CN114460072A - 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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- 229930182823 kanamycin A Natural products 0.000 title claims abstract description 69
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- 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
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Abstract
The invention discloses a colorimetric detection method of kanamycin based on nanoenzyme and application thereof, wherein potassium ferricyanide is coated in mesoporous silica, a nucleic acid aptamer is adsorbed on the surface of the mesoporous silica to be used as biological gating, the specific recognition reaction of a target object kanamycin and the nucleic acid aptamer causes the gating of the nucleic acid aptamer to be separated from the surface of the mesoporous silica, and potassium ferricyanide is released into a solution from the interior of the mesoporous silica, so that the colorimetric detection method of kanamycin based on nanoenzyme is applied to CuWO4The surface of the nano particle is combined to generate nano enzyme to induce color reaction; during the period, the aptamer in a product formed by combining kanamycin and the aptamer is cut by deoxyribonuclease I, the kanamycin is released into the solution again, and a new round of target substance/aptamer combination reaction and the release of potassium ferricyanide are induced again, so that a detection signal is amplified; the method for detecting kanamycin does not need biological molecular marking and fixation, and has the advantages of simple and convenient operation, low cost and good selectivity.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a method for colorimetric detection of kanamycin based on nanoenzyme and application thereof.
Background
Kanamycin is an important antibiotic applied to treatment and prevention of microbial infection, but excessive use of kanamycin can cause a series of adverse reactions including ototoxicity, nephrotoxicity, anaphylactic shock and the like. The european union has specified maximum residual levels of kanamycin in food products. Therefore, the detection of kanamycin has important significance for food safety supervision. At present, kanamycin is mainly detected by a microbiological method, a chromatographic method and an enzyme-linked immunosorbent assay. However, these methods often require expensive equipment, are time-consuming to operate, require specialized personnel to operate, and are not suitable for screening large quantities of samples. In contrast, the colorimetric method has the advantages of simplicity, low cost, capability of being recognized by naked eyes and the like, and has potential advantages in the aspect of screening of large-batch 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 obtaining, more stable property, easy storage, easy regulation and control of activity and the like compared with natural enzyme, and is expected to replace the natural enzyme to play a role in the fields of catalysis, bacteriostasis, biosensing, disease treatment and the like. Biosensing, Zhu et al [ Xu Zhu, Lin Tang, Jianjia Wang, Bo Peng, Xilian Ouyang, Jianui Tan, Jiangfang Yu, Haopeng Feng, Jianin Tang&Actuators:B.Chemical 330(2021)129318]Using CeO2BN quantum dots are fixed on the surface of the nanorod to form nanoenzyme, so that high-sensitivity detection of kanamycin is realized. However, this method is a signal-reduction type detection, and is susceptible to interference by various factors, resulting in false positive signals. In addition, the use of gold nanoparticles [ Tarun Kumar Sharma, Rajesh Ramanathan, Pabudi Werathunge, Mahsa Mohammadtaheri, Hemant Kumar Daima, Ravi Shukla and Vipul Bansal, chem. Commun.,2014,50,15856- -15859 has 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-]The kanamycin determination is realized by utilizing the dissociation of the aptamer on the surface of the nanoenzyme due to the binding of kanamycin and the aptamer as the nanoenzyme, thereby influencing the activity of the nanoenzyme. However, these methods are not sufficiently sensitive because there is no signal amplification reaction other than the nanoenzyme reaction. In addition, the nucleic acid aptamer may be irreversibly bound on the surface of the nanoenzyme, so that the target kanamycin and the aptamer cannot be smoothly bound, and the measurement effect is influenced.
Disclosure of Invention
In view of the above problems in the prior art, the present invention providesA colorimetric kanamycin detection method based on nano enzyme and application thereof. The invention is disclosed in CuWO4The nano-particle surface in situ forms copper hexacyanoferrate (CuHCF) nano-enzyme which can pass through CuWO4The direct reaction of the nanoparticles with potassium ferricyanide in solution occurs rapidly. Potassium ferricyanide is coated inside mesoporous silica through design, a nucleic acid aptamer is adsorbed on the surface of the mesoporous silica to serve as biological gating, the specific recognition reaction of a target kanamycin and the nucleic acid aptamer causes the nucleic acid aptamer gating to be separated from the surface of the mesoporous silica, potassium ferricyanide is released into a solution from the inside of the mesoporous silica, and therefore the potassium ferricyanide is coated inside the CuWO4The nano-particle surface is combined to generate nano-enzyme to induce color reaction. During this period, the aptamer in the product of kanamycin and aptamer binding is cleaved by dnase i (dnase i), kanamycin is released into the solution again, and a new round of target/aptamer binding reaction and release of potassium ferricyanide are induced again, so that the detection signal is amplified.
The technical scheme of the invention is as follows:
a colorimetric kanamycin detection method based on nano enzyme comprises the following steps:
(1)CuWO4and (3) synthesis of nano materials: preparation of CuWO by conventional method4A nanomaterial powder;
(2) synthesizing PMSN: preparing mesoporous silica MSN, and then carrying out surface modification on the MSN by using silane containing amino to obtain positively charged MSN, namely PMSN;
(3) preparation of Supported [ Fe (CN)6]3-The aptamer-encapsulated PMSN: dispersing the prepared PMSN into K3[Fe(CN)6]Adding KANA aptamer after incubation in the solution, and wrapping to obtain a load [ Fe (CN)6]3-The aptamer-encapsulated PMSN suspension of (a); the sequence of KANA aptamer is SEQ ID NO.1 ═ 5'-TGG GGG TTG AGG CTA AGC CGA-3';
(4) measurement of absorbance: loading the above-mentioned material [ Fe (CN)6]3-The aptamer-encapsulated PMSN suspension is mixed with kanamycin solutions of different known concentrations, incubated and addedReacting DNase I reaction buffer solution with DNase I; then adding the CuWO prepared in the step (1)4Suspending the nanometer material powder, and sequentially adding HAc-NaAc buffer solution, TMB and H2O2Measuring the absorbance at the position of 640-660nm after the reaction is finished;
(5) construction of a linear model: absorbance values A obtained by the step (4) of different known kanamycin concentrations, and absorbance values A obtained by the sample with the kanamycin concentration of 00Calculating to obtain the absorbance difference A-A corresponding to different concentrations0(ii) a Then, constructing a linear model by using logarithmic values of different known kanamycin concentrations and corresponding absorbance differences;
(6) and (4) measuring the concentration of the unknown kanamycin solution by repeating the step (4) by using the linear model obtained in the step (5).
Further, in the step (1), the CuWO4The synthesis of the nano material comprises the following specific steps:
adding Cu (NO)3)2·3H2Dissolving O in deionized water, adjusting pH to 4.0-6.0 with alkaline solution, and stirring at 50-80 deg.C for 10-50 min; adding Na2WO4·2H2O, continuously stirring for 30-60 minutes at 70-100 ℃; cooling to room temperature, washing with water and ethanol in sequence, drying at 50-70 deg.C for 8-16 hr, annealing at 500 deg.C for 1-3 hr to obtain CuWO4A nanomaterial powder.
Further, the Cu (NO)3)2·3H2O and Na2WO4·2H2The mass ratio of O is 1:1-2: 7.
Further, in the step (2), the synthesis of PMSN comprises the following specific steps:
firstly, dissolving Cetyl Trimethyl Ammonium Bromide (CTAB) in deionized water, then adding a NaOH solution into the solution, and stirring for 10-50 minutes at 40-70 ℃;
then, tetraethyl orthosilicate TEOS is slowly dropped into the solution, and after stirring for 4-8 hours at 70-90 ℃, the mixture is washed with water and ethanol for several times respectively; then, drying the washed product at 50-70 ℃ for 8-16 hours, and then annealing at 500-600 ℃ for 4-8 hours to remove redundant surfactant in the crude MSN sample to prepare the mesoporous silica MSN;
and then, carrying out surface modification on the MSN by using silane containing amino to obtain the MSN with positive electricity, which is named as PMSN.
Furthermore, the mass ratio of CTAB to TEOS is 1: 4-6;
the method for surface modification of MSN comprises the following steps: dispersing MSN in absolute ethyl alcohol, performing ultrasonic treatment, then slowly dropping 3-aminopropyl triethoxysilane APTES, and continuously stirring at room temperature for 6-10 hours; then, the mixture was washed alternately 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 supported [ Fe (CN) ]is prepared6]3-The aptamer-wrapped PMSN comprises the following specific steps: dispersing PMSN to K3[Fe(CN)6]In the solution, the mixture is shaken for 8-16 hours at 20-30 ℃, and then KANA aptamer is added into the suspension; shaking at 20-30 deg.C for 2-6 hr, washing with Tris-HCl buffer solution; subsequently, the load [ Fe (CN)6]3-(ii) resuspending the aptamer-encapsulated PMSN in Tris-HCl buffer; PMSN K3[Fe(CN)6]The mass ratio of (A) to (B) is as follows: 25-15: 1; the concentration of PMSN re-suspension is 4-8 mg/mL.
Further, in the step (4), the measurement of the absorbance comprises the following specific steps: will carry [ Fe (CN) ]6]3-The aptamer-encapsulated PMSN suspension of (a) was mixed with kanamycin solutions of different known concentrations and incubated at 30-40 ℃ for 10-50 minutes with shaking; adding DNase I reaction buffer solution and DNase I, and continuously shaking and incubating for 0.5-2 hours at the temperature of 30-40 ℃; subsequently, CuWO was added4Suspending the solution, reacting for 1-10 min, and then adding HAc-NaAc buffer solution, TMB and H in sequence2O2(ii) a Finally, the reaction was carried out at 40-50 ℃ for 10-50 minutes, and the absorbance was measured at 640-660 nm.
The application of the method in food safety detection.
A colorimetric kanamycin detection kit based on nano-enzyme comprises a load [ Fe (CN) ] in the method6]3-The aptamer-encapsulated PMSN suspension of (a).
The application of the kit is applied to food safety detection.
The beneficial technical effects of the invention are as follows:
the invention firstly provides the following idea for detecting kanamycin: as shown in attached figure 1, when a target kanamycin exists, an aptamer recognition reaction is generated to cause the detachment of an aptamer from the surface of mesoporous silica, and ferricyanide ions are released from the silica and are bound to CuWO4The generation of a chromogenic reaction is induced by the hexacyanoferrate nanoenzyme formed in situ on the surface of the nanoparticle, and an increased absorption signal is measured; during the process, DNase I is utilized to shear the aptamer in the aptamer/kanamycin complex, and the target is released again to cause more separation of the aptamer from the surface of the mesoporous silica and release of more ferricyanide ions, so that the signal is greatly enhanced. When the target kanamycin does not exist, the aptamer cannot be separated from the surface of the mesoporous silica to release ferricyanide ions, and as the free ferricyanide ions in the solution are greatly reduced, nano enzyme cannot be generated and color reaction is not induced, and 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 cyclic amplification reaction to improve the sensitivity of the reaction, does not need biomolecule marking and fixation during measurement, is convenient to operate, has low cost and good selectivity, has wide linear range (0.1-100nM) and low detection limit (3.5pM), and has certain potential in food safety detection application.
Drawings
FIG. 1 is a diagram of the biological reaction and detection principle for kanamycin detection based on nanoenzyme;
FIG. 2 is a schematic representation of CuWO prepared in example 14Sweeping of nanomaterialsDrawing an electron microscope image;
FIG. 3 is CuWO prepared in example 14An X-ray diffraction pattern of the nanomaterial;
FIG. 4 is the absorbance response obtained for example 1 (Δ A ═ A-A)0) A linear plot of the logarithm of kanamycin versus different concentrations;
FIG. 5 is a comparison of the absorbance responses of the detection method obtained in example 1 to kanamycin and other possible interferents.
Wherein the concentration of Chloramphenicol (CAP), Tetracycline (TC), Streptomycin (STR), Gentamicin (GEN), Tobramycin (TOB), Doxycycline (DC), Erythromycin (ERY), Ciprofloxacin (CIP), Norfloxacin (NFX), and Ofloxacin (OFLX) is 500 nM; kanamycin (KANA) concentration is 50 nM.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
The reagents or instruments used in the examples of the present invention are not indicated by manufacturers, and are all conventional reagent products available from commercial sources.
Example 1
A colorimetric kanamycin detection method based on nano enzyme comprises the following steps:
(1)CuWO4and (3) synthesis of nano materials: 0.34g of Cu (NO)3)2·3H2O was dissolved in 50mL of deionized water. Then, the pH of the solution was adjusted to 5.0 with 2.0M NaOH solution and stirred at 60 ℃ for 30 minutes. Then, 0.60g of Na was added2WO4·2H2O, stirring was continued at 70 ℃ for 40 minutes. Cooling to room temperature, washing with water and ethanol for 3 times, drying at 60 deg.C for 12 hr, annealing at 400 deg.C for 1 hr to obtain black CuWO4The powder has a scanning electron micrograph shown in figure 2, and an X-ray diffraction pattern shown in figure 3.
As can be seen from the scanning electron micrograph of FIG. 2, the synthesized CuWO4The nanometer material has irregular morphology and the size of the nanometer material is 50-100 nm. FIG. 3 shows all diffraction peaks and triclinic CuWO4The standard cards of (JCPDS No. 72-0616) are identical. Among them, 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 of cetyltrimethylammonium bromide (CTAB) was dissolved in 150mL of 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 Tetraethylorthosilicate (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. Next, the washed product was 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 (named PMSN). Briefly, 50mg of MSN was dispersed in 5.0mL of absolute ethanol and sonicated for 2 hours. Subsequently, 1.0mL of 3-Aminopropyltriethoxysilane (APTES) was slowly dropped, and stirring was continued at room temperature for 8 hours. Then, the mixture was washed alternately with water and ethanol. Finally, drying at 60 ℃ for 12 hours gave white powder PMSN.
Next, a support [ Fe (CN) ]is prepared6]3-The aptamer-encapsulated PMSN of (1). Briefly, 30mg of prepared PMSN was dispersed in 5.0mL of 1.0mM K3[Fe(CN)6]To the solution, the mixture was gently shaken at 25 ℃ for 12 hours, and then 100. mu.L of 10. mu.M KANA aptamer was added to the above suspension. After gentle shaking at 25 ℃ for 4 hours, the column was washed 3 times with Tris-HCl buffer (10mM, containing 10mM NaCl, pH 7.4). Subsequently, the bottom of the centrifugal tube was loaded with [ Fe (CN) ]6]3-The aptamer-encapsulated PMSN of (1) was resuspended in 5mL Tris-HCl buffer (10mM, containing 10mM NaCl, pH 7.4).
Finally, 160. mu.L of [ Fe (CN) ]was loaded6]3-The aptamer-encapsulated PMSN suspension of (a) was mixed with 20 μ L of different concentrations of KANA solution and incubated at 37 ℃ for 30 minutes with gentle shaking. Then 10. mu.L of 1 XDNase I reaction buffer and 1.0U of DNase I were added and incubated at 37 ℃ for 1 hour with constant shaking.
(3) Measurement of absorbance: to the mixture was added 5.0. mu.L of 1.0mg/mL CuWO4The 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 added in this order2O2. 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 a 0.1891 × log CKANA]+0.5373, linear correlation coefficient R20.989, a linear range of 0.1nM to 100nM, and a detection limit of 3.5 pM.
FIG. 5 shows a comparison of the absorbance responses of the test method obtained in example 1 to kanamycin and other possible interferents. Wherein the concentration of Chloramphenicol (CAP), Tetracycline (TC), Streptomycin (STR), Gentamicin (GEN), Tobramycin (TOB), Doxycycline (DC), Erythromycin (ERY), Ciprofloxacin (CIP), Norfloxacin (NFX), and Ofloxacin (OFLX) is 500 nM; kanamycin (KANA) concentration is 50 nM.
Example 2
A colorimetric kanamycin detection method based on nano enzyme comprises the following steps:
(1)CuWO4and (3) synthesis of nano materials: 0.34g of Cu (NO)3)2·3H2O was dissolved in 50mL of deionized water. Then, the pH of the solution was adjusted to 5.0 with 2.0M NaOH solution and stirred at 60 ℃ for 50 minutes. Then, 0.60g of Na was added2WO4·2H2O, stirring was continued at 80 ℃ for 50 minutes. Cooling to room temperature, washing with water and ethanol for 3 times, drying at 60 deg.C for 12 hr, annealing at 500 deg.C for 1 hr to obtain black CuWO4And (3) powder.
(2) Biological reaction: mesoporous Silica (MSN) was first prepared and 0.3g of cetyltrimethylammonium bromide (CTAB) was dissolved in 150mL of 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 Tetraethylorthosilicate (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. Next, the washed product was 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 (named PMSN). Briefly, 50mg of MSN was dispersed in 5.0mL of absolute ethanol and sonicated for 2 hours. Subsequently, 1.0mL of 3-Aminopropyltriethoxysilane (APTES) was slowly dropped, and stirring was continued at room temperature for 8 hours. Then, the mixture was washed alternately with water and ethanol. Finally, drying at 60 ℃ for 12 hours gave white powder PMSN.
Next, a support [ Fe (CN) ]is prepared6]3-The aptamer-encapsulated PMSN of (1). Briefly, 30mg of prepared PMSN was dispersed in 5.0mL of 1.0mM K3[Fe(CN)6]To the solution, the mixture was gently shaken at 25 ℃ for 12 hours, and then 100. mu.L of 10. mu.M KANA aptamer was added to the above suspension. After gently shaking at 25 ℃ for 4 hours, the column was washed 3 times with Tris-HCl buffer (10mM, containing 10mM NaCl, pH 7.4). Subsequently, the bottom of the centrifugal tube was loaded with [ Fe (CN) ]6]3-The aptamer-encapsulated PMSN of (1) was resuspended in 5mL Tris-HCl buffer (10mM, containing 10mM NaCl, pH 7.4).
Finally, 160. mu.L of [ Fe (CN) ]was loaded6]3-The aptamer-encapsulated PMSN suspension of (a) was mixed with 20 μ L of KANA solutions of different concentrations and incubated at 37 ℃ for 30 minutes with gentle shaking. Then 10. mu.L of 1 XDNase I reaction buffer and 1.0U of DNase I were added and incubated at 37 ℃ for 1 hour with constant shaking.
(3) Measurement of absorbance: to the mixture was added 5.0. mu.L of 1.0mg/mL CuWO4The 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 added in this order2O2. Finally, the reaction was carried out at 45 ℃ for 25 minutes, and the absorbance was measured at 652 nm.
It can be seen from the above embodiments that the invention provides a rapid and highly sensitive method for detecting kanamycin by colorimetry without marking or fixing, and the method has the advantages of simple operation, low cost, good selectivity, high sensitivity and the like.
The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> colorimetric kanamycin detection method based on nano enzyme and application thereof
<130> 2022
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> Artificial
<400> 1
tgggggttga ggctaagccg a 21
Claims (10)
1. A colorimetric kanamycin detection method based on nano-enzyme is characterized by comprising the following steps:
(1)CuWO4and (3) synthesis of nano materials: preparation of CuWO by conventional method4A nanomaterial powder;
(2) synthesizing PMSN: preparing mesoporous silica MSN, and then carrying out surface modification on the MSN by using silane containing amino to obtain positively charged MSN, namely PMSN;
(3) preparation of Supported [ Fe (CN)6]3-The aptamer-encapsulated PMSN: dispersing the prepared PMSN into K3[Fe(CN)6]Adding KANA aptamer after incubation in the solution, and wrapping to obtain a load [ Fe (CN)6]3-The aptamer-encapsulated PMSN suspension of (a); the KANA aptamer sequence is shown in SEQ ID NO. 1;
(4) measurement of absorbance: loading the above-mentioned material [ Fe (CN)6]3-The aptamer-encapsulated PMSN suspension is mixed with kanamycin solutions with different known concentrations, and DNase I reaction buffer and DNase I reverse are added after incubationThe preparation method comprises the following steps of; then adding the CuWO prepared in the step (1)4Suspending the nanometer material powder, and sequentially adding HAc-NaAc buffer solution, TMB and H2O2Measuring the absorbance at the position of 640-660nm after the reaction is finished;
(5) construction of a linear model: absorbance values A obtained by the step (4) of different known kanamycin concentrations, and absorbance values A obtained by the sample with the kanamycin concentration of 00Calculating to obtain the absorbance difference A-A corresponding to different concentrations0(ii) a Then, constructing a linear model by using logarithmic values of different known kanamycin concentrations and corresponding absorbance differences;
(6) and (4) measuring the concentration of the unknown kanamycin solution by repeating the step (4) by using the linear model obtained in the step (5).
2. The nanoenzyme-based colorimetric kanamycin detection method according to claim 1, wherein in the step (1), the CuWO is applied to4The synthesis of the nano material comprises the following specific steps:
adding Cu (NO)3)2·3H2Dissolving O in deionized water, adjusting pH to 4.0-6.0 with alkaline solution, and stirring at 50-80 deg.C for 10-50 min; adding Na2WO4·2H2O, continuously stirring for 30-60 minutes at 70-100 ℃; cooling to room temperature, washing with water and ethanol in sequence, drying at 50-70 deg.C for 8-16 hr, annealing at 500 deg.C for 1-3 hr to obtain CuWO4A nanomaterial powder.
3. The nanoenzyme-based colorimetric kanamycin detection method according to claim 2, wherein the Cu (NO) is3)2·3H2O and Na2WO4·2H2The mass ratio of O is 1:1-2: 7.
4. The nanoenzyme-based colorimetric kanamycin detection method according to claim 1, wherein the step (2) of synthesizing PMSN comprises the following specific steps:
firstly, dissolving Cetyl Trimethyl Ammonium Bromide (CTAB) in deionized water, then adding a NaOH solution into the solution, and stirring for 10-50 minutes at 40-70 ℃;
then, tetraethyl orthosilicate TEOS is slowly dropped into the solution, and after stirring for 4-8 hours at 70-90 ℃, the mixture is washed with water and ethanol for several times respectively; then, drying the washed product at 50-70 ℃ for 8-16 hours, and annealing at 500-600 ℃ for 4-8 hours to remove the redundant surfactant in the crude MSN sample to prepare the mesoporous silica MSN;
and then, carrying out surface modification on the MSN by using silane containing amino to obtain the MSN with positive electricity, which is named as PMSN.
5. The nanoenzyme-based colorimetric kanamycin detection method as claimed in claim 4, wherein the mass ratio of CTAB to TEOS is 1: 4-6;
the method for surface modification of MSN comprises the following steps: dispersing MSN in absolute ethyl alcohol, performing ultrasonic treatment, then slowly dropping 3-aminopropyl triethoxysilane APTES, and continuously stirring at room temperature for 6-10 hours; then, the mixture was washed alternately 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 nanoenzyme-based colorimetric kanamycin detection method according to claim 1, wherein in the step (3), a load [ Fe (CN) ]isprepared6]3-The aptamer-wrapped PMSN comprises the following specific steps: dispersing PMSN to K3[Fe(CN)6]In the solution, the mixture is shaken for 8-16 hours at 20-30 ℃, and then KANA aptamer is added into the suspension; shaking at 20-30 deg.C for 2-6 hr, washing with Tris-HCl buffer solution; subsequently, the load [ Fe (CN)6]3-(ii) resuspending the aptamer-encapsulated PMSN in Tris-HCl buffer; PMSN K3[Fe(CN)6]The mass ratio of (A) to (B) is as follows: 25-15: 1; the concentration of PMSN re-suspension is 4-8 mg/mL.
7. The nanoenzyme-based colorimetric kanamycin detection method according to claim 1, wherein the determination of absorbance in step (4) comprises the following specific steps: will carry [ Fe (CN) ]6]3-The aptamer-encapsulated PMSN suspension of (a) was mixed with kanamycin solutions of different known concentrations and incubated at 30-40 ℃ for 10-50 minutes with shaking; adding DNase I reaction buffer solution and DNase I, and continuously shaking and incubating for 0.5-2 hours at the temperature of 30-40 ℃; subsequently, CuWO was added4Suspending the solution, reacting for 1-10 min, and then adding HAc-NaAc buffer solution, TMB and H in sequence2O2(ii) a Finally, the reaction was carried out at 40-50 ℃ for 10-50 minutes, and the absorbance was measured at 640-660 nm.
8. Use of the method according to any one of claims 1 to 7 for food safety testing.
9. A kit for colorimetric detection of kanamycin based on nanoenzyme, characterized in that it comprises the load [ Fe (CN) ] in the method of any one of claims 1 to 76]3-The aptamer-encapsulated PMSN suspension of (a).
10. Use of the kit according to claim 9 for food safety testing.
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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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