time of the sample to be detected is 40 min.

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