CN116555395A - Label-free enzyme-free fluorescent aptamer sensing method for detecting kanamycin content - Google Patents
Label-free enzyme-free fluorescent aptamer sensing method for detecting kanamycin content Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6813—Hybridisation assays
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention belongs to the field of biosensors, and provides a label-free enzyme-free fluorescent aptamer sensing method for detecting kanamycin content. First, a recognition probe consisting of a blocking aptamer sequence and a trigger sequence is designed. Kanamycin specifically recognizes the aptamer and then initiates the conformational transition process of the recognition probe, generating an aptamer-kanamycin complex with an activating trigger sequence. The activated trigger sequence initiates the alternate hybridization of the two partially complementary assembly probes to produce long double-stranded DNA with many complete G-quadruplex structures for kanamycin amplification, enzyme-free detection. Fluorescent dye (NMM) is inserted into the complete G-quadruplex structure, and emits strong fluorescent signals for amplifying kanamycin and detecting without marking. Fluorescent aptamer sensors have higher selectivity for kanamycin than other antibiotics. The fluorescent aptamer sensor has a wide application prospect in food safety detection.
Description
Technical Field
The invention belongs to the field of biosensors, and particularly relates to a label-free enzyme-free fluorescent aptamer sensing method for detecting kanamycin content.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Kanamycin is a broad-spectrum aminoglycoside antibiotic. It effectively inhibits the biological activity of gram-negative and gram-positive bacteria by interfering with protein synthesis. Kanamycin has good antibacterial properties, but excessive use or even abuse of kanamycin can lead to its residues in animal-derived foods. Residual kanamycin accumulates in the human body through the food chain, and seriously damages the human health. To avoid harm, both China and European Union have stipulated maximum residual limits (200. Mu.g/kg and 150. Mu.g/kg) for kanamycin in animal derived foods. Therefore, the method for sensitively detecting kanamycin residues in animal-derived foods has important significance for guaranteeing the food safety.
Various kanamycin detection methods have been established to date, including liquid chromatography-mass spectrometry, capillary electrophoresis, and enzyme-linked immunosorbent assay (ELISA). They have their own advantages, but also suffer from the disadvantages of expensive instruments, complex sample preparation, expensive antibodies, etc., which limit their wide application. Thus, there is an urgent need to establish a cost-effective, sensitive method for detecting kanamycin residues in food products to ensure food safety.
Nucleic acid aptamer refers to single stranded oligonucleotides selected by the in vitro exponential enrichment (SELEX) technique. The method can specifically identify target molecules with high affinity, and the nucleic acid adaptation is embodied in the construction of aptamer sensors for the detection of various target molecules. Compared with antibodies, the aptamer has the characteristics of easy synthesis, simple modification, wide target molecules, high stability and low cost. In 2011, kanamycin aptamers were successfully screened by SELEX technology. Recently, a variety of aptamer sensors for kanamycin detection have been constructed based on the specific biological recognition of the aptamer and kanamycin, including electrochemical, colorimetric and fluorescent aptamer sensors. Among these aptamer sensors, fluorescent aptamer sensors have been continuously sought because of their high detection speed and high detection sensitivity. Fluorescent aptamer sensors based on nanomaterials have been constructed for kanamycin detection due to the specific optical properties of the nanomaterials. However, synthesis and modification of nanomaterials such as graphene oxide, gold nanoparticles, and the like are complicated. And the heterogeneous interface of the nanomaterial affects the reaction rate. Due to the effective amplifying capability of the signal amplifying technology, the uniform fluorescent aptamer sensor based on the signal amplifying technology is successively constructed for kanamycin detection so as to improve the detection performance. The detection sensitivity of the sensor is effectively improved, but there are limitations in that most signal amplification techniques require tool enzymes and labeled fluorophore probes. The synthesis of tool enzymes is expensive and the reaction conditions of tool enzymes are harsh. The synthesis and modification of the labeled probes is complex and expensive. Furthermore, the large fluorophores of the labeled probes affect nucleic acid hybridization. Therefore, a fluorescent aptamer sensor with high cost performance and high sensitivity is established to detect kanamycin residues so as to ensure that food safety is urgent.
The hybridization chain reaction first proposed in 2004 is an enzyme-free, efficient, entropy-driven amplification process, which can avoid the above drawbacks. In recent years, hybridization chain reactions have been applied to the construction of aptamer sensors with excellent detection properties for the detection of proteins, metal ions, pesticides and metabolites. However, there is no fluorescent aptamer sensor for detecting kanamycin constructed based on hybridization chain reaction.
Disclosure of Invention
In order to solve the problems, the invention provides an enzyme-free and label-free fluorescent aptamer sensor which is used for sensitively detecting kanamycin in milk samples. Fluorescent aptamer sensors are based on the specific biological recognition and hybridization chain reaction of an aptamer with kanamycin. The process of recognition of kanamycin and aptamer initiates the conformational transition of the recognition probe. The assembly process of the two assembly probes was then initiated to generate long double-stranded DNA with many complete G-quadruplex structures for amplification of kanamycin, enzyme-free detection. N-methylporphyrin dipropionate IX (NMM) selectively binds to the complete G-quadruplex structure, emitting a strong fluorescent signal, significantly higher than when free NMM alone is present. The G-quadruplex/NMM output format was used for kanamycin amplification and label-free detection. The fluorescent aptamer sensor has wide application prospect in food safety detection.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a label-free, enzyme-free fluorescent aptamer sensor for detecting kanamycin content, comprising: recognition probes, assembly probes, and fluorescent dyes;
the recognition probe consists of an aptamer sequence, a trigger blocking sequence and an aptamer blocking sequence.
In a second aspect, the invention provides the use of the label-free enzyme-free fluorescent aptamer sensor in kanamycin detection.
In a third aspect of the present invention, there is provided the above label-free enzyme-free fluorescent aptamer sensing method for detecting kanamycin content, comprising:
mixing kanamycin and a recognition probe, reacting in TNaK buffer solution at 37 ℃ for 30 minutes, adding an assembly probe, and starting hybridization chain reaction at 37 ℃ for 30 minutes to form a G-quadruplex structure;
adding fluorescent dye into the system of the G-quadruplex structure, inserting the fluorescent dye into the whole G-quadruplex structure, reacting for 20 minutes at 37 ℃, and detecting fluorescent signals to obtain the fluorescent dye.
The beneficial effects of the invention are that
(1) Based on hybridization chain reaction, the invention constructs an enzyme-free and label-free fluorescent aptamer sensor for sensitively detecting kanamycin in milk samples. The kanamycin and aptamer recognition process initiates the assembly process of the two assembly probes, and then the G-quadruplex/NMM signal output process. The kanamycin-initiated hybridization chain reaction realizes amplification and enzyme-free detection of kanamycin, and the detection limit is 0.562nM. The G-quadruplex/NMM signal output process realizes the marker-free detection of kanamycin. The aptamer sensor exhibited higher selectivity for kanamycin than other antibiotics, and the sensor successfully detected labeled kanamycin in milk samples. The result proves that the aptamer sensor has wide application prospect in food safety detection. In addition, by simply changing the aptamer sequence, the fluorescent aptamer sensor can be expanded to detect other antibiotic residues in food, which effectively widens the application range of the aptamer sensor.
(2) The preparation method is simple, has strong practicability and is easy to popularize.
Drawings
Fig. 1: the principle that a fluorescent aptamer sensor is used for kanamycin determination, (A) the identification process of kanamycin and the conformational transition process of an identification probe; (B) an assembly process initiated by an activated trigger sequence;
fig. 2: the feasibility of using fluorescent aptamer sensor for kanamycin detection;
fig. 3: Δf change plot for fluorescent aptamer sensor;
fig. 4: identifying a probe R concentration optimization graph, (A) a relation between CR and delta F, (B) a relation between Cp1 and delta F, (C) a relation between Cp2 and delta F, and (D) a relation between T and delta F;
fig. 5: fluorescent intensity of fluorescent aptamer sensor at different kanamycin concentrations, (a) relationship between fluorescent intensity and kanamycin concentration, (B) relationship between Δf and kanamycin concentration logarithm.
Fig. 6: selectivity of fluorescent aptamer sensor for kanamycin.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
A label-free enzyme-free fluorescent aptamer sensor, comprising: recognition probes, assembly probes, and fluorescent dyes;
the recognition probe consists of an aptamer sequence, a trigger blocking sequence and an aptamer blocking sequence.
In some embodiments, the sequence of the recognition probe is one of the following sequences:
R1：5’-CCA GCA ATACTC GAA CAC GTT ACC TAT TGC TGG GGG TTG AGG CTA AGC CGA-3’；
R2：5’-CCC AGC AAT ACT CGAACA CGT TAC CTA TTG CTG GGG GTT GAG GCT AAG CCG A-3’；
R3：5’-CCC CAG CAA TAC TCG AAC ACG TTA CCT ATT GCT GGG GGT TGAGGC TAA GCC GA-3’；
R4：5’-CCC AGC AAT ACT CGA ACA CGT TAC CAT TGC TGG GGG TTG AGG CTA AGC CGA-3’；
R5：5’-CCC AGC AAT ACT CGA ACA CGT TAC CGT ATT GCT GGG GGT TGAGGC TAAGCC GA-3’。
in some embodiments, the assembly probe comprises the following sequence:
P1：5’-CTC GAACAC GTT ACC GGG TAG GGC GTT AGG AGG TAA CGT GTT CGA GTA TTG C-3’；
P2：5’-TGG GTT CCT AAC GCC CTA CCC GGT AAC GTG TTC GAG GCA ATACTC GAA CAC GTT ACC GGG TAG GGC GGG-3’。
in some embodiments, the fluorescent dye is N-methylporphyrin dipropionic acid IX.
The method for detecting kanamycin by using the label-free enzyme-free fluorescent aptamer sensor comprises the following steps:
mixing kanamycin and a recognition probe, reacting in TNaK buffer solution at 37 ℃ for 30 minutes, adding an assembly probe, and starting hybridization chain reaction at 37 ℃ for 30 minutes to form a G-quadruplex structure;
adding fluorescent dye into the system of the G-quadruplex structure, inserting the fluorescent dye into the whole G-quadruplex structure, reacting for 20 minutes at 37 ℃, and detecting fluorescent signals to obtain the fluorescent dye.
In some embodiments, the amount of recognition probe is 25 to 250nM, with a preferred amount of recognition probe of 200nM.
In some embodiments, the assembly probe comprises: 100-500 nM P1 and 100-500 nM P2, with 400nM P1 and 400nM P2 being the preferred assembly probes, respectively.
In some embodiments, the fluorescent dye is used in an amount of 1 μm.
In some embodiments, the excitation and emission slit width is 10nm, the voltage is 700V, and the emission spectrum is scanned from 590nm to 640nm.
The invention will now be described in further detail with reference to the following specific examples, which should be construed as illustrative rather than limiting.
Example 1
1. Experimental part
1.1 materials
All oligonucleotide sequences (Table 1) were synthesized by the company Shanghai, inc. of biological engineering, and purified by Ultra-PAGE. Kanamycin aptamer was selected according to literature reports. Various antibiotics, including ampicillin sodium (Amp), chloramphenicol (Cap), gentamicin sulfate (Gen), kanamycin sulfate (Kan), streptomycin sulfate (Str), and tetracycline hydrochloride (Tet), were purchased from aladine biochemical technologies, inc. Milk samples were purchased from local supermarkets. Dye NMM was purchased from the beijing carboline technologies limited (china). The invention adopts an analysis reagent and ultrapure water to prepare TNaK buffer solution (20 mM Tris-HCl,5mM KCl,0.14M NaCl,pH 7.5).
TABLE 1 all oligonucleotide sequences in fluorescent aptamer sensors
1.2 fluorescent aptamer sensor assay for kanamycin
The identification probe and the assembly probe were heated at 95℃for 10 minutes. They were then cooled to 25 ℃ to form a secondary hairpin structure, which remained stable for 2 hours. In the kanamycin assay, different amounts of kanamycin and 200nM recognition probe were added and reacted in TNaK buffer at 37℃for 30 min. Then, assembly probes (400 nM P1 and 400nM P2) were added, which initiated the hybridization chain reaction at 37℃for 30 minutes to generate a number of complete G-quadruplex structures. Subsequently, 1. Mu.M dye NMM was added and inserted into the complete G-quadruplex structure and reacted at 37℃for 20 minutes to generate a detectable fluorescent signal. All experiments were performed in triplicate. The resulting fluorescence signals were collected on an F-7000 fluorescence spectrometer (Japanese Hitachi Co., ltd.). The excitation and emission slit width was 10nm, the voltage was 700V, and the emission spectrum was scanned from 590nm to 640nm. The maximum values of excitation and emission probes were 399nm and 610nm, respectively.
1.3 selectivity of fluorescent aptamer sensor
The selectivity of the fluorescent aptamer sensor is detected by selecting a plurality of antibiotics such as ampicillin sodium (Amp), chloramphenicol (Cap), gentamicin sulfate (Gen), kanamycin sulfate (Kan), streptomycin sulfate (Str), tetracycline hydrochloride (Tet) and the like. The selectivity of fluorescent aptamer sensors was studied by comparing their emitted fluorescence intensities.
1.4 milk sample detection
Different concentrations of kanamycin (10 nM,100nM,200 nM) were added to milk samples to study the practical application of fluorescent aptamer sensors. First, according to previous reports, a milk sample was pretreated. The following are specific experimental procedures. 10 ml of milk was diluted with 10 ml of ultrapure water to obtain a diluted milk sample. The pH was then titrated to 4.6 by adding 20% acetic acid (pka=4.75) dropwise to promote protein denaturation in the milk and precipitation around the isoelectric point. As the dripping process proceeded, the proteins in the milk sample were precipitated therewith and separated by centrifugation at 10,000rpm for 25 minutes, leaving the supernatant. The pH of the supernatant was titrated to 7.0 and filtered with a 0.22 μm filter to obtain a pretreated milk sample. Kanamycin was then added at various concentrations. And finally, detecting kanamycin added in milk by using a fluorescent aptamer sensor so as to examine the practical application prospect of the aptamer sensor.
2 results and discussion
2.1 principle of fluorescent aptamer sensor for kanamycin assay
The principle of the fluorescent aptamer sensor for sensitively detecting kanamycin in milk samples is shown in fig. 1. The sensor is based on the specific binding of an aptamer to kanamycin and hybridization chain reaction. First, a kanamycin recognition probe (R) consisting of an aptamer sequence (blue), a trigger sequence (red), a trigger blocking sequence (yellow) and an aptamer blocking sequence (dark green) was designed. A portion (II) of the trigger sequence is designed to be embedded in the stem of a recognition probe with a rigid hairpin structure. In the absence of kanamycin, the trigger sequence was not activated. In the presence of kanamycin, the aptamer sequence is specifically recognized by kanamycin and binds to kanamycin to generate an aptamer-kanamycin complex, resulting in shortening of the stem of the recognition probe and lower melting temperature. The recognition process of kanamycin and the conformational transition process of the recognition probe are shown in FIG. 1A. During these processes, the recognition probe undergoes a conformational transition, accompanied by the generation of an activated trigger sequence, which is used to initiate the assembly process. Thus, detection of kanamycin was successfully converted to detection of an activated trigger sequence. The assembly process initiated by the activated trigger sequence is shown in fig. 1B. We designed two partially complementary assembly probes (P1 and P2), where the same colored regions are complementary sequences. G-quadruplex subunits were designed in the stem and both terminal regions of P2 (green). In the rigid hairpin structure of P2, the complete G-quadruplex structure cannot be formed due to the blocking of the stem region and the steric hindrance of the two terminal regions. During assembly, the activated trigger sequence hybridizes to the assembly sequence of P1 (red), opening the hairpin structure of P1 by a strand displacement reaction and exposing the other assembly sequence (black). The exposed assembly sequence hybridizes to P2, opening the hairpin structure of P2 by a strand displacement reaction and exposing the same sequence as the trigger sequence. The newly exposed trigger sequence initiates alternate hybridization between P1 and P2, forming long double-stranded DNA with many complete G-quadruplex structures. As a fluorescent dye, NMM is selectively combined with a complete G-quadruplex structure to emit a strong fluorescent signal, so that amplification, enzyme-free and label-free detection of kanamycin are effectively realized.
2.2 feasibility of fluorescent aptamer sensor for kanamycin assay
The feasibility of the aptamer sensor for kanamycin detection was verified by fluorescence spectroscopy (fig. 2). In the presence of P2 alone, the fluorescence intensity of the aptamer sensor is extremely low (curve a) as the G-quadruplex subunit locks well into the stalk and both end regions of P2. In the presence of both P1 and P2, a small amount of intact G-quadruplexes are produced due to the non-specific hybridization of P1 and P2, and the fluorescence intensity increases slightly (curve b). After addition of the recognition probe, the fluorescence intensity as a background signal was further slightly increased, indicating that the recognition probe and the assembly probe can coexist relatively (curve c). After kanamycin addition, the fluorescence intensity increased significantly (curve d) due to the fact that kanamycin triggered the recognition and assembly process continuously, many complete G-quadruplex structures were generated. The results demonstrate that aptamer sensors based on the hybridization chain reaction can be successfully applied to kanamycin detection.
2.3 detection Condition optimization
Detection conditions including recognition probe sequence design, recognition probe concentration, assembly probe P1 and P2 concentration, and Hybridization Chain Reaction (HCR) time should be optimized to improve detection performance of the aptamer sensor for kanamycin detection.
A portion of the aptamer sequence (I) and a portion of the trigger sequence (II) are designed to be embedded in the stem of a recognition probe with a rigid hairpin structure.The length of the I sequence is directly related to the recognition event and the length of the II sequence is directly related to the assembly process. We designed 5 recognition probes (R1-R5) of different I and II sequence lengths using ΔF (ΔF=F Kanamycin -F No kanamycin ) The change in fluorescence intensity of the fluorescent aptamer sensor with and without kanamycin is shown. As the I sequence length increases from 3 bases to 5 bases, the Δf of the fluorescent aptamer sensor increases and then decreases (fig. 3), as a shorter I sequence length promotes specific binding of kanamycin and aptamer sequences, but induces a higher background signal. In contrast, a longer I sequence length is advantageous for shielding the trigger sequence, but is detrimental to the specific binding of kanamycin to the aptamer sequence, and therefore an I sequence length of 4 bases is preferred. As the II sequence length increases from 5 bases to 7 bases, the Δf of the fluorescent aptamer sensor increases and then decreases. The reason for this is that shorter II sequence lengths promote the hybridization chain reaction, but induce higher background signals. In contrast, longer II sequence lengths favor shielding trigger sequences, but favor hybridization chain reactions. Thus, a sequence length of 6 bases for II is suitable. In summary, R3 has a 4 base I and a 6 base II are the best recognition probe designs.
The recognition process is directly related to the recognition efficiency of the fluorescent aptamer sensor. The concentration of the recognition probe R affecting the recognition process is optimized to obtain higher recognition efficiency. As the R concentration increased from 25nM to 250nM, Δf gradually increased and stabilized at 200nM, 200nM was used for the subsequent experiments (a in fig. 4). The assembly process is directly related to the amplification efficiency of the fluorescent aptamer sensor. The concentration of the assembly probes P1 and P2 affecting the assembly process and the hybridization chain reaction time are optimized to obtain higher amplification efficiency. As the P1 concentration increased from 100nM to 500nM, Δf gradually increased and stabilized at 400nM, 400nM was used for the subsequent experiments (B in fig. 4). As the P2 concentration increased from 100nM to 500nM, Δf gradually increased and stabilized at 400nM, 400nM was used for the subsequent experiments (C in fig. 4). As the reaction time increased from 10 minutes to 40 minutes, Δf gradually increased and stabilized at 30 minutes, 30 minutes was used for the subsequent experiments (D in fig. 4).
2.4 detection Performance of fluorescent aptamer sensor to detect kanamycin
Under the selected conditions, the detection performance of the aptamer sensor was studied by measuring the fluorescence intensity of the fluorescent aptamer sensor at different kanamycin concentrations. The fluorescence intensity of the fluorescent aptamer sensor increased gradually with kanamycin concentration from 2.0 to 200nM (FIG. 5A). And Δf is linearly related to the kanamycin concentration logarithm (B in fig. 5), the correlation equation is Δf=13.19+43.95 lgc (R 2 = 0.9956). Kanamycin has a detection limit of 0.562nM, according to the principle of triple standard deviation, superior to or comparable to the results reported in the prior art (Table 2). Satisfactory results benefit from the high amplification efficiency of the hybridization chain reaction. The accuracy of the fluorescent aptamer sensor was checked by detecting kanamycin concentration in the same batch over the same day under the same conditions. The relative standard deviations (RSD%) obtained at kanamycin concentrations of 10nM,100nM and 200nM were 3.24%, 2.02% and 1.27%, respectively. The reproducibility of the fluorescent aptamer sensor was checked by detecting kanamycin concentration in three batches over three days under the same conditions. RSD% obtained at kanamycin concentrations of 10nM,100nM and 200nM were 4.10%, 3.27% and 2.20%, respectively. The RSD% obtained above are all within reasonable limits. The results demonstrate that fluorescent aptamer sensors have acceptable accuracy and reproducibility.
TABLE 2 comparison of detection Performance of different aptamer sensors for kanamycin detection
The selectivity of fluorescent aptamer sensors was detected with various antibiotics. Antibiotics include ampicillin sodium (Amp), chloramphenicol (Cap), gentamicin sulfate (Gen), kanamycin sulfate (Kan), streptomycin sulfate (Str), and tetracycline hydrochloride (Tet). Kanamycin (200 nM) induces high fluorescence intensity, comparable to that induced by the mixture samples. However, the other antibiotics (2 μm) induced only low fluorescence intensity, comparable to the background intensity induced by the blank sample (fig. 6). The results demonstrate that the fluorescent aptamer sensor has high selectivity for kanamycin.
Wherein [1] Wang, W.Z., yin, Y.Q., gunasekaran, S.,2022.Oxygen-terminated feed-delayed Ti3C2Tx MXene nanosheets as peroxidase-mimic nanozyme for colorimetric detection of kanamycin. Biosens. Bioelectron.218,114774.Https:// doi. Org/10.1016/j. Bios.2022.114774.
[2]Yin,M.,Zhang,L.,Wei,X.X.,Sun,Y.W.,Qi,S.Y.,Chen,Y.,Tian,X.X.,Qiu,J.X.,Xu,D.P.,2022.Spongy Co/Ni-Bio-MOF-based electrochemical aptasensor for detection of kanamycin based on coral-like ZrO2@Au as an amplification platform.Electroanal.Chem.920,116647.https://doi.org/10.1016/j.jelechem.2022.116647.
[3]Liu,X.P.,Cheng,J.L.,Mao,C.J.,Wu,M.Z.,Chen,J.S.,Jin,B.K.,2022.Highly sensitive electrochemiluminescence aptasensor based on a g-C3N4-COOH/ZnSe nanocomposite for kanamycin detection.Microchem.J.172,106928.https://doi.org/10.1016/j.microc.2021.106928.
[4]Liu,Y.,Guan,B.L.,Xu,Z.Q.,Wu,Y.H.,Wang,Y.H.,Ning,G.,2023.Afluorescent assay for sensitive detection of kanamycin by split aptamers and DNA-based copper/silver nanoclusters.Spectrochim.Acta A 286,121953.https://doi.org/10.1016/j.saa.2022.121953.
[5]Wang,C.K.,Li,J.Y.,2021.Fluorescence method for kanamycin detection based on the conversion of G-triplex and G-quadruplex.Anal.Bioanal.Chem.413,7073–7080.https://doi.org/10.1007/s00216-021-03676-y.
[6]Liu,S.S.,Chen,Y.M.,Ruan,Z.J.,Lin,J.Q.,Kong,W.,2022.Development of label-free fluorescent biosensor for the detection of kanamycin based on aptamer capped metal-organic framework.Environ.Res.206,112617.https://doi.org/10.1016/j.envres.2021.112617.
2.5 milk sample detection
The practical application of the aptamer sensor is examined by detecting the content of labeled kanamycin in a milk sample. Two brands of milk (milk 1 and milk 2) were selected for this experiment. The recovery rate of the labeled kanamycin is 98.66% -100.7%, and the RSD% is 1.33% -4.19%. The specific results of this experiment are shown in Table 3. The result proves that the fluorescent aptamer sensor is feasible for detecting kanamycin residues in real milk samples, and has a great application prospect in food safety detection.
TABLE 3 fluorescent aptamer sensor for detecting labeled kanamycin in milk samples
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A label-free, enzyme-free fluorescent aptamer sensor for detecting kanamycin content, comprising: recognition probes, assembly probes, and fluorescent dyes;
the recognition probe consists of an aptamer sequence, a trigger blocking sequence and an aptamer blocking sequence.
2. The label-free enzyme-free fluorescent aptamer sensor for detecting kanamycin content according to claim 1, wherein the sequence of the recognition probe is one of the following sequences:
R1：5’-CCAGCAATACTC GAA CAC GTT ACC TAT TGC TGG GGG TTG AGG CTA AGC CGA-3’；
R2：5’-CCC AGC AAT ACT CGAACA CGT TAC CTA TTG CTG GGG GTT GAG GCT AAG CCG A-3’；
R3：5’-CCC CAG CAATAC TCG AAC ACG TTA CCT ATT GCT GGG GGT TGAGGC TAAGCC GA-3’；
R4：5’-CCC AGC AAT ACT CGA ACA CGT TAC CAT TGC TGG GGG TTG AGG CTAAGC CGA-3’；
R5：5’-CCC AGC AAT ACT CGA ACA CGT TAC CGT ATT GCT GGG GGT TGA GGC TAA GCC GA -3’。
3. the label-free, enzyme-free fluorescent aptamer sensor for detection of kanamycin content of claim 1, wherein the assembly probe comprises the following sequence:
P1：5’-CTC GAA CAC GTT ACC GGG TAG GGC GTT AGG AGG TAA CGT GTT CGA GTA TTG C-3’；
P2：5’-TGG GTT CCT AAC GCC CTA CCC GGT AAC GTG TTC GAG GCA ATA CTC GAACAC GTT ACC GGG TAG GGC GGG-3’。
4. the label-free enzyme-free fluorescent aptamer sensor for detection of kanamycin content of claim 1, wherein the fluorescent dye is N-methylporphyrin dipropionic acid IX.
5. Use of a label-free, enzyme-free fluorescent aptamer sensor for detection of kanamycin content according to any one of claims 1-4 in kanamycin detection.
6. A label-free, enzyme-free fluorescent aptamer sensing method for detecting kanamycin content according to any one of claims 1-4, comprising:
mixing kanamycin and a recognition probe, reacting in TNaK buffer solution at 37 ℃ for 30 minutes, adding an assembly probe, and starting hybridization chain reaction at 37 ℃ for 30 minutes to form a G-quadruplex structure;
adding fluorescent dye into the system of the G-quadruplex structure, inserting the fluorescent dye into the whole G-quadruplex structure, reacting for 20 minutes at 37 ℃, and detecting fluorescent signals to obtain the fluorescent dye.
7. The method for detecting kanamycin content of label-free enzyme-free fluorescent aptamer according to claim 6, wherein the amount of the recognition probe is 25-250 nM or 200nM.
8. The method for detecting kanamycin content by using label-free enzyme-free fluorescent aptamer according to claim 6, wherein the assembling probe comprises: 100-500 nM P1 and 100-500 nM P2, or 400nM P1 and 400nM P2 for the assembly probes, respectively.
9. The method for detecting kanamycin content of label-free enzyme-free fluorescent aptamer of claim 6, wherein the amount of fluorescent dye is 1 μm.
10. The method for detecting kanamycin content of label-free enzyme-free fluorescent aptamer of claim 6, wherein the width of the exciting and emitting slit is 10nm, the voltage is 700V, and the emission spectrum is scanned from 590nm to 640nm.
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