Dual-signal aptamer sensor based on siO2 and fe3o4@au@pt, preparation method and application thereof
By utilizing a dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt, and leveraging aptamer recognition and enzyme catalytic activity, a sensor with both colorimetric and fluorescence dual-signal outputs is constructed. This solves the problems of expensive equipment, complex operation, and insufficient sensitivity in acetamiprid detection, achieving rapid and efficient detection results.
Patent Information
- Application Number
- CN202310701681.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-14
- Publication Date
- 2026-02-27
- Estimated Expiration
- 2043-06-14
AI Technical Summary
Existing methods for detecting acetamiprid residues suffer from problems such as expensive equipment, complex operation, long processing time, and insufficient sensitivity. There is an urgent need for a rapid, efficient, and sensitive detection method.
A dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt was constructed, using Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM as recognition probes. Combining the specific recognition of the aptamers and the enzymatic catalytic activity of Fe3O4@Au@Pt with magnetic separation technology, a sensor with colorimetric and fluorescence dual signal outputs was constructed.
It achieves highly sensitive detection of acetamiprid, with a fluorescence detection limit of 0.42 μg/mL and a colorimetric detection limit of 0.39 μg/mL, exhibiting good specificity and sensitivity, and is suitable for detection of practical samples.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of food safety rapid detection, and particularly relates to a high-sensitivity aptamer sensor with colorimetric and fluorescent dual-signal output based on SiO2 and Fe3O4@Au@Pt as well as a preparation method and application thereof. BACKGROUND
[0002] Acetamiprid (ACE) is a new type of chlorinated nicotinamide insecticide with contact and stomach toxicity, and excellent systemic activity. Due to its rapid insecticidal effect, low dosage, high activity, wide insecticidal spectrum, long persistence and other characteristics, it is widely used in vegetables to control aphids, whitefly, thrips, striped flea beetle and other pests. Although ACE is a low-toxicity pesticide, current studies have shown that it has certain genotoxicity and cytotoxicity, and has adverse effects on the nervous system and reproductive system of animals, posing a threat to human health. In recent years, due to the overuse of ACE and its own characteristics of being difficult to degrade in the environment, events of ACE residue exceeding the standard in vegetables have occurred frequently. According to the National Food Safety Standard Maximum Residue Limits of Pesticides in Foods (GB 2763-2021), the maximum residue limit of ACE in common cabbage, Chinese cabbage, cucumber, tomato, eggplant, sweet pepper and other vegetables is 1 mg / kg, and the maximum residue limit of ACE in cabbage, bitter gourd, broad bean, radish, bean and other vegetables is 0.5 mg / kg. Therefore, monitoring the ACE residue level in vegetables is of great significance to ensure food safety.
[0003] Currently reported methods for detecting ACE residues mainly include instrumental methods, among which chromatographic methods are more common, such as high performance liquid chromatography (HPLC), gas chromatography (GC), and chromatography-mass spectrometry (GC-MS). Instrumental methods have high sensitivity, good selectivity, accuracy and reliability, but they are expensive, complex to operate, require professional operators, and take a long time. Immunological methods involve a complex antibody preparation process, and antibodies are much less stable than deoxyribonucleic acid (DNA) during operation. Therefore, there is an urgent need for a rapid, efficient and sensitive method for identifying ACE residue levels. SUMMARY
[0004] To solve the above technical problems, the application provides a dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt as well as a preparation method and application thereof, which has good sensitivity and accuracy.
[0005] The technical solutions provided by the application are as follows:
[0006] The double-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt has Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM as recognition probes; the Fe3O4@Au@Pt@Apt is aptamer functionalized Fe3O4@Au@Pt, and the SiO2@cDNA-FAM is SiO2 functionalized with a fluorescently labeled aptamer complementary chain.
[0007] The application further provides a preparation method of the above-mentioned double-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt, and the preparation method of the Fe3O4@Au@Pt@Apt is as follows: after Apts are incubated with TCEP, Fe3O4@Au@Pt is added, and reaction is carried out at 4 DEG C for 3 hours to obtain Fe3O4@Au@Pt@Apt.
[0008] Further, the preparation method of the Fe3O4@Au@Pt is as follows: Fe3O4@Au is placed in a methanol-water solution, H2PtCl4.4H2O is added, reaction is carried out in an ice bath with stirring, NaBH4 aqueous solution is added, and after stirring, magnetic separation is carried out, and after washing and drying, Fe3O4@Au@Pt is obtained.
[0009] Further, the preparation method of the Fe3O4@Au is as follows: Fe3O4-NH2 is placed in a methanol-water solution, H2AuCl4.4H2O is added, reaction is carried out in an ice bath with stirring, NaBH4 aqueous solution is added, and after stirring, magnetic separation is carried out, and after washing and drying, Fe3O4@Au is obtained.
[0010] Further, the preparation method of the Fe3O4-NH2 is as follows: after dry Fe3O4 is dispersed in ethanol and ultrasonic treatment, APTES is added for reaction in an oil bath, and after reaction, magnetic separation is carried out, and after ethanol washing and drying, Fe3O4-NH2 is obtained.
[0011] Further, the preparation method of the SiO2@cDNA-FAM is as follows: SiO2-COOH is dispersed in pure water, cDNA-FAM and NHS and EDC are added to activate the carboxyl group, and SiO2@cDNA-FAM is obtained; the SiO2-COOH is obtained by carboxyl modification of SiO2.
[0012] Further, the preparation method of the SiO2-COOH is as follows: succinic anhydride is added to DMF to prepare mixed solution A, and ultrasonic treatment is carried out; then SiO2-NH2 is added to DMF to prepare mixed solution B, and ultrasonic treatment is carried out; after mixed solution A and mixed solution B are uniformly mixed, deionized water is added, and magnetic stirring is carried out in an oil bath overnight, centrifugation is carried out, and multiple repeated washing and centrifugation are carried out with distilled water to obtain SiO2-COOH.
[0013] Further, the preparation method of SiO2-NH2 is as follows: first, CTAB is added to pure water and ultrasonic treatment is performed, then TEOS is dissolved in ethanol, the solution is added dropwise to the above-mentioned CTAB solution by using a syringe, and stirring is performed. Then, ethanol containing APTES is added dropwise to the above-mentioned solution, and finally ammonia water is slowly added, and the mixture is allowed to react in an oil bath. After the reaction is completed, centrifugation is performed, the mixture is washed with a 2% hydrochloric acid-methanol solution, then the mixture is allowed to react in an oil bath, and the process is repeated twice. Finally, the mixture is washed with ethanol and pure water and dried to obtain SiO2-NH2.
[0014] The application further provides application of the above-mentioned aptasensor based on SiO2 and Fe3O4@Au@Pt in detection of acetamiprid.
[0015] Further, Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM are used as recognition probes, and after reaction with a sample to be detected, magnetic separation is performed, the fluorescence intensity of supernatant is detected, and the precipitate is resuspended in PBS for colorimetric analysis, and the absorbance value at 450 nm is determined.
[0016] Further, the volume ratio of Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM is 1:2, the quenching time is 60 min, and the incubation time of ACE is 80 min.
[0017] Further, the sequences of the aptamer and the fluorescently labeled complementary strand are as follows:
[0018] SH-(CH2)6-TGT AAT TTG TCT GCAGCG GTT CTT GAT CGC TGACAC CAT ATT ATGAAG A;
[0019] NH2-(CH2)6-CAT AAT ATG GTG TCA GCG-FAM.
[0020] Advantages
[0021] In this experiment, SiO2 was modified by carboxylation to obtain SiO2-COOH, and an aminofluorescein-labeled cDNA (cDNA-FAM) molecular recognition probe was synthesized. Through group combination, cDNA-FAM was successfully bound to the surface of SiO2-COOH to form SiO2@cDNA-FAM. Au NPs and Pt NPs were modified on the surface of Fe3O4 to obtain Fe3O4@Au@Pt, and thiolated Apts were combined with Fe3O4@Au@Pt through groups to form Fe3O4@Au@Pt@Apt. Using SiO2 and Fe3O4@Au@Pt as materials, Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM as recognition probes, using the specific recognition of aptamers to target substances, the enzyme catalytic activity of Fe3O4@Au@Pt and its quenching effect on fluorescence, and combining with magnetic separation technology, a colorimetric and fluorescent dual signal output high-sensitivity aptamer sensor for ACE detection was constructed.
[0022] Silicon dioxide (SiO2) nanoparticles have adjustable particle and pore size, particle shape, and porous structure, can be surface modified, have good biocompatibility, and large surface area and pore volume. By modifying the surface of SiO2 to improve its reactivity, the interfacial bonding between SiO2 and the polymer matrix can be significantly improved, and the performance of the composite material can be improved.
[0023] Ferroferric oxide (Fe3O4) nanoparticles have good magnetic response performance and peroxidase-like activity, and have been widely used in biological sensing, food safety, disease treatment and many other fields. Gold nanoparticles (Au NPs) have been widely used in the construction of fluorescent sensors and colorimetric sensors due to their easy-to-control particle size, good biocompatibility, excellent fluorescence quenching ability and peroxidase activity. Platinum nanoparticles (Pt NPs) have catalytic activity similar to natural enzymes, among which peroxidase activity is the most representative and is most widely used in food safety detection. In the presence of oxidants such as hydrogen peroxide, Au NPs and Pt NPs can catalyze the oxidation of certain organic substrates, promoting the color change of the chromogenic substrate.
[0024] The application uses Fe3O4@Au@Pt functionalized by aptamer (Fe3O4@Au@Pt@Apt) and SiO2 functionalized by fluorescently labeled complementary strand of aptamer (SiO2@cDNA-FAM) as recognition probes, uses specific recognition of aptamer to target and enzyme catalytic activity of Fe3O4@Au@Pt and its quenching effect on fluorescence, and combines with magnetic separation technology for acetamiprid detection. The fluorescence signal detection limit (LOD) is 0.42 μg / mL, and the colorimetric signal LOD is 0.39 μg / mL, and there is no cross reaction with thiamethoxam (THI), nitenpyram (NIT), imidacloprid (IMI) and clothianidin (CLO). The method can be used for detection of acetamiprid in Chinese cabbage and cucumber samples. The average recovery rate of the colorimetric signal is 85.59%-104.37%, and CV is less than 2.91%. The average recovery rate of the fluorescence signal is 92.69%-103.32%, and CV is less than 3.67%. It is shown that the sensor has good specificity and sensitivity, and can be used for actual sample detection. BRIEF DESCRIPTION OF DRAWINGS
[0025] Figure 1 It is a flow chart of the application.
[0026] Figure 2 It is a TEM diagram of SiO2-COOH.
[0027] Figure 3 It is FTIR spectrum diagram (A) and potential analysis diagram (B) of SiO2-NH2 and SiO2-COOH.
[0028] Figure 4 It is XPS diagram of SiO2-COOH.
[0029] Figure 5 It is XRD diagram of SiO2 and SiO2-COOH.
[0030] Figure 6 It is TEM diagram of Fe3O4 (A) and Fe3O4@Au@Pt (B), SEM diagram of Fe3O4 (C) and Fe3O4@Au@Pt (D), particle size distribution diagram of Fe3O4 (E) and Fe3O4@Au@Pt (F), and mapping diagram of Fe3O4@Au@Pt (G).
[0031] Figure 7 It is XRD diagram of Fe3O4 and Fe3O4@Au@Pt.
[0032] Figure 8The ratio of the contents of Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM (A) (without ACE), the reaction time of Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM (B) (without ACE), the incubation time of ACE (C) (the concentration of ACE is 6 μg / mL). (△F = F-F0, F and F0 are the fluorescence intensities in the presence or absence of ACE, respectively).
[0033] Figure 9 The fluorescence spectrum of different ACE concentrations (10, 8, 6, 4, 2, 1, 0.5, 0.25, 0 μg / mL).
[0034] Figure 10 The absorption spectrum of different ACE concentrations (10, 8, 6, 4, 2, 1, 0.5, 0.25, 0 μg / mL).
[0035] Figure 11 The specificity (A) and reproducibility (B) of the fluorescence detection method.
[0036] Figure 12 The specificity (A) and reproducibility (B) of the colorimetric detection method. DETAILED DESCRIPTION
[0037] The reagents and medium formulations involved in the examples:
[0038] (1) PBS buffer: weigh 8.0 g of NaCl, 0.2 g of KCl, 2.9 g of Na2HPO4·12H2O and 0.2 g of KH2PO4, dissolve in double distilled water and make up to 1 L, adjust the pH to 7.4.
[0039] (2) PBST: add 0.05% Tween-20 to the PBS buffer.
[0040] (3) 3% MPBS: weigh 1.2 g of skimmed milk powder, dissolve in 40 mL of double distilled water.
[0041] (4) CPBS: weigh 10.5 g of citric acid and 35.8 g of Na2HPO4·12H2O, dissolve in double distilled water and make up to 1 L, adjust the pH to 5.5.
[0042] (5) TMB stock solution: weigh 50 mg of TMB and dissolve in 5 mL of dimethyl sulfoxide, store at 4°C for short-term.
[0043] (6) Color developing solution: take 100 μL of TMB stock solution (10 mg / mL) and 25 μL of 0.65% H2O2, dissolve in 10 mL of CPBS, prepare fresh and use immediately.
[0044] (7) 2M H2SO4: 11.8 mL of concentrated sulfuric acid (98%) was slowly added into 80 mL of distilled water with constant stirring during the process. After cooling, the volume was made up to 100 mL.
[0045] (8) 2% H2AuCl4·4H2O: 1 g of H2AuCl4·4H2O was dissolved in 50 mL of H2O and stored at 4°C in the dark.
[0046] (9) 2% H2PtCl4·4H2O: 1 g of H2PtCl4·4H2O was dissolved in 50 mL of H2O and stored at 4°C in the dark.
[0047] (10) Standard solution of acetamiprid (1 mg / mL): 0.05 g of acetamiprid was dissolved in 50 mL of 5% methanol-PBS and stored at 4°C.
[0048] Sources of materials involved in the examples:
[0049] N,N-dimethylformamide (DMF, 99%), acetic acid (99%), copper chloride dihydrate (CuCl2·2H2O, 99%), methanol (99%), acetone (99%), cetyltrimethylammonium bromide (CTAB), ethyl silicate (TEOS), 3-aminopropyltrimethoxysilane (APTES), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) were all from Titan Kogyo; tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was from Adams Reagent Co., Ltd.; monoclonal antibody of acetamiprid (1.23 mg / mL) was from Shandong Lvdou Biotechnology Co., Ltd.; acetamiprid (ACE 99%), thiamethoxam (THI 98%), nitenpyram (NIT 98%), imidacloprid (IMI 98%), clothianidin (CLO 98%) were from Shanxi Qixing Pesticide Co., Ltd.; trisodium citrate dihydrate, anhydrous ferric chloride, succinic anhydride were from National Pharmaceutical Group Chemical Reagent Co., Ltd.; ethylene glycol, sodium peroxoacetate (NaOAc) were from Tianjin Kemio Chemical Reagent Co., Ltd.; sodium borohydride (NaBH4), 3,3',5,5'-tetramethylbenzidine (TMB) were from Shanghai McLean Biotechnology Co., Ltd.; sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate, potassium chloride, Tween-20, hydrogen peroxide were from Xilong Scientific Co., Ltd.; DNA fragments were from Shanghai Sangon Biotech Co., Ltd.
[0050] Aptamer (Apts) and fluorescently labeled complementary strand (cDNA-FAM) sequences involved in the examples:
[0051] SEQ ID NO. 1:
[0052] SH-(CH2)6-TGTAATTGTCTGCAGCGGTTCTTGATCGCTGACACCATATTATGAAGA
[0053] SEQ ID NO. 2:
[0054] NH2-(CH2)6-CAT AAT ATG GTG TCA GCG-FAM
[0055] Preparation of SiO2-NH2and SiO2-COOH
[0056] Preparation of SiO2-NH2: First, 0.5 g CTAB was added to 350 mL pure water, and ultrasonic vibration was performed for 20 min to completely dissolve it. Then, 0.5 mL TEOS was dissolved in 25 mL ethanol, which was slowly added dropwise to the above-mentioned CTAB solution by using a syringe, and stirring was performed to ensure the uniformity of the mixture. Then, 25 mL of ethanol containing 250 μL APTES was added dropwise to the above-mentioned solution, and finally 2.5 mL of ammonia water was slowly added. The mixture was allowed to react in a 30 °C oil bath for 6 h. After the reaction was completed, centrifugation was performed, and washing was performed three times with a 2% hydrochloric acid-methanol solution. Then, the mixture was allowed to react in a 60 °C oil bath for 5 h, and the process was repeated twice. Finally, washing was performed twice with ethanol and pure water, and vacuum drying was performed to obtain SiO2-NH2. The infrared spectrum (FTIR) and potential analysis results are shown in Figure 3 .
[0057] Preparation of SiO2-COOH: First, 0.5 g of succinic anhydride was weighed and added to 20 mL of DMF to prepare a mixed solution A, and ultrasonic vibration was performed. Then, 150 g of SiO2-NH2was weighed and added to 20 mL of DMF to prepare a mixed solution B, and ultrasonic vibration was performed. A and B were mixed, 2 mL of deionized water was added, and magnetic stirring was performed in a 60 °C oil bath overnight. Centrifugation was performed, and washing was performed multiple times with distilled water to obtain SiO2-COOH. The transmission electron microscopy (TEM) results are shown in Figure 2 . The prepared SiO2-COOH has a regular spherical morphology, good particle dispersion, uniform particle size, and a typical mesoporous structure, and the average particle size is 370 nm. The infrared spectrum (FTIR) and potential analysis results are shown in Figure 3 . The X-ray photoelectron spectroscopy (XPS) results are shown in Figure 4 . The X-ray diffraction (XRD) results are shown in Figure 5 . There is a relatively wide hill-shaped peak at 2θ = 22°, which is a typical peak of SiO2. The XRD spectrum of the carboxyl-modified SiO2 is not different from that of SiO2, indicating that the carboxylation has no effect on the structure of SiO2.
[0058] Preparation of SiO2@cDNA-FAM
[0059] 6 mg SiO2-COOH was dispersed in 1 mL pure water, 500 μL cDNA-FAM (1 μM) (SEQ ID NO. 2) and 250 μL NHS (10 mg / mL), 250 μL EDC (15 mg / mL) were added to activate the carboxyl group, and the mixture was reacted at 4 °C for 3 h.
[0060] Preparation of Fe3O4 and Fe3O4@Au@Pt
[0061] Preparation of Fe3O4: 1.66 g of anhydrous ferric chloride was dissolved in 80 mL of ethylene glycol under magnetic stirring, 7.2 g of NaOAc was added, and the mixture was stirred magnetically for 30 min, ultrasonicated for 10 min, transferred to a 50 mL high-pressure reactor, reacted at 200 °C for 8 h, magnetically separated, washed with pure water for 3 times, and dried in an oven.
[0062] Amination of dried Fe3O4: 160 mg of Fe3O4 was dispersed in 80 mL of ethanol, ultrasonicated for 5 min, 2.4 mL of APTES was added, and the mixture was reacted in a 50 °C oil bath for 12 h, magnetically separated, washed with ethanol for 3 times, and dried in an oven to obtain Fe3O4-NH2.
[0063] Preparation of Fe3O4@Au: 120 mg of Fe3O4-NH2 was placed in 30 mL of 50% methanol-water, 750 μL of 2% H2AuCl4·4H2O was added, and the mixture was stirred in an ice bath for 1.5 h, 30 mL of an aqueous solution containing 60 mg of NaBH4 was added, and the mixture was stirred for 0.5 h, magnetically separated, washed with pure water for 3 times, and dried in an oven to obtain Fe3O4@Au.
[0064] Preparation of Fe3O4@Au@Pt: 100 mg of Fe3O4@Au was placed in 25 mL of 50% methanol-water, 625 μL of 2% H2PtCl4·4H2O was added, and the mixture was stirred in an ice bath for 1.5 h, 25 mL of an aqueous solution containing 50 mg of NaBH4 was added, and the mixture was stirred for 0.5 h, magnetically separated, washed with pure water for 3 times, and dried in an oven to obtain Fe3O4@Au@Pt. The TEM results of Fe3O4 and Fe3O4@Au@Pt are shown in Figure 6 A and Figure 6 B, and the SEM results of Fe3O4 and Fe3O4@Au@Pt are shown in Figure 6 C and Figure 6 D. From Figure 6 A, C we can see that the prepared Fe3O4 is uniform spherical particles. As Figure 6B, D show that there are many bright particles on Fe3O4, and the shape structure changes little, which is Au NPs and Pt NPs. The particle size distribution results of Fe3O4 and Fe3O4@Au@Pt are as shown in Figure 6 E and Figure 6 F show that the average particle size of Fe3O4 is 297 nm, and the average particle size of Fe3O4@Au@Pt is 318 nm, which is slightly larger than that of Fe3O4. The mapping element analysis results of Fe3O4@Au@Pt are as shown in Figure 6 G show that Fe, O, Au, Pt are uniformly distributed on the surface of Fe3O4, which confirms the successful preparation of Fe3O4@Au@Pt. Figure 7 The XRD patterns of Fe3O4 and Fe3O4@Au@Pt are shown in the figure. In the curve of Fe3O4, seven typical diffraction peaks (30.3°, 35.6°, 43.4°, 53.7°, 57.2°, 62.8° and 73.8°) are observed, which correspond to (220), (311), (400), (422), (511), (440) and (533) crystal planes, respectively. After coating Au NPs and Pt NPs on the surface of Fe3O4, four new peaks of 38.4°, 44.6°, 64.9° and 77.8° appear on Fe3O4@Au@Pt, which are called (111), (200), (220) and (311) crystal planes. These four new peaks are the typical peaks of Au NPs and Pt NPs, which confirm the successful preparation of Fe3O4@Au@Pt.
[0065] Example 4 Preparation of Fe3O4@Au@Pt@Apt
[0066] After 200 μL of Apts (1 mM) (SEQ ID NO. 1) was incubated with 200 μL of TCEP (1 mM) for 1 h, 1 mL of Fe3O4@Au@Pt (1 mg / mL) was added, and the reaction was carried out at 4°C for 3 h to obtain Fe3O4@Au@Pt@Apt.
[0067] Example 5 Operating steps of double signal detection
[0068] The double signal detection is fluorescence detection and colorimetric detection respectively. After Fe3O4@Au@Pt@Apt reacts with SiO2@cDNA-FAM, it reacts with different concentrations of ACE. After the reaction is completed, magnetic separation is carried out, and the supernatant is directly subjected to fluorescence detection to measure the fluorescence intensity. The precipitate is resuspended in PBS for colorimetric reaction, and the colorimetric detection steps are as follows:
[0069] (1) Coating: dilute the anti-acetamiprid monoclonal antibody with PBS to 2.46 μg / mL, 100 μL / well is added to the 96-well plate, and the coating is carried out at 4°C overnight.
[0070] (2) Blocking: 300 μL / well PBST wash plate 3 times, 200 μL / well 3% MPBS added to the 96-well plate, 37°C incubation for 2 h.
[0071] (3) Sample addition: 300 μL / well PBST wash plate 3 times, 100 μL / well of the above-mentioned resuspension of the precipitate added to the 96-well plate, 37°C incubation for 1 h.
[0072] (4) Color development: 300 μL / well PBST wash plate 3 times, 100 μL / well TMB color development solution added to the 96-well plate, 37°C incubation for 1 h.
[0073] (5) Measurement: 50 μL / well 2M H2SO4 added to the 96-well plate to terminate color development, enzyme-labeled instrument reading of the OD 450 value of each well.
[0074] Example 6 Optimization of the detection method
[0075] In order to improve the fluorescence detection performance, through single factor experiment, the volume ratio of Fe3O4@Au@Pt@Apt (0.7 mg / mL) and SiO2@cDNA-FAM (3 mg / mL) (1:1, 1:1.5, 1:2, 1:2.5, 1:3), the quenching time of Fe3O4@Au@Pt@Apt to SiO2@cDNA-FAM fluorescence (0, 20, 40, 60, 80, 100, 120, 140 min), and the incubation time of the mixture with ACE (0, 20, 40, 60, 80, 100, 120, 140 min) on the detection sensitivity were investigated. The results are shown in Figure 8 A, after mixing Fe3O4@Au@Pt@Apt (0.7 mg / mL) and SiO2@cDNA-FAM (3 mg / mL) in different volume ratios, it was found that when the volume ratio of Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM was 1:2, the fluorescence intensity reached a maximum value and almost stopped changing, therefore, 1:2 was selected as the best volume ratio. In addition, as shown in Figure 8 B, after mixing Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM in a volume ratio of 1:2, it was found that when the quenching time reached 60 min, the fluorescence intensity almost did not decrease, indicating that the fluorescence quenching efficiency had reached saturation at this time. Therefore, 60 min was selected as the best quenching time.
[0076] Subsequently, the effect of ACE incubation time on fluorescence intensity was optimized. After determining the optimal volume ratio and quenching time, Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM were mixed at a volume ratio of 1:2 and reacted at 4°C for 60 min, then ACE (6 μg / mL) was added and incubated for different times. The results are shown in Figure 8 C, when the incubation time of ACE (6 μg / mL) in the whole system reached 80 min, the fluorescence intensity returned to the maximum value. Therefore, 80 min was selected as the optimal incubation time of ACE.
[0077] Example 7 Establishment of Standard Curve
[0078] After optimizing the experimental parameters, under the optimal conditions determined in Example 6, the concentrations of ACE standards were diluted to 10, 8, 6, 4, 2, 1, 0.5, 0.25, and 0 μg / mL, respectively, and determined according to the above method. The concentration of acetamiprid was taken as the horizontal coordinate, and the fluorescence difference ΔF (ΔF = F-F0, F and F0 are the fluorescence intensities in the presence and absence of ACE, respectively) and the absorbance value A of TMB color development were taken as the vertical coordinates, and standard curves were established respectively. The fluorescence detection results are shown in Figure 9 As the ACE concentration increased from 0 μg / mL to 10 μg / mL, the fluorescence intensity gradually increased. ACE in the concentration range of 0.25-6 μg / mL showed a good linear correlation with the substrate fluorescence difference ΔF. The linear equation was ΔF = 233.68*C + 34.48, R 2 = 0.9937 (where C is the concentration of ACE), and the LOD was 0.42 μg / mL (S / N = 3, S / N is the signal-to-noise ratio).
[0079] The colorimetric detection results are shown in Figure 10 As the ACE concentration increased, the absorbance value (A) increased. ACE in the concentration range of 0.25-6 μg / mL showed a good linear correlation with the absorbance value A. The linear equation was A = 0.0385*C + 0.0486, R 2 = 0.9966 (where C is the concentration of ACE). The LOD was 0.39 μg / mL (S / N = 3, S / N is the signal-to-noise ratio).
[0080] Example 8 Specificity and Reproducibility of Detection Method
[0081] To verify the specificity of the established method, THI, NIT, IMI, and CLO, four structural analogs of neonicotinoids, were selected at a concentration of 6 μg / mL, and the fluorescence intensity and absorbance value were measured after reaction according to the operation steps of Example 5. The results are shown in Figure 11As shown in Fig. 4A, the fluorescence intensity and absorbance of the whole system were significantly enhanced in the presence of ACE. When detecting other interfering pesticides, the increase of fluorescence intensity and absorbance was negligible. Therefore, only ACE had a significant fluorescence signal response, indicating that the fluorescence signal had good specificity; at the same time, the color of the TMB oxidation product was the deepest in the presence of ACE, indicating that the colorimetric signal also had good specificity for ACE, which could accurately detect ACE under complex environmental conditions. Figure 12 A).
[0082] To verify the reproducibility of the established method, the fluorescence intensity and absorbance values were measured after the same volume of ACE solution (6 μg / mL) (n = 5) was fully reacted with the mixture of Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM. The results showed that the relative standard deviation (RSD) of 5 experiments in fluorescence detection was 2.67% (Fig. 4B), and the relative standard deviation (RSD) in colorimetric detection was 5.84% (Fig. 4B), indicating that the dual signal method had good reproducibility. Figure 11 B). Figure 12 B).
[0083] Example 9 Recovery Test
[0084] To determine the analysis performance of the method in the detection of actual samples, the established method was applied to the detection of ACE in cabbage and cucumber samples. First, the sample was pretreated, and 50.0 g of cabbage and cucumber samples were accurately weighed, and different concentrations of ACE standard solution were added to configure low (0.4 mg / kg), medium (1.6 mg / kg), and high (4 mg / kg) concentration gradients. The sample was ground thoroughly and placed in a centrifuge tube, and centrifuged at 8000 rpm for 6 min. The supernatant was placed in a 70°C water bath for 30 min, then centrifuged at 8000 rpm for 6 min, filtered with a 0.22 μM membrane to obtain 20 mL of solution, and stored at 4°C for standby. Under the optimal conditions determined in Example 6, the fluorescence intensity and absorbance were measured according to the operation method of Example 5. All experiments were performed simultaneously for 3 times, and all data were expressed as the mean ± standard deviation of the data, and the recovery rate was calculated. The results are shown in Table 1, the average recovery rate of the colorimetric signal was 85.59%-104.37%, CV<2.91%; the average recovery rate of the fluorescence signal was 92.69%-103.32%, CV<3.67%. It is proved that the dual signal detection method based on SiO2 and Fe3O4@Au@Pt has good accuracy and can be used for the detection of actual samples.
[0085] Table 1 Results of the addition recovery experiment of acetamiprid
[0086]
Claims
1. A dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt, characterized in that, The dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt is used for acetamiprid detection, with Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM as recognition probes; Fe3O4@Au@Pt@Apt is aptamer-functionalized Fe3O4@Au@Pt, and SiO2@cDNA-FAM is fluorescently labeled aptamer-complementary strand-functionalized SiO2; the dual signals are colorimetric and fluorescent dual signals.
2. The dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt according to claim 1, characterized in that, The preparation method of Fe3O4@Au@Pt@Apt is as follows: after incubating Apts with TCEP, Fe3O4@Au@Pt is added, and the reaction is carried out at 4 ℃ for 3 h to obtain Fe3O4@Au@Pt@Apt.
3. The dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt according to claim 1, characterized in that, The preparation method of Fe3O4@Au@Pt is as follows: Fe3O4@Au is placed in a methanol-water solution, then H2PtCl4·4H2O is added, the reaction is stirred in an ice bath, then NaBH4 aqueous solution is added, the mixture is stirred and magnetically separated, washed and dried to obtain Fe3O4@Au@Pt.
4. The dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt according to claim 1, characterized in that, The preparation method of Fe3O4@Au is as follows: Fe3O4-NH2 is placed in a methanol-water solution, then H2AuCl4·4H2O is added, the reaction is stirred in an ice bath, then an aqueous solution of NaBH4 is added, the mixture is stirred and magnetically separated, washed and dried to obtain Fe3O4@Au.
5. The dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt according to claim 1, characterized in that, The preparation method of Fe3O4-NH2 is as follows: dried Fe3O4 is dispersed in ethanol, ultrasonically treated, APTES is added and reacted in an oil bath, magnetic separation is performed after reaction, and Fe3O4-NH2 is obtained after washing with ethanol and drying.
6. The dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt according to claim 1, characterized in that, The preparation method of SiO2@cDNA-FAM is as follows: SiO2-COOH is dispersed in pure water, and cDNA-FAM, NHS, and EDC are added to activate the carboxyl groups to obtain SiO2@cDNA-FAM; the SiO2-COOH is obtained by SiO2 carboxylation modification.
7. The application of the dual-signal aptamer sensor based on SiO2 and Fe3O4@Au@Pt as described in claim 1 in the detection of acetamiprid.
8. The application according to claim 7, characterized in that, Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM were used as recognition probes. After reacting with the sample, magnetic separation was performed, and the fluorescence intensity of the supernatant was detected. The precipitate was resuspended in PBS for colorimetric analysis, and the absorbance value at 450 nm was measured.
9. The application according to claim 8, characterized in that, The volume ratio of Fe3O4@Au@Pt@Apt and SiO2@cDNA-FAM was 1:2; the quenching time was 60 min; and the ACE incubation time was 80 min.
10. The application according to claim 9, characterized in that, The sequences of the aptamer and the fluorescently labeled complementary strand are as follows: SH-(CH2)6-TGT AAT TTG TCT GCA GCG GTT CTT GAT CGC TGA CAC CAT ATT ATG AAGA; NH2-(CH2)6-CAT AAT ATG GTG TCA GCG-FAM.