A dual-mode rapid detection method for acrylamide using Fe / PDA-Au-janusAg@AuNPs combined with aptamers

Abstract

The present invention discloses a method for rapid detection of acrylamide by dual-mode Fe / PDA-Au-janusAg@AuNPs combined with an aptamer. The method synthesizes Au-Ag Janus nanoparticles were further reduced with sodium borohydride to prepare Au‑janusAg@AuNPs, and Fe / PDA and Au‑janusAg@AuNPs complexes (Fe / PDA‑Au‑janusAg@AuNPs) were prepared by self-assembly. The complex was incubated with acrylamide aptamer to obtain Fe / PDA‑Au‑janusAg@AuNPs‑Apt, which has the dual activity of mimicking enzymes and increasing surface enhanced Raman scattering (SERS). In the presence of hydrogen peroxide, the colorless substrate 3,3',5,5'‑tetramethylbenzidine (TMB) was oxidized to the SERS-active oxidized blue oxTMB, while the addition of glutathione (GSH) inhibited the oxidation of TMB. Then, based on the thiol‑ene Michael addition reaction between GSH and acrylamide (AA), the increase in AA concentration accelerated the oxidation of TMB and restored the deep blue color. Based on this principle, a new selective and strong AA colorimetric-SERS detection method was established, with a detection limit of 0.012µg / kg, which can meet the relevant national food safety requirements.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis and detection, and in particular to a method for rapid detection of acrylamide by dual-mode using Fe / PDA-Au-janusAg@AuNPs combined with an aptamer. Background Art

[0002] Acrylamide (AA, C3H5NO) is a nerve agent and a replicative toxin that is commonly produced in thermally processed foods. In 1994, the International Agency for Research on Cancer (IARC) classified AA as "category 2A" (possibly carcinogenic to humans); at the same time, in 2002, the European Commission classified AA as a category 2 carcinogen and category 2 mutagen. High concentrations of AA have been detected in various thermally processed foods, such as potato chips, breakfast cereals, and coffee. Considering the toxicity of AA, it is essential to develop simple and efficient methods to sensitively detect AA in thermally processed foods. At present, the determination methods of AA mainly include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), high performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA). Enzyme-based colorimetric detection methods show excellent sensitivity and strong operability due to their inherent exponential signal amplification. However, natural enzymes are often degraded and have the characteristics of high cost and instability, which limits their application. Compared with natural enzymes, nanomaterials with enzyme-like properties (nanozymes) have the advantages of high environmental tolerance, low cost, and flexible structural design, which has aroused great research interest.

[0003] Surface-enhanced Raman spectroscopy (SERS) is a powerful fingerprint spectroscopy technique that can detect small molecules at the single-molecule level without complex sample processing procedures. One of the two widely accepted theories of the enhancement effect is the electromagnetic mechanism, which predicts that the local amplification of the electromagnetic field originates from the unique plasmon coupling between adjacent nanoparticles. Therefore, SERS usually requires noble metal nanoparticles with strong surface plasmon resonance (SPR) effect as substrate materials. However, the SERS signal of a single nanoparticle is too weak to be widely used in ultrasensitive detection. Au-Ag Janus NPs can integrate the advantages of both materials in one nanostructure, but overly large nanoparticles are prone to aggregation, large Ag islands are easily oxidized, which affects the stability of the nanoparticles, and the SERS intensity of Au-Ag Janus nanoparticles is only limited. The study of combining nanozymes with SERS for analytical detection provides great application space.

[0004] The present invention uses a metal-ligand cross-linking method to prepare a chrysanthemum-shaped Fe / PDA coordination polymer, uses sodium citrate as a reducing agent and a template to prepare gold nanoparticles (AuNPs), and synthesizes Au-Ag under the mediation of 2-mercaptobenzimidazole-5-carboxylic acid (MBIA). Janus nanoparticles were further reduced with sodium borohydride to prepare Au-janusAg@AuNPs, and Fe / PDA and Au-janusAg@AuNPs complexes (Fe / PDA-Au-janusAg@AuNPs) were prepared by self-assembly. The complex was incubated with acrylamide aptamer to prepare Fe / PDA-Au-janusAg@AuNPs-Apt. Fe / PDA-Au-janusAg@AuNPs-Apt has the dual activity of mimicking enzymes and increasing surface enhanced Raman scattering (SERS). In the presence of hydrogen peroxide, the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) was oxidized to the surface enhanced Raman spectroscopy active oxidized blue oxTMB, while the addition of glutathione (GSH) inhibited the oxidation of TMB. Then, based on the thiol-ene Michael addition reaction between glutathione and acrylamide (AA), the increase in AA concentration accelerated the oxidation of TMB and restored the deep blue color. Based on this principle, a new highly sensitive and selective AA colorimetric-SERS detection method was established, with a detection limit of 0.012 µg / kg, which can meet the relevant national food safety requirements. In the actual sample determination, the introduction of aptamers improved the specificity of the method. This method has the characteristics of simple operation, high sensitivity and rapidity. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for rapid detection of acrylamide by dual mode of Fe / PDA-Au-janusAg@AuNPs combined with aptamers, and utilizes the principle of thiol-ene Michael addition reaction between glutathione and acrylamide (AA) and the increase of AA concentration to accelerate the oxidation of TMB, thereby establishing a colorimetric and SERS rapid detection method of AA.

[0006] The method of the present invention for rapid detection of acrylamide by dual-mode Fe / PDA-Au-janusAg@AuNPs combined with aptamers is as follows:

[0007] (1) Mix 20-30 mL of ethylene glycol, 5-10 mL of ammonia water, and 10-20 mL of ultrapure water, place in a 60-70°C water bath and stir for 10-15 min, drop 20-40 mL of 50 mg / mL dopamine hydrochloride solution into the mixture while stirring, then slowly add 25-35 mL of 18-20 mg / mL ferric nitrate solution, react at 60-70°C for 10-12 h, cool naturally to room temperature after the reaction is completed, centrifuge at 8000-10000 r / min for 5-10 min, and wash the solid with ethanol and water alternately for 3-4 times to obtain Fe / PDA;

[0008] (2) Add 200-300 μL of 0.05 mol / L chloroauric acid to 50-60 mL of pure water, heat to boiling, add 100-150 μL of 1 mol / L sodium citrate, heat and boil for 15-20 min, and cool naturally to room temperature to obtain AuNPs;

[0009] (3) Add 100-150 μL of 1 mmol / L 2-mercaptobenzimidazole-5-carboxylic acid MBIA to 5-6 mL of AuNPs solution, stir evenly, incubate in a 60°C water bath for 2-3 h, cool to room temperature, slowly drop 150-200 μL of 10 mmol / L hydroquinone and 150-200 μL of 1 mmol / L AgNO3 under vigorous stirring, shake well, and let stand for 4-5 h to obtain Au-Ag-Janus NPs; then add 20-50 μL of 0.05 mol / L chloroauric acid under stirring, add 20-50 μL of 0.05 mol / L sodium borohydride aqueous solution, and continue stirring for 4-5 h to obtain Au-janusAg@AuNPs;

[0010] (4) Mix 0.5-1 mL of 5 mg / mL Fe / PDA dispersion with 2-5 mL of 0.5 mg / mL Au-janusAg@AuNPs and stir at room temperature for 12-15 h to obtain Fe / PDA-Au-janusAg@AuNPs nanozyme;

[0011] (5) Mix 400-500 μL Fe / PDA-Au-janusAg@AuNPs with 10-15 μL acrylamide aptamer and incubate at 37°C for 12-15 h to obtain the nanozyme and aptamer complex Fe / PDA-Au-janusAg@AuNPs-Apt;

[0012] (6) Add glutathione GSH solution to the acrylamide AA standard solution, adjust the solution pH to 12 with 0.1 mol / L sodium hydroxide solution, and place in a 50°C water bath for 40-60 min to obtain GSH-AA reactant. Add pH 4 acetate buffer solution, then add Fe / PDA-Au-janusAg@AuNPs-Apt, TMB solution and H2O2, and place for 5-10 min to generate blue oxTMB. Then use a portable Raman instrument and UV photometer to perform Raman spectroscopy and absorbance detection on the mixture, respectively. According to the molecular structure of oxTMB and the assignment of Raman peaks, the peak at 1609 cm -1 The characteristic peak at 654 nm and the wavelength of 654 nm were used as the basis for the detection of AA by surface enhanced Raman scattering spectroscopy and photometry, and the linear relationship between AA concentration and the peak area and absorbance of the characteristic peak was determined.

[0013] (7) Take the sample solution of the AA to be tested and determine the AA in the sample in the same manner as in step (6).

[0014] Preferably, the acrylamide aptamer nucleotide sequence is shown in SEQ ID NO: 1 (5'-ACC GCA TCA TGCCGA AAG GAC TAC CGG AAA CGG CAA ATC CTC G-3').

[0015] The concentration of the GSH solution is 1 mg / mL, and the amount added is 10-30 μL; the concentration of the acetate buffer solution at pH 4 is 0.1 mol / L, and the amount added is 1-2 mL; the amount added of the Fe / PDA-Au-janusAg@AuNPs-Apt solution is 20-30 μL; the concentration of the TMB solution is 5 mmol / L, and the amount added is 100-150 μL; the concentration of H2O2 is 50 mmol / L, and the amount used is 50-100 μL.

[0016] Advantages and technical effects of the present invention:

[0017] 1. The Fe / PDA coordination polymer prepared by the metal-ligand cross-linking strategy of the present invention is 3+ By changing the dielectric constant of the reaction solution, a daisy-shaped Fe / PDA polymer was prepared. 3+ The PDA polymer can be considered as the active center and cofactor of Fe / PDA, showing ultra-high peroxidase-like activity.

[0018] 2. The AuNPs prepared by the present invention can form abundant "hot spots" due to the presence of interstitial metal nano-islands. Due to the strong plasma coupling effect between Au dots and Au-janusAg NPs, Au-janusAg@AuNPs exhibit enhanced SERS activity. Due to the daisy-like structure of Fe / PDA, it is compounded with Au-janusAg@AuNPs to obtain a complex with a mimetic enzyme and higher SERS activity. In the presence of H2O2, the colorless substrate 3,3',5,5'-tetramethylbenzidine (TMB) is oxidized to oxTMB with surface enhanced Raman spectroscopy activity, while the addition of glutathione (GSH) inhibits the oxidation of TMB. Then, based on the thiol-ene Michael addition reaction between GSH and AA, the increase in AA concentration accelerates the oxidation of TMB and restores the deep blue color. Based on this principle, a new method of highly sensitive and selective AA colorimetric-SERS detection is established.

[0019] 3. The acrylamide detection method established by the present invention has high detection sensitivity and a detection limit of 0.012 µg / kg, which can meet the relevant national food safety requirements. The introduction of aptamers eliminates the interference of coexisting substances, and the method has good selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 SEM images of Fe / PDA (a) and Fe / PDA-Au-janusAg@AuNPs prepared in Example 1 of the present invention (b);

[0021] Figure 2 The ultraviolet absorption spectra of (Fe / PDA+TMB+H2O2) and (Fe / PDA-Au-janusAg@AuNPs-Apt+TMB +H2O2) in Example 1 of the present invention;

[0022] Figure 3 The ultraviolet absorption spectra of nanozyme + TMB + H2O2, nanozyme + TMB + H2O2 + AA, nanozyme + TMB + H2O2 + GSH and nanozyme + TMB + H2O2 + GSH + AA in Example 1 of the present invention are shown;

[0023] Figure 4 The colorimetric detection AA absorption spectrum and regression equation of the Fe / PDA-Au-janusAg@AuNPs-Apt+TMB+H2O2+GSH system in Example 1;

[0024] Figure 5The SERS detection AA absorption spectrum and regression equation of the Fe / PDA-Au-janusAg@AuNPs-Apt+TMB+H2O2+GSH system in Example 1;

[0025] Figure 6 The results of the influence of coexisting substances on the colorimetric detection of AA in Example 1;

[0026] Figure 7 This is the result of the effect of coexisting substances on SERS detection of AA in Example 1. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0028] Example 1: Determination of AA in roasted coffee

[0029] 1. Preparation of Fe / PDA-Au-janusAg@AuNPs-Apt nanozyme

[0030] (1) 20 mL of ethylene glycol, 5 mL of ammonia water, and 10 mL of ultrapure water were mixed and placed in a 60°C water bath and stirred for 15 min. 20 mL of 50 mg / mL dopamine hydrochloride solution was added dropwise under stirring, followed by slow addition of 25 mL of 18 mg / mL ferric nitrate solution. The mixture was reacted at 60°C for 12 h. After the reaction was completed, the mixture was naturally cooled to room temperature and centrifuged at 8000 r / min for 10 min. The solid was washed alternately with ethanol and water for 3-4 times to obtain Fe / PDA. SEM characterization showed that the synthesized nanomaterial had a similar daisy-like structure ( Figure 1 a).

[0031] (2) Add 200 μL of 0.05 mol / L chloroauric acid into 50 mL of pure water, heat to boiling, add 100 μL of 1 mol / L sodium citrate, heat and boil for 15 min, and naturally cool to room temperature to obtain AuNPs.

[0032] (3) Add 100 μL of 1 mmol / L 2-mercaptobenzimidazole-5-carboxylic acid MBIA to 5 mL of AuNPs solution, stir evenly, incubate in a 60°C water bath for 2 h, cool to room temperature, slowly drop 150 μL of 10 mmol / L hydroquinone and 150 μL of 1 mmol / L AgNO3 under vigorous stirring, shake well, and let stand for 4 h. Au-Ag-Janus NPs are obtained; then 20 μL of 0.05 mol / L chloroauric acid is added under stirring, and 20 μL of 0.05 mol / L sodium borohydride aqueous solution is added, and stirring is continued for 4 h to obtain Au-janusAg@AuNPs.

[0033] (4) 0.5 mL of 5 mg / mL Fe / PDA dispersion was mixed with 3 mL of 0.5 mg / mL Au-janusAg@AuNPs and stirred at room temperature for 12 h to obtain Fe / PDA-Au-janusAg@AuNPs nanozyme. SEM characterization showed that the synthesized nanozyme had a chrysanthemum shape similar to that of Fe / PDA. This structure showed more SERS "hot spots" ( Figure 1 b).

[0034] (5) Take 400 μL Fe / PDA-Au-janusAg@AuNPs and mix it with 10 μL acrylamide aptamer. The preferred acrylamide aptamer sequence is shown in SEQ ID NO: 1 (5'-ACC GCA TCA TGC CGA AAG GAC TAC CGG AAA CGGCAA ATC CTC G-3'). Incubate at 37 °C for 12 h to obtain the nanozyme and aptamer complex Fe / PDA-Au-janusAg@AuNPs-Apt.

[0035] 2. Evaluation of peroxidase activity of Fe / PDA-Au-janusAg@AuNPs-Apt nanozyme

[0036] (1) Nanozyme peroxidase activity: Take 100 μL of 5 mmol / L TMB, add 0.1 mg / mL Fe / PDA or Fe / PDA-Au-janusAg@AuNPs-Apt 100 μL and 50 mmol / L H2O2 100 μL, shake well, let stand for 10 min, and measure the absorbance at 654 nm. The results are as follows: Figure 2 , Fe / PDA-Au-janusAg@AuNPs-Apt exhibited stronger peroxidase-like activity;

[0037] (2) Effect of AA on the activity of nanozyme peroxidase: Take 10 μL of 1 mg / mL glutathione, add 1 mL of deionized water, adjust the solution pH to 12 with 0.1 mol / L sodium hydroxide solution, add 10 μL of 1 μg / mL acrylamide standard solution, and place in a 50°C water bath for 50 min to obtain GSH-AA reactant; add 100 μL of 0.1 mg / mL Fe / PDA-Au-janusAg@AuNPs-Apt, 1 mL of 0.1 mol / L pH 4 acetate buffer solution, 100 μL of 50 mmol / L H2O2, and 100 μL of 5 mmol / L TMB, shake well and react for 10 min, add deionized water to 4 mL, and measure the absorbance at a wavelength of 654 nm. The results are shown in Figure 2. Figure 3, GSH inhibited the peroxidase-like activity of the nanozyme, while AA restored the peroxidase-like activity of the nanozyme.

[0038] 3. Preparation of AA working curve: Take 10 μL of 1 mg / mL glutathione, add 1 mL of deionized water, adjust the solution pH to 12 with 0.1 mol / L sodium hydroxide solution, add 0.05-10 μg / L acrylamide standard solution, place in a 50℃ water bath for 50 min to obtain GSH-AA reactant; add 100 μL of 0.1 mg / mL Fe / PDA-Au-janusAg@AuNPs-Apt, 1 mL of 0.1 mol / L pH 4 acetate buffer solution, 100 μL of 50 mmol / L H2O2 and 100 μL of 5 mmol / L TMB, shake well and react for 10 min, add deionized water to 3 mL, measure the absorbance at a wavelength of 654 nm, plot the standard curve with AA concentration as the horizontal axis and absorbance A as the vertical axis, and obtain the regression equation, see Figure 4 ; The regression equation, correlation coefficient, relative standard deviation, linear range, etc. are shown in Table 1;

[0039] At the same time, 10 μL of 0.4-20 μg / L acrylamide standard solution was taken and placed in a 50℃ water bath for 50 min to obtain GSH-AA reactant; 100 μL of 0.1 mg / mL Fe / PDA-Au-janusAg@AuNPs-Apt, 1 mL of 0.1 mol / L pH 4 acetate buffer solution, 100 μL of 50 mmol / L H2O2 and 100 μL of 5 mmol / L TMB were added, and the mixture was reacted for 10 min after shaking. Deionized water was added to 3 mL, and the mixture was scanned for 10 s under the conditions of 785 nm excitation light and 500 mW laser power. The mixed solution was detected by Raman spectroscopy using a portable Raman instrument. The Raman spectrum is shown in the figure. Figure 5 ; With AA concentration as the horizontal axis, at 1609 cm -1 The peak area of ​​the characteristic peak is used as the ordinate, and the standard curve is drawn to obtain the regression equation, see Table 1.

[0040]

[0041] 4. Method specificity investigation: The selectivity of AA determination was investigated. AA and other possible coexisting substances were mixed to detect the effect of coexisting substances on the detection system. The concentration of AA was 100 ng / kg and the concentration of other interfering substances was 500 ng / kg. Figure 6 , 7The results of the effects of coexisting substances (acrylic acid (Aa), L-asparagine (Asn), caffeic acid (CA), fumaric acid (FA), methacrylamide (MA), maleic acid (MaA), propionic acid (PA), potassium sorbate (PS), succinic acid (SA), glucose (Glu), starch (Sta), and glycine (Gly)) on AA. As can be seen from the figure, only AA has a significant enhancing effect on the catalytic activity of the nanozyme, while other substances have almost no effect. The method has good selection specificity.

[0042] 5. Determination of AA in roasted coffee

[0043] (1) Sample treatment: The roasted coffee sample was ground into powder and pretreated according to the QuEChERS method as follows: 3.0 g of powder was added to a centrifuge tube together with 20 mL of acetonitrile and 10 mL of deionized water and stirred rapidly for 5 min. The QuEChERS mixture (4 g MgSO4 and 0.5 g NaCl) was transferred to a centrifuge tube and ultrasonically treated for 10 min to obtain a uniform mixture. The mixed solution was centrifuged at 10,000 rpm for 8 min to obtain a supernatant, which was completely dried at 40 °C using a nitrogen flow to obtain the extracted AA. The extracted AA was redispersed in 1 mL of deionized water and filtered with a 0.22 μM organic membrane to obtain the test solution.

[0044] (2) Colorimetric determination of AA in roasted coffee: Take 10 μL of 1 mg / mL glutathione, add 1 mL of deionized water, adjust the solution pH to 12 with 0.1 mol / L sodium hydroxide solution, add 500 μL of the test solution prepared in step (1), and place in a 50°C water bath for 50 min to obtain GSH-AA reactant; add 100 μL of 0.1 mg / mL Fe / PDA-Au-janusAg@AuNPs-Apt, 1 mL of 0.1 mol / L pH 4 acetate buffer solution, 100 μL of 50 mmol / L H2O2, and 100 μL of 5 mmol / L TMB, shake well and react for 10 min, add deionized water to 4 mL, measure the absorbance at a wavelength of 654 nm, and substitute into the regression equation. The AA content is 6.52 μg / g.

[0045] (3) SERS determination of AA in roasted coffee: 20 μL of the test solution prepared in step (1) was pipetted into a 96-well plate, and 20 μL of 0.1 mol / L pH 4 acetate buffer solution, 10 μL of 0.1 mg / mL Fe / PDA-Au-janusAg@AuNPs-Apt, 5 μL of 50 mmol / L H2O2, and 10 μL of 5 mmol / L TMB were added. After mixing, the reaction was continued for 5-10 min. The sample was scanned for 10 s at 785 nm excitation light and 500 mW laser power. A portable Raman spectrometer was used to analyze the sample at 1609 cm -1 The mixed solution was detected by Raman spectroscopy and substituted into the regression equation, the content of AA was 6.98 μg / g.

[0046] Example 2: Determination of AA in potato chips and cookie samples

[0047] 1. Preparation of Fe / PDA-Au-janusAg@AuNPs-Apt nanozyme

[0048] (1) Mix 30 mL of ethylene glycol, 10 mL of ammonia water, and 20 mL of ultrapure water, place in a 70°C water bath and stir for 10 min. Add 40 mL of 50 mg / mL dopamine hydrochloride solution dropwise while stirring, and then slowly add 35 mL of 20 mg / mL ferric nitrate solution. React at 70°C for 10 h. After the reaction is completed, cool naturally to room temperature, centrifuge at 10,000 r / min for 5 min, and wash the solid alternately with ethanol and water for 3-4 times to obtain Fe / PDA.

[0049] (2) Add 300 μL of 0.05 mol / L chloroauric acid into 60 mL of pure water and heat to boiling. Add 150 μL of 1 mol / L sodium citrate and boil for 20 min. Then cool naturally to room temperature to obtain AuNPs.

[0050] (3) Add 150 μL of 1 mmol / L 2-mercaptobenzimidazole-5-carboxylic acid MBIA to 6 mL of AuNPs solution, stir evenly, incubate in a 60°C water bath for 3 h, cool to room temperature, slowly drop 200 μL of 10 mmol / L hydroquinone and 200 μL of 1 mmol / L AgNO3 under vigorous stirring, shake well, and let stand for 5 h. Au-Ag-Janus NPs are obtained; then, 50 μL of 0.05 mol / L chloroauric acid is added under stirring, and 50 μL of 0.05 mol / L sodium borohydride aqueous solution is added, and stirring is continued for 5 h to obtain Au-janusAg@AuNPs.

[0051] (4) Mix 1 mL of 5 mg / mL Fe / PDA dispersion with 5 mL of 0.5 mg / mL Au-janusAg@AuNPs and stir at room temperature for 15 h to obtain Fe / PDA-Au-janusAg@AuNPs nanozyme.

[0052] (5) Mix 500 μL Fe / PDA-Au-janusAg@AuNPs with 15 μL acrylamide aptamer and incubate at 37 °C for 15 h to obtain the nanozyme and aptamer complex Fe / PDA-Au-janusAg@AuNPs-Apt.

[0053] 2. Preparation of AA working curve: same as in Example 1.

[0054] 3. Determination of AA in potato chips and cookies samples

[0055] (1) Sample processing: same as in Example 1.

[0056] (2) Sample determination: As in Example 1, the AA contents of potato chips and cookie samples detected by colorimetry were 7.19 μg / g and 3.20 μg / g, respectively. The AA contents of potato chips and cookie samples detected by SERS were 7.64 μg / g and 3.11 μg / g, respectively.

[0057] The samples of Example 1 and Example 2 were subjected to recovery and precision experiments: 3 different concentrations of AA standard solutions were added to the samples respectively; each concentration was measured in parallel 3 times, the spiked recovery was calculated, and the relative standard deviation RSD was calculated, and the results are shown in Table 2; the spiked recovery of AA was 93.7% to 106.1%, and the RSD was 1.23% to 4.89%. This method has good accuracy and precision.

[0058]

[0059] The AA determination method established in the present invention has the advantages of fewer processing steps, shorter time, lower processing cost, easier operation, and no need for large-scale instruments and equipment, thus having great advantages in actual detection.

Claims

1. A method for rapid detection of acrylamide by dual-mode Fe / PDA-Au-janusAg@AuNPs combined with aptamers, characterized in that: The following steps are involved: (1) 20-30 mL of ethylene glycol, 5-10 mL of aqueous ammonia, and 10-20 mL of ultrapure water were mixed, placed in a 60-70° C. water bath and stirred for 10-15 min, 20-40 mL of a 50 mg / mL dopamine hydrochloride solution was added dropwise under stirring, and then 25-35 mL of a 18-20 mg / mL ferric nitrate solution was slowly added, and the mixture was reacted at 60-70° C. for 10-12 h. After the reaction was completed, the mixture was naturally cooled to room temperature, centrifuged at 8000-10000 r / min for 5-10 min, and the solid was washed alternately with ethanol and water for 3-4 times to obtain Fe / PDA; (2) adding 200-300 μL of 0.05 mol / L chloroauric acid to 50-60 mL of pure water, heating to boiling, adding 100-150 μL of 1 mol / L sodium citrate, heating and boiling for 15-20 min, and naturally cooling to room temperature to obtain AuNPs; (3) Add 100-150 μL of 1 mmol / L 2-mercaptobenzimidazole-5-carboxylic acid MBIA to 5-6 mL of AuNPs solution, stir evenly, incubate in a 60°C water bath for 2-3 h, cool to room temperature, slowly drop 150-200 μL of 10 mmol / L hydroquinone and 150-200 μL of 1 mmol / L AgNO3 under vigorous stirring, shake well, and let stand for 4-5 h to obtain Au-Ag-Janus NPs; then add 20-50 μL of 0.05 mol / L chloroauric acid under stirring, add 20-50 μL of 0.05 mol / L sodium borohydride aqueous solution, and continue stirring for 4-5 h to obtain Au-janusAg@AuNPs; (4) Mix 0.5-1 mL of 5 mg / mL Fe / PDA dispersion with 2-5 mL of 0.5 mg / mL Au-janusAg@AuNPs, and stir at room temperature for 12-15 h to obtain Fe / PDA-Au-janusAg@AuNPs nanozyme; (5) 400-500 μL Fe / PDA-Au-janusAg@AuNPs was mixed with 10-15 μL acrylamide aptamer, and incubated at 37°C for 12-15 h to obtain the nanozyme and aptamer complex Fe / PDA-Au-janusAg@AuNPs-Apt, and the nucleotide sequence of the acrylamide aptamer was as shown in SEQ ID NO: 15'-ACC GCA TCA TGC CGA AAG GAC TAC CGG AAA CGG CAA ATCCTC G-3'; (6) Glutathione GSH solution was added to the acrylamide AA standard solution, and the solution pH was adjusted to 12 with 0.1 mol / L sodium hydroxide solution. The solution was placed in a 50°C water bath for 40-60 min to obtain GSH-AA reactant. A pH 4 acetate buffer solution was added, and then Fe / PDA-Au-janusAg@AuNPs-Apt, TMB solution and H2O2 were added. The mixture was placed for 5-10 min to generate blue oxTMB. The mixture was then subjected to Raman spectroscopy and absorbance detection using a portable Raman spectrometer and a UV photometer, respectively. According to the molecular structure of oxTMB and the Raman peak position, the peak at 1609 cm -1 The characteristic peak at 654nm and the wavelength of 654nm were used as the basis for the detection of AA by surface enhanced Raman scattering spectroscopy and photometry, and the linear relationship between AA concentration and the peak area and absorbance of the characteristic peak was determined; (7) Take the sample solution of AA to be tested and determine the AA in the sample in the same manner as in step (6).

2. The method according to claim 1, characterized in that: The concentration of GSH solution is 1 mg / mL, and the amount added is 10-30 μL; the concentration of pH 4 acetate buffer solution is 0.1 mol / L, and the amount added is 1-2 mL; the amount added of Fe / PDA-Au-janusAg@AuNPs-Apt solution is 20-30 μL; the concentration of TMB solution is 5 mmol / L, and the amount added is 100-150 μL; the concentration of H2O2 is 50 mmol / L, and the amount used is 50-100 μL.

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