CN114460072B - Colorimetric detection method for kanamycin based on nano enzyme and application thereof - Google Patents
Colorimetric detection method for kanamycin based on nano enzyme and application thereof Download PDFInfo
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- 229930027917 kanamycin Natural products 0.000 title claims abstract description 70
- 229960000318 kanamycin Drugs 0.000 title claims abstract description 70
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- 229930182823 kanamycin A Natural products 0.000 title claims abstract description 70
- 238000001514 detection method Methods 0.000 title claims abstract description 30
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- 238000000034 method Methods 0.000 claims abstract description 26
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 10
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
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- 229930182566 Gentamicin Natural products 0.000 description 4
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 4
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- 238000004737 colorimetric analysis Methods 0.000 description 3
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- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 2
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- PANJMBIFGCKWBY-UHFFFAOYSA-N iron tricyanide Chemical compound N#C[Fe](C#N)C#N PANJMBIFGCKWBY-UHFFFAOYSA-N 0.000 description 2
- UETZVSHORCDDTH-UHFFFAOYSA-N iron(2+);hexacyanide Chemical compound [Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] UETZVSHORCDDTH-UHFFFAOYSA-N 0.000 description 2
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a colorimetric detection method of kanamycin based on nano enzymeAnd the application thereof, which coats potassium ferricyanide inside mesoporous silica, and the aptamer is adsorbed on the surface of the mesoporous silica as biological gating, wherein the specific recognition reaction of kanamycin as a target substance and the aptamer leads the aptamer to be separated from the surface of the mesoporous silica, so that the potassium ferricyanide is released into solution from the inside of the mesoporous silica, thereby in CuWO 4 The surface of the nano-particle is combined to generate nano-enzyme to induce a color reaction; during the period, the aptamer in the combined product of kanamycin and the aptamer is sheared by deoxyribonuclease I, kanamycin is released into the solution again, a new round of target/aptamer combination reaction and release of potassium ferricyanide are induced again, and the detection signal is amplified; the method for detecting kanamycin does not need a biological molecular marker and is fixed, and the method is simple and convenient to operate, low in cost and good in selectivity.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a colorimetric detection method for kanamycin based on nano-enzyme and application thereof.
Background
Kanamycin is an important antibiotic for treating and preventing microbial infection, but excessive use can cause a series of adverse reactions including ototoxicity, renal toxicity, anaphylactic shock and the like. The European Union has specified the maximum residual amount of kanamycin in foods. Therefore, detection of kanamycin is of great significance for food safety supervision. Currently, kanamycin is mainly detected by a microbiological method, a chromatographic method and an enzyme-linked immunosorbent assay. However, these methods often require expensive instrumentation, are time consuming to operate, require specialized personnel to operate, are not suitable for screening large volumes of samples, and the like. In contrast, the colorimetric method has the advantages of simplicity, low cost, visual recognition and the like, and has potential advantages in screening of large-scale samples.
The nano enzyme is a nano material with enzyme-like catalytic activity, and researches show that the nano enzyme has the advantages of low price, easy acquisition, more stable property, easy storage, easy activity regulation and control and the like compared with the natural enzyme, and is hopeful to replace the natural enzyme in the fields of catalysis, bacteriostasis, biological sensing, disease treatment and the likeThe domain functions. In terms of biosensing, zhu et al [ Xu Zhu, lin Tang, jiajia Wang, bo Peng, xilian Ouyang, jisui Tan, jiangafang Yu, haopeng Feng, jialin Tang. Sensors&Actuators:B.Chemical 330(2021)129318]By CeO 2 BN quantum dots are fixed on the surface of the nanorods to form nano enzyme, so that high-sensitivity detection of kanamycin is realized. However, this method is a signal-weakening type detection, and is susceptible to interference by various factors, producing false positive signals. In addition, gold nanoparticles [ Tarun Kumar Sharma, rajesh Ramanathan, pabudi Weerathunge, mahsa Mohammadtaheri, hemant Kumar Daima, ravi Shukla and Vipul bansal.chem.Commun.,2014,50,15856-15859 have also been reported]Or WS2 nanorods [ Yue Tang, yang Hu, pei Zhou, chunxiao Wang, han Tao, and Yuangen Wu J. Agric. Food chem.2021,69,2884-2893 ]]As the nano-enzyme, the combination of kanamycin and the nucleic acid aptamer is utilized to lead the dissociation of the nucleic acid aptamer on the surface of the nano-enzyme, thereby affecting the activity of the nano-enzyme to realize the determination of kanamycin. However, these methods have not had a signal amplification reaction other than the nanoenzyme reaction, and the sensitivity of measurement was not high enough. In addition, as the nucleic acid aptamer can be irreversibly combined on the surface of the nano-enzyme, the target kanamycin and the aptamer cannot be successfully combined, so that the measurement effect is affected.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a colorimetric detection method for kanamycin based on nano-enzyme and application thereof. The invention is disclosed in CuWO 4 In-situ formation of copper hexacyanoferrate (CuHCF) nanoenzyme on nanoparticle surface, which can be used for preparing the nanoparticle by CuWO 4 The direct reaction of the nanoparticle with potassium ferricyanide in solution results in rapid production. Through designing to coat potassium ferricyanide inside mesoporous silica, adsorbing a nucleic acid aptamer on the surface of mesoporous silica as biological gating, and specifically identifying target kanamycin and the nucleic acid aptamer to react to cause the gating of the nucleic acid aptamer to be separated from the surface of the mesoporous silica, so that the potassium ferricyanide is released into solution from the inside of the mesoporous silica, thereby in CuWO 4 The nanoparticle surface binds to produce a nanoenzyme to induce a chromogenic reaction. During this period, through the deoxidizing nucleusThe aptamer in the combined product of kanamycin and the aptamer is sheared by the sugar nuclease I (DNase I), the kanamycin is released into the solution again, a new round of target/aptamer combination reaction and release of potassium ferricyanide are induced again, and the detection signal is amplified.
The technical scheme of the invention is as follows:
a method for colorimetric detection of kanamycin based on nano-enzymes, comprising the following steps:
(1)CuWO 4 synthesis of nano materials: preparation of CuWO by conventional methods 4 A nanomaterial powder;
(2) Synthesizing PMSN: preparing mesoporous silica MSN, and then carrying out surface modification on the MSN by using silane containing amino groups to obtain positively charged MSN, namely PMSN;
(3) Preparation of Supports [ Fe (CN) 6 ] 3- PMSN of aptamer-coated of (a): dispersing the prepared PMSN into K 3 [Fe(CN) 6 ]Adding KANA aptamer into the solution after incubation, and packaging to obtain load [ Fe (CN) 6 ] 3- Is a suspension of aptamer-coated PMSN; KANA aptamer sequence SEQ ID No. 1= 5'-TGG GGG TTG AGG CTA AGC CGA-3';
(4) Measurement of absorbance: the above-mentioned load [ Fe (CN) 6 ] 3- Mixing the aptamer-coated PMSN suspension of (2) with kanamycin solutions with different known concentrations, and adding DNase I reaction buffer solution and DNase I for reaction after incubation; followed by addition of CuWO prepared in step (1) 4 Suspension of nanomaterial powder, sequentially adding HAc-NaAc buffer solution, TMB and H 2 O 2 Measuring absorbance at 640-660nm after the reaction is completed;
(5) Construction of a linear model: an absorbance value A obtained by the step (4) of a sample having a kanamycin concentration of 0 and an absorbance value A obtained by the step of obtaining an absorbance value A of a different known kanamycin concentration 0 Calculating to obtain absorbance difference A-A corresponding to different concentrations 0 The method comprises the steps of carrying out a first treatment on the surface of the Then constructing a linear model by utilizing the logarithmic value of different known kanamycin concentrations and the corresponding absorbance difference value;
(6) Determination of the concentration of unknown kanamycin solution Using the linear model obtained in step (5), the concentration of unknown kanamycin solution was determined by repeating step (4).
Further, in step (1), the CuWO 4 The synthesis of the nano material comprises the following specific steps:
cu (NO) 3 ) 2 ·3H 2 Dissolving O in deionized water, regulating pH to 4.0-6.0 with alkali solution, and stirring at 50-80deg.C for 10-50 min; adding Na 2 WO 4 ·2H 2 O, stirring continuously at 70-100deg.C for 30-60 min; cooling to room temperature, washing with water and ethanol sequentially, drying at 50-70deg.C for 8-16 hr, and annealing at 300-500deg.C for 1-3 hr to obtain CuWO 4 Nanomaterial powder.
Further, the Cu (NO 3 ) 2 ·3H 2 O and Na 2 WO 4 ·2H 2 The mass ratio of O is 1:1-2:7.
Further, in the step (2), the synthesis of PMSN includes the following specific steps:
firstly, cetyl trimethyl ammonium bromide CTAB is dissolved in deionized water, then NaOH solution is added into the solution, and stirring is carried out for 10-50 minutes at 40-70 ℃;
then, tetraethyl orthosilicate TEOS is slowly dripped into the solution, and after stirring is carried out for 4-8 hours at 70-90 ℃, the mixture is respectively washed with water and ethanol for several times; then, drying the washed product at 50-70 ℃ for 8-16 hours, and then annealing at 500-600 ℃ for 4-8 hours to remove superfluous surfactant in the crude MSN sample, so as to obtain mesoporous silica MSN;
the MSN was then surface modified with an amine-containing silane to give a positively charged MSN, designated PMSN.
Further, the mass ratio of CTAB to TEOS is 1:4-6;
the method for carrying out surface modification on MSN comprises the following steps: dispersing MSN in absolute ethyl alcohol, carrying out ultrasonic treatment, then slowly dripping 3-aminopropyl triethoxysilane APTES, and continuously stirring for 6-10 hours at room temperature; then, the mixture was alternately washed with water and ethanol; finally, drying at 50-70 ℃ for 8-16 hours to obtain PMSN powder; the mass ratio of MSN to APTES is 1:15-25.
Further, in the step (3), a load [ Fe (CN) ] is produced 6 ] 3- The aptamer-coated PMSN of (a) comprises the following specific steps: dispersing PMSN to K 3 [Fe(CN) 6 ]Shaking the mixture at 20-30deg.C for 8-16 hr, and adding KANA aptamer to the suspension; shaking at 20-30deg.C for 2-6 hr, and washing with Tris-HCl buffer solution; subsequently, the load [ Fe (CN) 6 ] 3- The aptamer-coated PMSN of (2) is resuspended in Tris-HCl buffer; PMSN: K 3 [Fe(CN) 6 ]The mass ratio of (3) is as follows: 25-15:1; the concentration of PMSN re-suspension was 4-8mg/mL.
Further, in the step (4), the measurement of absorbance includes the following specific steps: load [ Fe (CN) 6 ] 3- Mixing the aptamer-coated PMSN suspension of (2) with kanamycin solution of different known concentrations, and incubating for 10-50 minutes at 30-40 ℃ with shaking; adding DNase I reaction buffer solution and DNase I, and continuously shaking and incubating for 0.5-2 hours at 30-40 ℃; subsequently, cuWO is added 4 The suspension is reacted for 1 to 10 minutes, and then HAc-NaAc buffer solution, TMB and H are added in sequence 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the Finally, the reaction is carried out at 40-50℃for 10-50 minutes, and the absorbance is measured at 640-660 nm.
Use of the method in food safety detection.
Kit for colorimetric detection of kanamycin based on nanoenzyme, comprising the load [ Fe (CN) in the method 6 ] 3- Is a suspension of aptamer-coated PMSN.
The application of the kit is applied to food safety detection.
The beneficial technical effects of the invention are as follows:
the invention firstly proposes the following ideas for detecting kanamycin: as shown in FIG. 1, when kanamycin as a target is present, an aptamer recognition reaction is generated to cause the aptamer to be separated from the surface of mesoporous silica, and ferricyanide ions are released from the silica so as to be bound to CuWO 4 Formed in situ on the surface of the nanoparticleThe copper hexacyanoferrate nano enzyme induces the occurrence of chromogenic reaction, and the increased absorption signal is measured; during this period, the aptamer in the aptamer/kanamycin complex is sheared by DNase I, and the target is released again, so that more aptamer is separated from the surface of mesoporous silica and more ferricyanide ions are released, and the signal is greatly enhanced. When kanamycin which is a target object does not exist, the aptamer cannot be separated from the surface of mesoporous silicon dioxide to release iron cyanide ions, and as the free iron cyanide ions in the solution are greatly reduced, nano-enzyme cannot be generated and a chromogenic reaction is induced, so that a weak absorption signal is obtained.
Compared with the prior art, the invention has the following advantages: the invention provides a colorimetric analysis method for detecting kanamycin with high sensitivity, which combines the wrapping effect of mesoporous silica and DNase I-mediated target object circulation amplification reaction to improve the sensitivity of the reaction, does not need the labeling of biomolecules and the fixation thereof during the detection, has convenient operation, low cost and good selectivity, has wide linear range (0.1-100 nM), low detection limit (3.5 pM) and has certain potential in food safety detection application.
Drawings
FIG. 1 is a schematic diagram of a nanoenzyme-based biological reaction for kanamycin detection;
FIG. 2 is a CuWO prepared in example 1 4 Scanning electron microscope pictures of nano materials;
FIG. 3 is a CuWO prepared in example 1 4 An X-ray diffraction pattern of the nanomaterial;
FIG. 4 shows the absorbance response (ΔA=A-A) obtained in example 1 0 ) A plot of linear relationship to the logarithm of kanamycin for different concentrations;
FIG. 5 is a comparison of absorbance response of the detection method obtained in example 1 for kanamycin and other possible interferents.
Wherein, the concentration of Chloramphenicol (CAP), tetracycline (TC), streptomycin (STR), gentamycin (GEN), tobramycin (TOB), doxycycline (DC), erythromycin (ERY), ciprofloxacin (CIP), norfloxacin (NFX) and Ofloxacin (OFLX) is 500nM; kanamycin (KANA) was found to be 50nM.
Detailed Description
The present invention will be described in detail below with reference to the drawings and examples.
The reagents or instruments used in the examples of the present invention are conventional reagent products commercially available, without the manufacturer's knowledge.
Example 1
A method for colorimetric detection of kanamycin based on nano-enzymes, comprising the following steps:
(1)CuWO 4 synthesis of nano materials: 0.34g Cu (NO) 3 ) 2 ·3H 2 O was dissolved in 50mL deionized water. The pH of the solution was then adjusted to 5.0 with 2.0M NaOH solution and stirred at 60℃for 30 minutes. Next, 0.60g of Na was added 2 WO 4 ·2H 2 O, stirring was continued for 40 minutes at 70 ℃. Cooling to room temperature, washing with water and ethanol for 3 times, drying at 60deg.C for 12 hr, annealing at 400deg.C for 1 hr to obtain black CuWO 4 The powder has a scanning electron microscope image shown in figure 2 and an X-ray diffraction image shown in figure 3.
As can be seen from the scanning electron microscope image of FIG. 2, the synthesized CuWO 4 The nano material has irregular morphology and the size is 50-100nm. FIG. 3 shows all diffraction peaks and triclinic CuWO 4 (JCPDS No. 72-0616) standard cards are identical. Wherein diffraction peaks at 15.2, 19.0, 28.6, 30.2, and 31.5 ° correspond to crystal planes (010), (100), (-1-11), (111), and (020).
(2) Biological reaction: mesoporous Silica (MSN) was first prepared and 0.3g cetyltrimethylammonium bromide (CTAB) was dissolved in 150mL deionized water. Then, 1.0mL of 2.0M NaOH solution was added to the above solution, and stirred at 50℃for 30 minutes. Subsequently, 1.5mL of tetraethyl orthosilicate (TEOS) was slowly dropped into the above solution. After stirring vigorously at 80℃for 6 hours, the mixture was washed 3 times with water and ethanol, respectively. The washed product was then dried at 60 ℃ for 12 hours and then annealed at 550 ℃ for 6 hours to remove excess surfactant from the crude MSN sample. The MSN was then surface modified with an amine group-containing silane to give a positively charged MSN (designated PMSN). Briefly, 50mg of MSN was dispersed in 5.0mL of absolute ethanol and sonicated for 2 hours. Subsequently, 1.0mL of 3-aminopropyl triethoxysilane (APTES) was slowly added dropwise, and stirring was continued at room temperature for 8 hours. The mixture was then washed alternately with water and ethanol. Finally, drying at 60℃for 12 hours gave PMSN as a white powder.
Next, a supported [ Fe (CN) was prepared 6 ] 3- PMSN of aptamer-coated of (a). Briefly, 30mg of the prepared PMSN was dispersed to 5.0mL of 1.0mM K 3 [Fe(CN) 6 ]In solution, the mixture at 25 ℃ gently rock 12 hours, then to the suspension added to 100 u L10 u M KANA aptamer. After 4 hours of gentle shaking at 25℃the cells were washed 3 times with Tris-HCl buffer (10 mM, 10mM NaCl, pH 7.4). Subsequently, the bottom of the centrifuge tube was loaded with [ Fe (CN) 6 ] 3- The aptamer-coated PMSN of (A) was resuspended in 5mL Tris-HCl buffer (10 mM, containing 10mM NaCl, pH 7.4).
Finally, 160. Mu.L of the loaded [ Fe (CN) 6 ] 3- The aptamer-coated PMSN suspension of (2) was mixed with 20 μl of KANA solution of different concentrations and incubated for 30 min at 37 ℃ with gentle shaking. Then 10. Mu.L of 1 XDNase I reaction buffer and 1.0U of DNase I were added and incubated with continuous shaking at 37℃for 1 hour.
(3) Measurement of absorbance: to the above mixture was added 5.0. Mu.L of 1.0mg/mL CuWO 4 The suspension was reacted for 5 minutes, then 20. Mu.L of HAc-NaAc buffer (0.2M, pH 4.0), 20. Mu.L of 5.0mM TMB and 20. Mu.L of 5.0mM H were sequentially added 2 O 2 . Finally, the reaction was carried out at 45℃for 25 minutes, and the absorbance was measured at 652 nm.
The results are shown in FIG. 4, which shows a sensitive response to KANA with a linear equation of ΔA= 0.1891 ×log [ C ] KANA ]+0.5373, linear correlation coefficient R 2 0.989, a linear range of 0.1nM to 100nM, a limit of detection of 3.5pM.
FIG. 5 shows a comparison of absorbance responses of the test methods obtained in example 1 for kanamycin and other potential interferents. Wherein, the concentration of Chloramphenicol (CAP), tetracycline (TC), streptomycin (STR), gentamycin (GEN), tobramycin (TOB), doxycycline (DC), erythromycin (ERY), ciprofloxacin (CIP), norfloxacin (NFX) and Ofloxacin (OFLX) is 500nM; kanamycin (KANA) was found to be 50nM.
Example 2
A method for colorimetric detection of kanamycin based on nano-enzymes, comprising the following steps:
(1)CuWO 4 synthesis of nano materials: 0.34g Cu (NO) 3 ) 2 ·3H 2 O was dissolved in 50mL deionized water. The pH of the solution was then adjusted to 5.0 with 2.0M NaOH solution and stirred at 60℃for 50 minutes. Next, 0.60g of Na was added 2 WO 4 ·2H 2 O, stirring was continued for 50 minutes at 80 ℃. Cooling to room temperature, washing with water and ethanol for 3 times, drying at 60deg.C for 12 hr, and annealing at 500deg.C for 1 hr to obtain black CuWO 4 And (3) powder.
(2) Biological reaction: mesoporous Silica (MSN) was first prepared and 0.3g cetyltrimethylammonium bromide (CTAB) was dissolved in 150mL deionized water. Then, 1.0mL of 2.0M NaOH solution was added to the above solution, and stirred at 50℃for 30 minutes. Subsequently, 1.5mL of tetraethyl orthosilicate (TEOS) was slowly dropped into the above solution. After stirring vigorously at 80℃for 6 hours, the mixture was washed 3 times with water and ethanol, respectively. The washed product was then dried at 60 ℃ for 12 hours and then annealed at 550 ℃ for 6 hours to remove excess surfactant from the crude MSN sample. The MSN was then surface modified with an amine group-containing silane to give a positively charged MSN (designated PMSN). Briefly, 50mg of MSN was dispersed in 5.0mL of absolute ethanol and sonicated for 2 hours. Subsequently, 1.0mL of 3-aminopropyl triethoxysilane (APTES) was slowly added dropwise, and stirring was continued at room temperature for 8 hours. The mixture was then washed alternately with water and ethanol. Finally, drying at 60℃for 12 hours gave PMSN as a white powder.
Next, a supported [ Fe (CN) was prepared 6 ] 3- PMSN of aptamer-coated of (a). In brief, 30mg of the preparationIs dispersed to 5.0mL of 1.0mM K 3 [Fe(CN) 6 ]In solution, the mixture at 25 ℃ gently rock 12 hours, then to the suspension added to 100 u L10 u M KANA aptamer. After 4 hours of gentle shaking at 25℃the cells were washed 3 times with Tris-HCl buffer (10 mM, 10mM NaCl, pH 7.4). Subsequently, the bottom of the centrifuge tube was loaded with [ Fe (CN) 6 ] 3- The aptamer-coated PMSN of (A) was resuspended in 5mL Tris-HCl buffer (10 mM, containing 10mM NaCl, pH 7.4).
Finally, 160. Mu.L of the loaded [ Fe (CN) 6 ] 3- The aptamer-coated PMSN suspension of (2) was mixed with 20 μl of KANA solution of different concentrations and incubated for 30 min at 37 ℃ with gentle shaking. Then 10. Mu.L of 1 XDNase I reaction buffer and 1.0U of DNase I were added and incubated with continuous shaking at 37℃for 1 hour.
(3) Measurement of absorbance: to the above mixture was added 5.0. Mu.L of 1.0mg/mL CuWO 4 The suspension was reacted for 5 minutes, then 20. Mu.L of HAc-NaAc buffer (0.2M, pH 4.0), 20. Mu.L of 5.0mM TMB and 20. Mu.L of 5.0mM H were sequentially added 2 O 2 . Finally, the reaction was carried out at 45℃for 25 minutes, and the absorbance was measured at 652 nm.
As can be seen from the above examples, the invention provides a colorimetric method for detecting kanamycin without labeling, fixing, rapidly and highly sensitively, which has the advantages of simple operation, low cost, good selectivity, high sensitivity and the like.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, i.e. the present invention is not limited to the above embodiments, but is capable of being modified and varied in all ways according to the following claims and the detailed description.
SEQUENCE LISTING
<110> university of Jiangnan
<120> method for colorimetric detection of kanamycin based on nano-enzyme and application thereof
<130> 2022
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial work
<400> 1
tgggggttga ggctaagccg a 21
Claims (10)
1. A colorimetric detection method for kanamycin based on nano-enzyme, which is characterized by comprising the following steps:
(1)CuWO 4 synthesis of nano materials: preparation of CuWO by conventional methods 4 A nanomaterial powder;
(2) Synthesizing PMSN: preparing mesoporous silica MSN, and then carrying out surface modification on the MSN by using silane containing amino groups to obtain positively charged MSN, namely PMSN;
(3) Preparation of Supports [ Fe (CN) 6 ] 3- PMSN of aptamer-coated of (a): dispersing the prepared PMSN into K 3 [Fe(CN) 6 ]Adding KANA aptamer into the solution after incubation, and packaging to obtain load [ Fe (CN) 6 ] 3- Is a suspension of aptamer-coated PMSN; the KANA aptamer sequence is shown as SEQ ID NO. 1;
(4) Measurement of absorbance: the above-mentioned load [ Fe (CN) 6 ] 3- Mixing the aptamer-coated PMSN suspension of (2) with kanamycin solutions with different known concentrations, and adding DNase I reaction buffer solution and DNase I for reaction after incubation; followed by addition of CuWO prepared in step (1) 4 Suspension of nanomaterial powder, sequentially adding HAc-NaAc buffer solution, TMB and H 2 O 2 Measuring absorbance at 640-660nm after the reaction is completed;
(5) Construction of a linear model: an absorbance value A obtained by the step (4) of a sample having a kanamycin concentration of 0 and an absorbance value A obtained by the step of obtaining an absorbance value A of a different known kanamycin concentration 0 Calculating to obtain absorbance difference A-A corresponding to different concentrations 0 The method comprises the steps of carrying out a first treatment on the surface of the Then constructing a linear model by utilizing the logarithmic value of different known kanamycin concentrations and the corresponding absorbance difference value;
(6) Determination of the concentration of unknown kanamycin solution Using the linear model obtained in step (5), the concentration of unknown kanamycin solution was determined by repeating step (4).
2. The method of nano-enzyme based colorimetric detection of kanamycin according to claim 1, wherein in step (1), the CuWO 4 The synthesis of the nano material comprises the following specific steps:
cu (NO) 3 ) 2 ·3H 2 Dissolving O in deionized water, regulating pH to 4.0-6.0 with alkali solution, and stirring at 50-80deg.C for 10-50 min; adding Na 2 WO 4 ·2H 2 O, stirring continuously at 70-100deg.C for 30-60 min; cooling to room temperature, washing with water and ethanol sequentially, drying at 50-70deg.C for 8-16 hr, and annealing at 300-500deg.C for 1-3 hr to obtain CuWO 4 Nanomaterial powder.
3. The method of nanoenzyme-based colorimetric detection of kanamycin according to claim 2, wherein the Cu (NO 3 ) 2 ·3H 2 O and Na 2 WO 4 ·2H 2 The mass ratio of O is 1:1-2:7.
4. The method for colorimetrically detecting kanamycin based on nano enzymes according to claim 1, wherein in the step (2), the synthesis of PMSN comprises the following specific steps:
firstly, cetyl trimethyl ammonium bromide CTAB is dissolved in deionized water, then NaOH solution is added into the solution, and stirring is carried out for 10-50 minutes at 40-70 ℃;
then, tetraethyl orthosilicate TEOS is slowly dripped into the solution, and after stirring is carried out for 4-8 hours at 70-90 ℃, the mixture is respectively washed with water and ethanol for several times; then, drying the washed product at 50-70 ℃ for 8-16 hours, and then annealing at 500-600 ℃ for 4-8 hours to remove superfluous surfactant in the crude MSN sample, so as to obtain mesoporous silica MSN;
the MSN was then surface modified with an amine-containing silane to give a positively charged MSN, designated PMSN.
5. The method for colorimetrically detecting kanamycin based on nano enzymes according to claim 4, wherein the mass ratio of CTAB to TEOS is 1:4-6;
the method for carrying out surface modification on MSN comprises the following steps: dispersing MSN in absolute ethyl alcohol, carrying out ultrasonic treatment, then slowly dripping 3-aminopropyl triethoxysilane APTES, and continuously stirring for 6-10 hours at room temperature; then, the mixture was alternately washed with water and ethanol; finally, drying at 50-70 ℃ for 8-16 hours to obtain PMSN powder; the mass ratio of MSN to APTES is 1:15-25.
6. The method for colorimetrically detecting kanamycin based on nano enzymes according to claim 1, wherein in the step (3), a load [ Fe (CN) is prepared 6 ] 3- The aptamer-coated PMSN of (a) comprises the following specific steps: dispersing PMSN to K 3 [Fe(CN) 6 ]Shaking the mixture at 20-30deg.C for 8-16 hr, and adding KANA aptamer to the suspension; shaking at 20-30deg.C for 2-6 hr, and washing with Tris-HCl buffer solution; subsequently, the load [ Fe (CN) 6 ] 3- The aptamer-coated PMSN of (2) is resuspended in Tris-HCl buffer; PMSN: K 3 [Fe(CN) 6 ]The mass ratio of (3) is as follows: 25-15:1; the concentration of PMSN re-suspension was 4-8mg/mL.
7. The method for colorimetrically detecting kanamycin based on nano enzymes according to claim 1, wherein in the step (4), the determination of absorbance comprises the following specific steps: load [ Fe (CN) 6 ] 3- Mixing the aptamer-coated PMSN suspension of (2) with kanamycin solution of different known concentrations, and incubating for 10-50 minutes at 30-40 ℃ with shaking; adding DNase I reaction buffer solution and DNase I, and continuously shaking and incubating for 0.5-2 hours at 30-40 ℃; subsequently, cuWO is added 4 The suspension is reacted for 1 to 10 minutes, and then HAc-NaAc buffer solution, TMB and H are added in sequence 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the Finally, the reaction is carried out at 40-50℃for 10-50 minutes, and the absorbance is measured at 640-660 nm.
8. Use of the method according to any one of claims 1-7 in food safety detection.
9. Kit for colorimetric detection of kanamycin based on nanoenzymes, characterized in that it comprises a load [ Fe (CN) in the method according to any one of claims 1 to 7 6 ] 3- Is a suspension of aptamer-coated PMSN.
10. Use of a kit according to claim 9 for food safety detection.
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| CN105063200A (en) * | 2015-08-06 | 2015-11-18 | 常熟理工学院 | Method for determining kanamycin concentration of sample |
| CN107238699A (en) * | 2017-05-10 | 2017-10-10 | 江南大学 | A kind of colorimetric methods that magnetic bead and gold nano grain analogue enztme activity detection kanamycins are modified based on aptamers |
| CN107389568A (en) * | 2017-05-23 | 2017-11-24 | 宁波大学 | A kind of colorimetric detection probes and its detection method for detecting kanamycins |
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