Electrochemiluminescence aptamer sensor based on NH2-Luminol / Ag-coated SiO2NSs as well as preparation method and application of electrochemical luminescence aptamer sensor

Abstract

Aiming at potential hazards of organophosphorus pesticide residues in tea leaves, the invention constructs an electrochemical luminescence (ECL) aptamer biosensor based on a functionalized nano material. Firstly, nano gold (AuNPs) is electrodeposited on the surface of multi-walled carbon nanotubes (MWCNTs) at a constant potential, so that a good fixed site is provided for complementary single-stranded DNA (cDNA); and secondly, synthesizing aminated luminol / nano silver coated spherical nano silicon dioxide (NH2-Luminol / Ag coated SiO2NSs) and a composite nano material through a one-pot method. And finally, combining the NH2-Luminol / Ag-coated SiO2NSs with the malathion aptamer to obtain a signal probe (SPs) which is used for constructing the sensor. Under optimized conditions, the sensor has a wide linear range (110 <-3 >-1103 ng / mL) and a low detection limit (0.3 * 10 <-3 > ng / mL). The aptamer sensor is high in sensitivity, good in stability and high in specificity, and can be used for detecting malathion residues in tea leaves. The research provides an effective method for constructing a rapid, high-sensitivity and low-cost aptamer sensor.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid food safety detection, and in particular relates to an electrochemiluminescent aptamer sensor based on NH2-Luminol / Ag@SiO2NSs, and a preparation method and application thereof. Background Art

[0002] Malathion is an organophosphorus insecticide that is mainly used to control a variety of pests and diseases, including aphids, beetles, plant hoppers, spider mites, ladybugs, whiteflies, borers, etc. It can also be used for soil disinfection and seed treatment. In addition, malathion is also used as an insecticide in homes and public places to 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 of nerves, and malathion inhibits the activity of the enzyme, causing the neurotransmitter to accumulate in the synaptic cleft, thereby causing nerve conduction disorders and ultimately leading to paralysis and death of pests. And there is no cross-resistance with other pesticides. It is widely used in 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, developing highly sensitive and specific detection methods for detecting malathion residues in tea is of great significance for protecting human health.

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

[0004] In order to solve the above technical problems, the present invention provides an electrochemiluminescent 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 solution provided by the present invention is as follows.

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

[0007] Furthermore, the preparation method of the AuNPs / MWCNTs is: drop MWCNTs onto the surface of a glassy carbon electrode (GCE), dry at room temperature, and then perform constant potential electrodeposition in a HAuCl4·3H2O solution using a current it curve to obtain AuNPs / MWCNTs.

[0008] Furthermore, the preparation method of NH2-Luminol / Ag@SiO2NSs is as follows: add anhydrous ethanol, Luminol solution, PVP-AgNPs, and ultrapure water to a single-necked flask under light-proof conditions, and stir. Add NH3·H2O and perform ultrasound. Add 1 mL of C8H 20 O4Si, stir and then add 1 mL of C8H 20 O4Si, and continue stirring. Finally, add (3-Aminopropyl) triethoxysilane and stir at 1500 rpm. The obtained product is centrifuged and washed three times to obtain NH2-Luminol / Ag@SiO2NSs, which are stored at 4°C in the dark.

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

[0010] Furthermore, the sequences of the complementary strands of the thiol-modified malathion aptamer and the carboxyl-modified malathion aptamer are as follows: 5'-SH-(CH2)6-GGGAGCCAACACCAG-3'; 5'-COOH-ATCCGTCACAACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'.

[0011] The present invention provides application of the above-mentioned electrochemiluminescent aptamer sensor based on NH2-Luminol / Ag@SiO2NSs in malathion detection.

[0012] Furthermore, a glassy carbon electrode was used as the sensing platform, NH2-Luminol / Ag@SiO2NSs was used as the ECL luminescent body, and an aptamer was used as the recognition element. After adding bovine serum albumin to block the specific site, it reacted with the sample to be tested for detection.

[0013] Furthermore, the Luminol-hydrogen peroxide ECL luminescence system was used for electrochemiluminescence detection, the pH value of PBS was 9.0, the volume ratio of PBS to H2O2 was 1:500, the concentration of the aptamer was 200 nM, and the incubation time of the sample to be tested was 40 min. Beneficial Effects

[0014] In this experiment, SiO2NSs functionalized with Luminol and AgNPs were used as luminophores, and NH2-Luminol / Ag@SiO2NSs was connected to the carboxyl-modified aptamer through an amide bond to form Apt-Luminol / Ag@SiO2NSs as a signal probe. When malathion is not present, the connection between the signal probe and cDNA fixes the signal probe on the electrode surface through base complementary pairing and hydrogen bonding, and the ECL signal is strong. When malathion is present, Apt on the surface of the signal probe specifically binds to it and peels off from the electrode surface, and the ECL signal decreases. This is because the interaction between Apt and Malathion is stronger than the strength between the complementary double strands. Quantitative detection of malathion is achieved by monitoring the signal changes.

[0015] Luminol is a highly efficient luminescent 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 biomolecule labeling and molecular probes, especially showing significant advantages in the detection and imaging of low-concentration targets.

[0016] SiO2NSs is an inorganic compound material with the advantages of high specific surface area, controllable size, good biocompatibility and adjustable surface properties. It can be doped with a large amount of Luminol and AgNPs, providing a stable reaction environment for the ECL luminescence system. SiO2NSs can enhance the luminescence effect by immobilizing Luminol and AgNPs, and also improve the sensitivity and efficiency of the ECL reaction.

[0017] AgNPs can catalyze the decomposition of H2O2, enhance the luminescence efficiency and stability of the ECL luminescence system, and improve the sensitivity of the ECL system.

[0018] AuNPs can fix cDNA on the electrode surface through gold-sulfur bonds, and can catalyze the decomposition of H2O2 to enhance the luminescence efficiency and stability of the ECL luminescence system, thereby improving the sensitivity and efficiency of the ECL reaction. . `-

[0019] The present invention uses NH2-Luminol / Ag@SiO2NSs as an ECL luminophore, and uses aptamer complementary chains and aptamers as recognition elements. When malathion is present, the interaction between Apt and Malathion is stronger than the strength between the complementary double chains, resulting in the Apt on the surface of the signal probe specifically binding to it and peeling off from the electrode surface, and the ECL signal is reduced. The quantitative detection of malathion is achieved by monitoring the changes in the quenching signal. The detection range of the ECL aptamer sensor is 1´10 -3 -1´10 3ng / mL, detection limit is 0.3´10 -3 ng / mL, and has no cross-reaction to propyl bromide, phoxim, chlorpyrifos and phorate. It can be used for the detection of malathion residues in green tea, black tea and Pu'er tea samples with good stability, specificity and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the synthesis and assembly steps of the sensing material.

[0021] Figure 2 TEM images (a, b, c) and STEM-EDS elemental mapping (d, e, f, g) of NH2-Luminol / Ag@SiO2NSs.

[0022] Figure 3 SEM images (a, b) and EDS spectra (c) of NH2-Luminol / Ag@SiO2NSs.

[0023] Figure 4 Infrared spectra of NH2-Luminol / Ag@SiO2NSs and Luminol / Ag@SiO2NSs (A); UV-visible spectra of AgNPs, SiO2NSs and NH2-Luminol / Ag@SiO2NSs (B).

[0024] Figure 5 The XPS full spectrum of NH2-Luminol / Ag@SiO2NSs (a) and the fine spectra before and after Apt modification (b, c).

[0025] Figure 6Figure 2 shows the assembly diagram of the aptamer sensor, including CV characterization (A) and EIS characterization (B). a: AuNPs / MWCNTs / GCE; b: MWCNTs / GCE; c: bare 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 assembly 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: malathion / SPs / BSA / cDNA / AuNPs / MWCNTs / GCE; 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.

[0026] Figure 7 Experimental parameters optimized include: chloroauric acid concentration (A); aptamer concentration (B); test base solution pH (C); incubation time (D).

[0027] Figure 8 Response of the aptamer sensor to different concentrations of malathion (A); calibration curve of the aptamer sensor for detecting different concentrations of malathion (B).

[0028] Fig. 9 Stability evaluation (A); reproducibility evaluation (B); specificity evaluation (C) of aptamer sensors: (a) Probofos; (b) Phoxim; (c) Chlorpyrifos; (d) Phorate; (e) Malathion; (f) Probofos, Phoxim, Chlorpyrifos, Phorate; (g) Probofos, Phoxim, Chlorpyrifos, Phorate, Malathion. DETAILED DESCRIPTION

[0029] The reagents and culture medium formulations involved in the examples.

[0030] (1) Add 5 mL of luminol solution (0.01 M, stored at 4°C away from light 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. Then, add 1 mL of C8H20 O4Si, and finally add 500 μL (3-Aminopropyl) triethoxysilane.

[0031] (2) HAuCl4·3H2O: 2 mL HAuCl4·3H2O (1 mM, 2 mM, 3 mM, 4 mM, 5 mM).

[0032] (3) PBS: 5.3 nM NaH2PO4·2H2O, 94.7 nM Na2HPO4·12H2O, 99.9 nM KCl.

[0033] (4) 30% H2O2: The volume ratio of PBS to H2O2 is 1:500.

[0034] (5) Malathion standard (1 ng / mL): 0.5 mg malathion was dissolved in 500 mL 5% methanol and stored at 4°C.

[0035] Sources of materials mentioned in the examples.

[0036] BSA, KCl, NaH2PO4·2H2O, Na2HPO4·12H2O, ethanol absolute, methanol, and acetone were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). HAuCl4·3H2O and (3-Aminopropyl)triethoxysilane (98%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. H2O2 (30%) was purchased from Xi'an Chemical Reagent Factory. MWCNTs, AgNO3, Na3C6H5O7·2H2O, Polyvinyl pyrrolidone (PVP, High PurityGrade, K30), luminol (98%), NaOH, C8H 20 O4Si and NH3·H2O were purchased from Shanghai Aladdin Biotechnology Co., Ltd.; pesticide standards (analytical grade) were purchased from Beijing Putian Tongchuang Biotechnology Co., Ltd., and aptamers were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0037] The complementary chain sequence of the thiol-modified malathion aptamer is as follows.

[0038] 5'-SH-(CH2)6-GGGAGCCAACACCAG-3'.

[0039] The sequence of the carboxyl-modified malathion aptamer is as follows.

[0040] 5'-COOH-ATCCGTCACAACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'.

[0041] Example 1 Preparation of AuNPs / MWCNTs.

[0042] Add 10 mg MWCNTs to 5 mL ultrapure water, place on an oscillator for 1 h, and ultrasonicate for 24 h to completely dissolve it, to obtain a 2 mg / mL MWCNTs solution, which was stored at 4°C for later use. Before constant potential electrodeposition, the bare GCE was repeatedly polished on suede with 0.3 μm and 0.05 μm alumina polishing powders, then ultrasonically cleaned with anhydrous ethanol and ultrapure water, and dried with nitrogen. 5 μL of the prepared 2 mg / mL MWCNTs solution was added to the electrode surface and dried at room temperature. Electrodeposition was performed in HAuCl4·3H2O solution (2 mM) using the current it curve (initial potential: -0.2 V, sampling interval: 0.1 s, running time: 300 s) to assemble AuNPs onto MWCNTs, and the AuNPs / MWCNTs composite material was prepared.

[0043] Example 2 Preparation of NH2-Luminol / Ag@SiO2NSs.

[0044] Under light-proof conditions, add 40 mL of anhydrous ethanol, 5 mL of luminol solution (0.01 M, stored at 4°C in the dark for one week before use), 5 mL of PVP-AgNPs, and 5 mL of ultrapure water to a single-necked flask and stir at 400 rpm for 10 min. Add 1 mL of NH3·H2O and mix thoroughly under ultrasonication for 5 min. Slowly add 1 mL of C8H 20 O4Si, stirred for 4 h, and then 1 mL of C8H 20 O4Si, and continue stirring for 4 h. Finally, add 500 μL (3-Aminopropyl)triethoxysilane and stir at 1500 rpm for 12 h. The resulting product was centrifuged and washed three times in the order of ultrapure water-anhydrous ethanol-ultrapure water to obtain NH2-Luminol / Ag@SiO2NSs composite nanomaterials.

[0045] Example 3 Preparation of electrochemiluminescent aptamer sensor based on NH2-Luminol / Ag@SiO2NSs.

[0046] First, the glassy carbon electrode needs to be polished in alumina powder, then placed in an ethanol solution for ultrasonic treatment, and finally rinsed with ultrapure water and dried with nitrogen to obtain a mirror-like surface. 5 μL MWCNTs were dropped onto the GCE surface, dried at room temperature, and then electrochemical workstation was used to perform constant potential electrodeposition in HAuCl4·3H2O with a concentration of 2 mM. AuNPs were electrodeposited onto MWCNTs, gently rinsed with ultrapure water and blown dry. 5 μL of 100nM thiol-modified cDNA was dropped onto the electrode surface, and the cDNA was fixed by the Au-S bond formed between cDNA and AuNPs. The nonspecific binding sites exposed on the electrode surface were blocked by dropping 5 μL of BSA (0.05%) solution, and then 5 μL of signal probe was dropped onto the electrode for 30 min to fix the signal probe through hydrogen bonds between complementary double strands. The GCE was gently rinsed with ultrapure water to remove the unreacted signal probe. In order to evaluate the electrochemiluminescence effect of the aptasensor, 5 μL of 1 ng / mL malathion pesticide standard was dropped onto the modified GCE surface and incubated for 40 min.

[0047] Example 4 Detection principle.

[0048] When malathion is absent, the signal probe is connected to cDNA through base complementary pairing and hydrogen bonding, fixing the signal probe on the electrode surface, and the ECL signal is strong. When malathion is present, Apt on the surface of the signal probe specifically binds to it and peels off from the electrode surface, and the ECL signal decreases. This is because the interaction between Apt and Malathion is stronger than the strength between complementary double strands.

[0049] Example 5 Optimization of detection method.

[0050] In order to verify the performance of the aptamer sensor, optimization experiments were conducted on the concentration of chloroauric acid, aptamer concentration, pH value of the test base solution, and incubation time. In the optimization experiment, we used 1 ng / mL of malathion as the experimental condition. The concentration of chloroauric acid is the main condition that affects the conductivity of the sensor. This work verified the optimal concentration of chloroauric acid for malathion detection, such as Figure 7 As shown in A, the chloroauric acid concentration of 3 mM is the best choice. The concentration of aptamer is an important condition affecting the performance of aptamer sensor. This work verifies the optimal concentration of aptamer for malathion detection. Figure 7 As shown in B, the aptamer concentration of 200 nM is the optimal choice. Figure 7 C, the ECL signal intensity was strong when the pH value was between 8.0 and 10.0, and the best effect was obtained when the pH value was 9. Figure 7As shown in Figure D, the effect of incubation time (20 min, 30 min, 40 min, 50 min, 60 min) on the performance of the aptamer sensor was studied. 40 min was selected as the optimal incubation time.

[0051] Example 6 Establishment of standard curve.

[0052] Under the optimal conditions, different concentrations of malathion were tested. Figure 8 As shown in B, the malathion concentration is 1×10 -3 ~1×10 3 ng / mL range, and the ECL signal intensity showed good linearity, achieving quantitative detection of malathion. The regression equation is: ECL =5065.24-996.66LgC Malathion , R 2 The detection limit of the aptasensor is 0.3×10 -3 ng / mL.

[0053] Example 7 Stability, specificity and repeatability of the detection method.

[0054] like Fig. 9 As shown in A, 12 aptamer sensors were prepared under the same conditions and stored at 4°C. 1 ng / mL malathion was detected after 0, 1, 7, and 14 days, respectively. The aptamer sensor has good stability. Fig. 9 As shown in B, 7 aptamer sensors were developed under the same conditions to detect 1 ng / mL malathion, and the RSD of the 7 electrodes was 1.69%, indicating that the sensor has good repeatability. Figure 7 As shown in C, the specificity of the aptamer sensor was studied using propyl bromide, phoxim, chlorpyrifos, and phorate as interference sources, showing outstanding specificity.

[0055] Example 8 Addition recovery test.

[0056] In order to verify the performance of the aptamer sensor in actual scenarios, green tea, black tea and Pu'er tea were used as tea samples to detect malathion. Standard concentrations of malathion (0, 1, 10 and 100 ng / mL) were added to the tea samples for detection. After extraction, the aptamer sensor was used for detection, and the detection results were put into the regression equation for calculation. The calculation results are shown in Table 1. The aptamer sensor showed good performance in practical applications.

[0057] Table 1 Recovery results of tea samples spiked with different concentrations of malathion evaluated by ECL.

[0058] sample Addition concentration (ng / mL) Measured concentration (ng / mL) Relative standard deviation (%) Recovery rate (%) 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'er 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 electrochemiluminescent aptamer sensor based on NH2-Luminol / Ag@SiO2NSs, characterized in that: AuNPs / MWCNTs are used as substrates, and the aptamer complementary chain is fixed through gold-sulfur bonds. NH2-Luminol / Ag@SiO2NSs are used as ECL luminescent bodies, and the aptamer complementary chain and the aptamer are used as recognition elements; the NH2-Luminol / Ag@SiO2NSs are SiO2NSs functionalized with luminol and AgNPs.

2. The method for preparing the electrochemiluminescent aptamer sensor based on NH2-Luminol / Ag@SiO2NSs according to claim 1, characterized in that: The preparation method of the AuNPs / MWCNTs is as follows: MWCNTs are added dropwise onto the surface of a glassy carbon electrode (GCE), dried at room temperature, and then electro-deposited in a HAuCl4·3H2O solution using a current it curve to obtain AuNPs / MWCNTs. The preparation method of NH2-Luminol / Ag@SiO2NSs is as follows: under light-proof conditions, anhydrous ethanol, Luminol solution, PVP-AgNPs, and ultrapure water are added to a single-necked flask and stirred. NH3·H2O is added and ultrasound is applied. 1 mL of C8H is added to the mixed solution. 20 O4Si, stir and then add 1 mL of C8H 20 O4Si, continue stirring. Finally, add (3-Aminopropyl)triethoxysilane and stir at 1500 rpm. The obtained product is centrifuged and washed three times to obtain NH2-Luminol / Ag@SiO2NSs, which are stored at 4°C in the dark.

3. The method for preparing the electrochemiluminescent aptamer sensor based on NH2-Luminol / Ag@SiO2NSs according to claim 1, characterized in that: The AuNPs / MWCNTs are combined with the complementary chain of the thiol-modified aptamer through a gold-sulfur bond; and the NH2-Luminol / Ag@SiO2NSs are combined with the carboxyl-modified aptamer through an amide bond.

4. The use according to claim 5, characterized in that: The sequences of the complementary strands of the thiol-modified malathion aptamer and the carboxyl-modified malathion aptamer are as follows: 5′-SH-(CH2)6-GGGAGCCAACACCAG-3′; 5'-COOH-ATCCGTCACAACCTGCTCTTATACACAATTGTTTTTCTCTTAACTTCTTGACTGCTGGTGTTGGCTCCCGTAT-3'.

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

6. The use according to claim 5, characterized in that: A platinum electrode was used as the sensing platform, NH2-Luminol / Ag@SiO2NSs was used as the ECL luminescent body, and the aptamer and its complementary chain were used as the recognition elements. After adding bovine serum albumin to block the specific site, it reacted with the sample to be tested for detection.

7. The use according to claim 5, characterized in that: The Luminol-hydrogen peroxide ECL luminescence system was used for electrochemiluminescence detection. The pH value of PBS was 9, the volume ratio of PBS to H2O2 was 1:500, the concentration of the aptamer was 200 nM, and the incubation time of the sample to be tested was 40 min.

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