Fluorescent aptamer sensor for simultaneously detecting Hg < 2 + > and Pb < 2 + > in fruit and vegetable food and application method of fluorescent aptamer sensor

Abstract

The invention provides a fluorescent aptamer sensor for simultaneously detecting Hg < 2 + > and Pb < 2 + > in fruit and vegetable foods and an application method of the fluorescent aptamer sensor. The aptamer sensor takes a nucleic acid aptamer (Apt) for specifically recognizing the Hg < 2 + > and the Pb < 2 + > as a recognition unit, a fluorophore FAM labeled at the 5'end of the Apt as a donor, and a quenching group BHQ-1 labeled at the 3 'end of a complementary chain (CS) of the Apt as a receptor. When Apt and CS are hybridized, FAM and BHQ-1 are close to each other, fluorescence quenching is caused by a fluorescence resonance energy transfer effect, but when Hg < 2 + > or Pb < 2 + > exists, Apt forms a U-shaped structure or a G-quadruplex structure, hybridization of Apt and CS is prevented, the fluorescence resonance energy transfer effect is weakened, and fluorescence is recovered. The sensor has good linear response to Hg < 2 + > and Pb < 2 + > under the optimal condition, the detection limits are 11.40 nM and 1.15 nM respectively, Hg < 2 + > and Pb < 2 + > in fruit and vegetable food can be accurately and sensitively detected within 25 minutes, and the sensor has good accuracy, precision and specificity and is easy and convenient to operate, rapid and sensitive.

Description

Technical Field

[0001] The present invention belongs to the technical field of heavy metal detection, and particularly relates to a method for simultaneously detecting Hg in fruit and vegetable foods. 2+ and Pb 2+ Fluorescent aptamer sensor and its application method. Background Art

[0002] Mercury and lead are two common toxic heavy metal pollutants. 2+ ) can be enriched through the food chain, causing damage to the nervous system, immune system, endocrine system, etc., while lead ions (Pb 2+ ) is particularly harmful to children's growth and development, and can lead to decreased intelligence, abnormal behavior, etc. Hg 2+ and Pb 2+ Pollution has become a global problem for fruits and vegetables, and my country is no exception. Studies have shown that the average content of Hg and Pb in my country's soil is 0.067 mg / kg and 27 mg / kg respectively, and pollution is more serious in some local areas. In addition, some studies have found that the content of Hg and Pb in rice, vegetables, aquatic products and other foods in my country exceeds the standard, which seriously threatens the health of residents. Therefore, it is urgent to develop a highly sensitive, selective, fast and portable Hg 2+ and Pb 2+ Detection technology is used for early warning monitoring, source tracing analysis, control and remediation of pollution. Currently Hg 2+ and Pb 2+ Common detection methods include atomic absorption / emission spectroscopy, inductively coupled plasma mass spectrometry, fluorescence spectroscopy, etc., but these methods are highly sensitive, but the instruments are expensive and the operation is complicated, which makes it difficult to meet the needs of rapid on-site detection. In recent years, the development of nucleic acid aptamer technology has provided a new idea for heavy metal ion detection. Nucleic acid aptamers are a class of oligonucleotide sequences that can bind to target molecules with high affinity and high specificity. They are obtained by screening from random nucleic acid libraries through the systematic evolution of ligands by exponential enrichment (SELEX). Compared with antibodies, nucleic acid aptamers are easy to synthesize artificially, low in cost, and have stable chemical properties. Currently, a variety of Hg 2+ and Pb 2+ Specific aptamers can be combined with fluorescence resonance energy transfer (FRET), colorimetry, electrochemistry and other signal transduction methods to achieve sensitive detection of heavy metal ions. However, most of these nucleic acid aptamers are targeted at a single ion type and cannot detect Hg at the same time. 2+ and Pb 2+ There are few reports on Hg detection. Moreover, the actual sample matrix is ​​complex, and there may be interference such as competitive binding between ions, which affects the accuracy and reproducibility of detection. Therefore, it is necessary to develop a Hg detection method that has both sensitivity and selectivity, strong anti-interference ability, and wide applicability.2+ and Pb 2+ Meanwhile, testing new methods remains of great significance. Summary of the invention

[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for simultaneously detecting Hg in fruit and vegetable foods. 2+ and Pb 2+ A fluorescent aptamer sensor and an application method thereof, wherein the aptamer sensor specifically recognizes Hg 2+ and Pb 2+ The nucleic acid aptamer (Apt) is used as the recognition unit, the fluorescent group FAM is labeled at the 5' end of Apt as a donor, and the quenching group BHQ-1 is labeled at the 3' end of the complementary chain (CS) of Apt as a receptor. When Apt hybridizes with CS, FAM and BHQ-1 are close to each other, and the fluorescence resonance energy transfer effect occurs, resulting in fluorescence quenching. However, when Hg 2+ or Pb 2+ When present, Apt forms a "U-shaped" structure or a "G-quadruplex" structure, preventing it from hybridizing with CS, weakening the fluorescence resonance energy transfer effect, and restoring fluorescence. 2+ and Pb 2+ It has good linear response, with detection limits of 11.40nM and 1.15nM respectively, and can accurately and sensitively detect Hg in fruits and vegetables within 25 minutes. 2+ and Pb 2+ , has good accuracy, precision and specificity, is easy to operate, rapid and sensitive.

[0004] Technical solution: A method for simultaneously detecting Hg in fruits and vegetables 2+ and Pb 2+ The fluorescent aptamer sensor consists of two components. Component 1 is used to detect Hg 2+ and Pb 2+ The nucleic acid aptamer probe, component 2 is the complementary chain probe. Furthermore, the nucleic acid aptamer probe includes a specific nucleic acid aptamer Apt, the 5' end of Apt is labeled with a fluorescent group FAM, and the base sequence is 5'-GGTTGGTGTGGTTGG-3'. Furthermore, the complementary chain probe comprises a complementary nucleic acid chain CS, the 3' end of CS is labeled with a fluorescent quenching group BHQ-1, and the base sequence is 5'-CCAACCACACCAACC-BHQ1-3'. Furthermore, the application method of the fluorescent aptamer sensor comprises the following steps: S1. Fluorescence detection of standard samples: The aptamer probe FAM-Apt and the complementary chain probe CS-BHQ-1 were dissolved in HEPES buffer to prepare aptamer probe solution and complementary chain probe solution, and then different concentrations of Hg 2+ or Pb 2+ The standard is incubated to obtain a mixed solution for the first incubation, and then a complementary chain probe solution is added to the mixed solution for the first incubation, mixed evenly, and incubated for the second time to obtain a sensing system, and the fluorescence intensity F of the sensing system is detected; S2. Fluorescence detection of blank control: The aptamer probe FAM-Apt and the complementary chain probe CS-BHQ-1 were dissolved in HEPES buffer to prepare aptamer probe solution and complementary chain probe solution, and then Hg-free 2+ or Pb 2+ The mixture was incubated with HEPES buffer to obtain a first incubation mixture, and then the complementary chain probe solution was added to the first incubation mixture, mixed evenly, and incubated for a second time to obtain a sensing system. The fluorescence intensity F0 of the sensing system was detected, and the fluorescence intensity difference and the Hg content were calculated. 2+ or Pb 2+ The relationship curve between the concentration of the standard substance and the concentration of the standard substance was fitted to obtain the equation; S3. Fluorescence detection of the sample to be tested: the aptamer probe FAM-Apt and the complementary chain probe CS-BHQ-1 were dissolved in HEPES buffer respectively to prepare the aptamer probe solution and the complementary chain probe solution, and then the sample to be tested was added to the aptamer probe solution for incubation to prepare a mixed solution for the first incubation, and then the complementary chain probe solution was added to the mixed solution for the first incubation, mixed evenly, and incubated for the second time to prepare a sensing system, and the fluorescence intensity of the sensing system was detected, and the Hg in the sample to be tested was calculated according to the difference in fluorescence intensity. 2+ and Pb 2+ The content. Furthermore, the concentrations of Apt and CS in step S1, step S2 and step S3 are all 6.25-100 nM. Furthermore, the incubation time of step S1, step S2 and step S3 is 5-25 min. Furthermore, in the steps S1, S2 and S3, the excitation wavelength for detecting the fluorescence intensity of the sensor system is 480 nm, and the emission wavelength is 520 nm. Furthermore, the sample to be tested in step S3 is a sample of fruit and vegetable food after filtration, pH adjustment and constant volume. In the present invention, Hg is detected simultaneously 2+ and Pb 2+ The principle of the fluorescent aptamer sensor is: In the absence of target ions, Apt and CS hybridize to form a double-stranded structure, FAM and BHQ-1 are close to each other, and fluorescence resonance energy transfer occurs, resulting in fluorescence quenching. 2+ or Pb 2+ When present, Apt combines with the target ion to form a specific "U-shaped" structure or "G-quadruplex" structure, preventing it from hybridizing with CS, weakening the fluorescence resonance energy transfer effect, and restoring fluorescence. At the same time, by detecting the changes in the fluorescence signal, Hg in fruit and vegetable foods can be detected. 2 and Pb 2+ The quantitative detection principle diagram is shown in Figure 2 shown. Beneficial effects: 1. The aptamer sequence used in the present invention contains only 15 bases, the stem-loop structure is simple, the aptamer is easy to synthesize, the detection cost is low, and the sensor can accurately identify Hg in complex matrices. 2+ and Pb 2+ , it can detect two heavy metal ions at one time, effectively reducing the interference of other metal ions, and has high efficiency, high accuracy, high reliability and good specificity. 2. The fluorescence aptamer sensor of the present invention for Hg 2+ The linear range of detection is 30-1500nM, and for Pb 2+ The detection linear range is 3-900nM. The linear range of target ion detection is wide, which can be applied to the detection of samples at different concentration levels. The fluorescence resonance energy transfer technology can accurately detect Hg in fruits and vegetables within 25 minutes. 2+ and Pb 2+ , for Hg 2+ The detection limit was 11.40 nM for Pb 2+ The detection limit was 1.15 nM, showing high sensitivity. 3. Compared with the traditional instrumental analysis method, the present invention requires lower reagent and equipment costs, no large-scale instruments and equipment are required, suitable for on-site rapid detection, simple operation, rapid and sensitive, good selectivity, low cost and high efficiency, and can be used in the detection of Hg in fruit and vegetable food 2+ and Pb 2+ It provides a good theoretical research foundation in the field of pollution monitoring and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Hg 2+ and Pb 2+ The simulated secondary structure diagram of nucleic acid aptamer Apt and its complementary chain CS; Figure 2 Simultaneous detection of Hg by fluorescence aptasensor based on fluorescence resonance energy transfer 2+ and Pb 2+ Schematic diagram of the principle; Figure 3 Detection of Hg by fluorescent aptasensor 2+ and Pb 2+ Feasibility verification result diagram; Figure 4 Added scheme optimization result graph for reagents; Figure 5 This is the result diagram of the optimization of the concentrations of nucleic acid aptamer Apt and complementary chain CS; Figure 6 The result diagram of the optimization of the incubation time between the nucleic acid aptamer Apt and the complementary chain CS; Figure 7 Schematic diagram of the optimization results of the detection system buffer and pH; Figure 8 Hg 2+ and Pb 2+ Detection sensor sensitivity result diagram; Fig. 9 Hg 2+ and Pb 2+ Result graph of the sensitivity of the detection sensor. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: The nucleic acid aptamer Apt of the fluorescent aptamer sensor of the present invention has a base sequence of 5'-GGTTGGTGTGGTTGG-3', with the 5' end labeled with a fluorescent group FAM, and a complementary nucleic acid chain CS has a base sequence of 5'-CCAACCACACCAACC-BHQ1-3', with the 3' end of CS labeled with a fluorescent quenching group BHQ-1. Depend on Figure 1 It can be seen that Apt and its complementary chain CS have no complementary paired bases and cannot fold themselves, which reduces the interference of self-folding on their binding with the target metal ion. Example 1 A method for simultaneously detecting Hg in fruits and vegetables 2+ and Pb 2+ The application method of the fluorescent aptamer sensor comprises the following steps: S1. Fluorescence detection of standard samples (1) Solution preparation ① Nucleic acid aptamer FAM-Apt solution and complementary chain CS-BHQ-1 solution: Dissolve the aptamer probe FAM-Apt and the complementary chain probe CS-BHQ-1 in a 10 mM HEPES buffer at pH 8.0 to prepare 50 and 75 nM nucleic acid aptamer FAM-Apt solutions and 50 and 75 nM complementary chain CS-BHQ-1 solutions respectively; ②Contains Hg 2+ Standard solution containing Hg 2+ The standard was dissolved in 10 mM HEPES buffer at pH 8.0 and prepared into Hg-containing 2+ Standard solution of ③Contains Pb 2+ Standard solution containing Pb 2+ The standard was dissolved in 10 mM HEPES buffer at pH 7.6 and prepared into 0, 3, 30, 60, 90, 120, 150, 300, 600, 900, 1200 nM Pb-containing 2+ (2) Incubate with a standard solution of ①Hg 2+ Incubation: First, 15 μL of 75 nM aptamer FAM-Apt solution and 170 μL of (1) containing Hg 2+ The standard solution was incubated at 25°C for 7.5 min for the first time, and then 15 μL of 75 nM complementary chain CS-BHQ-1 solution was added and incubated for a second time for 7.5 min. The total volume of the reaction system was 200 μL. ②Pb 2+ Incubation: First, 10 μL of 50 nM nucleic acid aptamer FAM-Apt solution and 180 μL of (1) medium concentration of Pb 2+ The standard solution was incubated at 25°C for 10 min for the first time, and then 10 μL of 50 nM complementary chain CS-BHQ-1 solution was added and incubated for the second time for 10 min. The total volume of the reaction system was 200 μL. (3) Fluorescence intensity detection The fluorescence intensity F was measured using a multifunctional microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. S2. Fluorescence detection of blank control (1) Solution preparation The aptamer probe FAM-Apt and the complementary chain probe CS-BHQ-1 were dissolved in a HEPES buffer with a concentration of 10 mM and a pH of 8.0, respectively, to prepare 50 and 75 nM nucleic acid aptamer FAM-Apt solutions and 50 and 75 nM complementary chain CS-BHQ-1 solutions; (2) Incubation ①Hg 2+ Incubation: First, 15 μL of 75 nM aptamer FAM-Apt solution and 170 μL Hg-free 2+The HEPES buffer was incubated at 25°C for 7.5 min for the first time, and then 15 μL of 75 nM complementary chain CS-BHQ-1 solution was added for the second incubation for 7.5 min. The total volume of the reaction system was 200 μL. ②Pb 2+ Incubation: First, 10 μL of 50 nM aptamer FAM-Apt solution and 180 μL of Pb-free 2+ The cells were incubated for 10 min at 25°C with HEPES buffer, and then 10 μL of 50 nM complementary chain CS-BHQ-1 solution was added for a second incubation of 10 min. The total volume of the reaction system was 200 μL. (3) Fluorescence intensity detection The fluorescence intensity F0 was measured using a multifunctional microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. (4) Standard curve drawing Calculate the fluorescence intensity difference ΔF = F-F0, and according to the fluorescence intensity difference ΔF and Hg 2+ The linear fitting equation is y=1.85x+1227.86. According to the fluorescence intensity difference ΔF and the concentration of Pb 2+ The linear fitting equation is y=1.26x+15.93. In the linear equations, x represents the Hg content in the test sample. 2+ or Pb 2+ The concentration is nmol / L, and y represents the difference in fluorescence intensity of the samples ΔF; S3. Fluorescence detection of samples to be tested (1) Sample pretreatment Weigh 0.2 g of the sample to be tested, homogenize it, and treat it with 5 mL of concentrated HNO3 and 2.5 mL of 30% H2O2. Place the mixture in a graphite digestion furnace for digestion. The temperature program is to maintain it at 120°C for 30 min, then at 160°C for 30 min, and finally at 180°C for 30 min. Then, use 1 M NaOH solution to adjust the pH value of the obtained digest to 8.0 and 7.6, respectively, and use HEPES buffer to make the volume to 25 mL to prepare the sample solution to be tested. (2) Incubation ①Hg 2+ Incubation: First, 15 μL of 75 nM aptamer FAM-Apt solution and 170 μL of the sample solution at pH 8.0 were incubated at 25°C for 7.5 min, and then 15 μL of 75 nM complementary chain CS-BHQ-1 solution was added and incubated for a second time for 7.5 min. The total volume of the reaction system was 200 μL. ②Pb 2+Incubation: First, 10 μL of 50 nM aptamer FAM-Apt solution and 180 μL of the sample solution at pH 7.6 were incubated at 25°C for 10 min, and then 10 μL of 50 nM complementary chain CS-BHQ-1 solution was added and incubated for a second time for 10 min. The total volume of the reaction system was 200 μL. (3) Fluorescence intensity detection The fluorescence intensity F was measured by a multifunctional microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. 样 , calculate the fluorescence intensity difference of the sample to be tested, according to Hg 2+ and Pb 2+ The Hg content in the sample to be tested was calculated by the standard curve fitting equation 2+ and Pb 2+ The content. Performance Test: 1. Verification of the detection principle of fluorescent aptamer sensor The concentrations of nucleic acid aptamer Apt and complementary chain CS were both configured to 1 μM using pH = 7.4 10 mM HEPES buffer to verify the detection principle. (1) Detection of Hg by fluorescent aptasensor 2+ Proof-of-principle experiment Contains Hg 2+ Solution preparation: 10 μL of Apt was mixed with different volumes of 2000 nM Hg 2+ The solutions were mixed to make the Hg 2+ The concentrations were 0, 150, 300, 450, 600, and 750 nM, respectively. Each group was made up to 190 μL with the target buffer, and after oscillation and low-speed centrifugation, the mixture was incubated at room temperature for 10 min; Incubation: Add 10 μL 1 μM CS and continue incubation for 10 min. The total reaction volume is 200 μL. Fluorescence intensity determination: Samples were taken in black 96-well plates and the fluorescence intensity F of each group was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an Infinite M200 Pro microplate reader. 2+ The fluorescence of the control group was recorded as F0, and the fluorescence intensity difference of each concentration group was calculated as ΔF = F-F0. (2) Detection of Pb by fluorescent aptasensor 2+ Proof-of-principle experiment Contains Pb 2+ Solution preparation: 10 μL of Apt was mixed with different volumes of 2000 nM Pb-containing 2+ The solution was mixed to make the Pb in the final detection system 2+The concentrations were 0, 50, 100, 200, 400, and 800 nM, and each group was fixed to 190 μL using the target buffer. The mixture was incubated at room temperature for 10 min; Incubation: Add 10 μL of 1 μM CS and continue incubation for 10 min. The total reaction volume is 200 μL. Fluorescence intensity determination: Samples were taken in a black 96-well plate and the fluorescence intensity F of each group was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an Infinite M200 Pro microplate reader. 2+ The fluorescence of the control group was recorded as F0, and the fluorescence intensity difference of each concentration group was calculated as ΔF = F-F0. Depend on Figure 3 It can be seen that Figure 3 A is the aptamer sensor detecting Hg 2 The feasibility verification results are Figure 3 B is the aptamer sensor detecting Pb 2+ The feasibility verification results show that with the increase of Hg 2+ or Pb 2+ With the increase of Hg concentration, the fluorescence recovery value ΔF of the system gradually increased, indicating that 2+ or Pb 2+ The increase in the concentration can change the fluorescence signal of the fluorescent aptamer sensor, thereby proving the feasibility of the detection principle of the fluorescent aptamer sensor. 2. Optimization of reagent loading scheme The sample loading scheme of the fluorescent aptamer sensor application method in this experiment is shown in Table 1. Table 1 Sample addition scheme Contains Hg 2+ or Pb 2+ Solution preparation: Prepare Hg-containing solutions with concentrations of 200, 1000, 2000, and 20000 nM. 2+ Solution or Pb-containing solution with concentrations of 50, 100, 300, 600, 900 nM 2+ Solution; Incubation: Different concentrations of Hg 2+ or Pb 2+ The solutions were incubated according to the three loading schemes in Table 1; Fluorescence intensity determination: Samples were taken in black 96-well plates and the fluorescence intensity F of each group was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an Infinite M200 Pro microplate reader. 2+ or Pb 2+The fluorescence intensity of the control group was recorded as F0, and the fluorescence intensity difference of each concentration group was calculated as ΔF = F-F0. The experiment was repeated three times, and the average value of the fluorescence intensity difference ΔF was used as the ordinate. 2+ or Pb 2+ The solution concentration is plotted as the horizontal axis. Depend on Figure 4 It can be seen that with the increase of Hg 2+ or Pb 2+ As the concentration increases, the fluorescence recovery difference of the sensing system gradually increases, among which the fluorescence intensity difference ΔF corresponding to the sample addition scheme 1 is the highest. Therefore, the sample addition scheme 1 is selected as the sample addition scheme of this reaction system. 3. Optimization of FAM-Apt and CS-BHQ-1 concentrations The FAM-Apt and CS-BHQ-1 concentration optimization scheme for the fluorescent aptamer sensor application method in this experiment is shown in Table 2. Table 2 FAM-Apt and CS-BHQ-1 concentration optimization scheme Contains Hg 2+ or Pb 2+ Solution preparation: Prepare Hg-containing solution at a concentration of 2000nM 2+ Solution or containing Pb 2+ Solution; Incubation: According to Table 2, add Hg-containing 2+ or Pb 2+ The solution and the sample addition scheme of the corresponding concentration of FAM-Apt solution were mixed and incubated for 10 min, and then the corresponding concentration of CS-BHQ-1 solution was added and incubated for 10 min; Fluorescence intensity determination: Samples were taken in black 96-well plates and the fluorescence intensity F of each group was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an Infinite M200 Pro microplate reader. 2+ or Pb 2+ The fluorescence intensity of the control group was recorded as F0, and the fluorescence intensity difference ΔF=F-F0 was calculated. The experiment was repeated three times, and the average value of the fluorescence intensity difference ΔF was used as the ordinate and the concentration of the nucleic acid aptamer FAM-Apt solution or the concentration of the nucleic acid aptamer complementary chain CS-BHQ-1 solution was used as the abscissa. Depend on Figure 5 It can be seen that Figure 5 A represents Hg 2+ Detect the effect of different FAM-Apt solution concentrations on the fluorescence intensity difference. Figure 5 B stands for Hg 2+ Detect the effect of different CS-BHQ-1 solution concentrations on the fluorescence intensity difference. Figure 5 C stands for Pb 2+ Detect the effect of different FAM-Apt solution concentrations on the fluorescence intensity difference. Figure 5 D stands for Pb 2+ The effect of different CS-BHQ-1 solution concentrations on the fluorescence intensity difference was detected. Depend on Figure 5 A and Figure 5 B shows that for Hg 2+ The detection showed that with the increase of FAM-Apt and CS-BHQ-1 concentrations, the fluorescence recovery value of the system gradually increased, and both reached the highest value at 75 nM. Therefore, the concentration value corresponding to the maximum ΔF value (75 nM) was selected as the optimal concentration of the nucleic acid aptamer FAM-Apt and its complementary chain CS-BHQ-1 solution. Depend on Figure 5 C and Figure 5 D shows that for Pb 2+ The detection showed that with the increase of FAM-Apt and CS-BHQ-1 concentrations, the fluorescence recovery value of the system gradually increased, and both reached the highest value at 50 nM. Therefore, the concentration value corresponding to the maximum ΔF value (50 nM) was selected as the optimal concentration of the nucleic acid aptamer FAM-Apt and its complementary chain CS-BHQ-1 solution. 4. Optimization of incubation time The optimized incubation time scheme of FAM-Apt and CS-BHQ-1 for the fluorescent aptamer sensor application method in this experiment is shown in Table 3. Table 3 Optimization scheme of incubation time of FAM-Apt and CS-BHQ-1 Contains Hg 2+ or Pb 2+ Solution preparation: Prepare Hg-containing solution at a concentration of 2000nM 2+ Solution or containing Pb 2+ Solution; Incubation: Incubate according to the sample loading scheme and incubation time in Table 3; Fluorescence intensity determination: Samples were taken in black 96-well plates and the fluorescence intensity F of each group was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using an Infinite M200 Pro microplate reader. 2+ or Pb 2+ The fluorescence intensity of the control group was recorded as F0, and the fluorescence intensity difference ΔF=F-F0 was calculated. The experiment was repeated three times, and the average value of the fluorescence intensity difference ΔF was used as the ordinate and the incubation time of the nucleic acid aptamer FAM-Apt or the complementary chain CS-BHQ-1 solution was used as the abscissa. Depend on Figure 6 It can be seen that Figure 6 A represents Hg2+ Detect the effect of the first incubation time on the fluorescence intensity difference. Figure 6 B stands for Hg 2+ Detect the effect of the second incubation time on the fluorescence intensity difference. Figure 6 C stands for Pb 2+ Detect the effect of the first incubation time on the fluorescence intensity difference. Figure 6 D stands for Pb 2+ The effect of the second incubation time on the fluorescence intensity difference was detected. Depend on Figure 6 A and Figure 6 B shows that for Hg 2+ The fluorescence recovery value of the system gradually increased with the increase of the first incubation time and the second incubation time, and both reached the highest value at 7.5 min. Therefore, the incubation time corresponding to the maximum ΔF value (7.5 min) was selected as the optimal incubation time for the nucleic acid aptamer FAM-Apt and its complementary chain CS-BHQ-1 solution. Depend on Figure 6 C and Figure 6 D shows that for Pb 2+ The fluorescence recovery value of the system gradually increased with the increase of the first incubation time and the second incubation time. Both reached the highest value at 10 min. Therefore, the incubation time corresponding to the maximum ΔF value (10 min) was selected as the optimal incubation time for the nucleic acid aptamer FAM-Apt and its complementary chain CS-BHQ-1 solution. 5. Optimization of buffer type and system pH The buffer types and system pH optimization scheme for preparing the target ion detection solution in this experiment are shown in Table 4. Table 4 Optimization scheme for preparation of target ion detection solution Solution preparation: Prepare according to the solution preparation scheme in Table 4; Hg 2+ Incubation: Add 75nM nucleic acid aptamer FAM-Apt solution and 2000nM Hg-containing 2 + solution, incubate for 7.5 min; then add 75 nM complementary chain CS-BHQ-1 and continue incubation for 7.5 min, keeping the total reaction volume at 200 μL; Pb 2+ Incubation: Add 75nM nucleic acid aptamer FAM-Apt solution and 2000nM Pb-containing 2+Solution, incubate for 10 min; then add 75 nM complementary chain CS-BHQ-1 and continue incubation for 10 min, keeping the total reaction volume at 200 μL; fluorescence intensity measurement: sample in a black 96-well plate and measure the fluorescence intensity F of each group at 480 nm excitation wavelength and 520 nm emission wavelength using Infinite M200 Pro microplate reader, and the Hg-free 2+ or Pb 2+ The fluorescence intensity of the control group was recorded as F0, and the fluorescence intensity difference ΔF=F-F0 was calculated. The experiment was repeated three times, and the average value of the fluorescence intensity difference ΔF was used as the ordinate and the type of buffer or the pH value of the reaction system was used as the abscissa. Depend on Figure 7 It can be seen that Figure 7 A represents Hg 2+ The effect of the detection buffer type on the fluorescence intensity difference. Figure 7 B stands for Hg 2+ The effect of the pH of the detection reaction system on the fluorescence intensity difference was analyzed. Figure 7 C stands for Pb 2+ The effect of the detection buffer type on the fluorescence intensity difference. Figure 7 D stands for Pb 2+ The effect of the pH value of the reaction system on the fluorescence intensity difference was detected. Depend on Figure 7 A and Figure 7 B shows that for Hg 2+ The largest ΔF change was observed when HEPES buffer was used as the buffer, indicating that different buffer types may affect the performance of the aptamer sensor, so the buffer corresponding to the maximum ΔF value (HEPES buffer) was selected as the optimal buffer type for the system reaction; at the same time, the ΔF value gradually increased in the pH range of 7.6 to 8.0, reached a maximum value at 8.0, and then decreased as the pH continued to increase, indicating that changes in pH generally affect the binding state between Apt and the target ion, change the generation and stability of the fluorescence signal, and thus affect the ΔF value, so the pH corresponding to the maximum ΔF value (8.0) was selected as the optimal pH for the system reaction. Depend on Figure 7 C and Figure 7 D shows that for Pb 2+ The detection, buffer type and system pH value will affect the fluorescence difference ΔF of the detection system. When the buffer is HEPES buffer and the system pH is 7.6, the fluorescence recovery value of the system reaches the highest value. Therefore, the buffer type (HEPES) and pH value (7.6) corresponding to the maximum ΔF value are selected as the buffer type and incubation pH of the detection system. 6. Hg in different fruit and vegetable samples 2+ and Pb 2+ Spike recovery (1) Hg 2+ Detection standard curve: Use 10mM pH=8.0 HEPES buffer to dissolve each reagent, add 75nM nucleic acid aptamer FAM-Apt solution to a 1.5mL brown centrifuge tube, and then add 0, 30, 60, 90, 120, 150, 300, 600, 900, 1200, 1500nM Hg to the system respectively. 2+ The solution was incubated for 7.5 min, and then 75 nM complementary chain CS-BHQ-1 was added and incubated for another 7.5 min, keeping the total reaction volume at 200 μL; the sample was taken into a black 96-well plate and the fluorescence intensity was measured using an Infinite M200 Pro microplate reader to obtain F and F0 and calculate ΔF. The fluorescence intensity difference ΔF was used as the ordinate, and Hg 2+ The corresponding curve is drawn with concentration as the horizontal axis. (2) Pb 2+ Detection standard curve: Use 10mM pH=7.6 HEPES buffer to dissolve each reagent, add 50nM nucleic acid aptamer FAM-Apt solution to a 1.5mL brown centrifuge tube, and then add 0, 3, 30, 60, 90, 120, 150, 300, 600, 900, 1200nM Pb to the system respectively. 2+ The solution was incubated for 10 min, and then 50 nM complementary chain CS-BHQ-1 was added and incubated for another 10 min, keeping the total reaction volume at 200 μL. The samples were taken in a black 96-well plate and the fluorescence intensity was measured using an Infinite M200 Pro microplate reader. F and F0 were obtained and ΔF was calculated. The fluorescence intensity difference ΔF was used as the ordinate, and Pb 2+ The corresponding curve is drawn with concentration as the horizontal axis. (3) Preparation of sample detection system: Take 0.2 g of the sample to be tested and digest it according to the sample treatment method in GB5009.268-2016. 2+ and Pb 2+ Detection: adjust the pH of the sample system to 8.0 and 7.6, respectively, and dilute to 25 mL with HEPES buffer. Add the detection system prepared by methods (1) and (2) into the centrifuge tube to replace the Hg dissolved in the HEPES buffer in steps (1) and (2). 2+ and Pb 2+ Solution, after treatment according to (1) and (2) detection steps, ΔF is determined according to Hg 2+ and Pb 2+ Detect the standard curve and obtain the Hg content in the sample to be tested 2+ Content and Pb 2+ content. (4) Verification: Determine the Hg content using method (3) 2+Hg in fruit and vegetable samples at concentrations of 30, 750, and 1500 nM 2+ Content, fruits and vegetables include grapes, broccoli, cabbage; use (3) method to determine the content of Pb 2+ Pb in fruit and vegetable samples with concentrations of 10, 450 and 900 nM 2+ Fruits and vegetables include grapes, broccoli and cabbage. Depend on Figure 8 It can be seen that Figure 8 A is Hg 2+ Standard curve for Hg 2+ Detection, using the method of the present invention to determine Hg in food samples 2+ The concentration range is 30-1500nM, the linear fitting equation is y=1.85x+1227.86, and the minimum detection limit is 11.40nM, where x in the linear equation represents the Hg content in the test sample. 2+ concentration (nM), y represents the sample ΔF difference; Figure 8 B is Pb 2+ Standard curve for detection of Pb 2+ Detection, using the method of the present invention to determine Hg in food samples 2+ The concentration range is 3-900 nM, the linear fitting equation is y=1.26x+15.93, and the minimum detection limit is 1.45 nM, where x in the linear equation represents the Pb content in the test sample. 2+ The concentration of the sample is (nM), and y represents the ΔF difference of the samples. Table 5 Hg in different fruit and vegetable samples 2+ Detected spike recovery Table 6 Pb in different fruit and vegetable samples 2+ Detected spike recovery It can be seen from Tables 5 and 6 that the fluorescent aptamer sensor is effective for Hg 2+ The average recovery rates for Pb 2+ The average recovery rate obtained by the detection ranged from 89.23% to 105.46%, indicating that the application method of the fluorescent aptamer sensor has good accuracy, reliability and operability in actual sample determination. 7.Hg 2+ and Pb 2+ Detection sensitivity test (1) Hg 2+ Detection sensitivity test Set up in the presence of 1500nM Hg 2++ Test groups of various interfering heavy metal cations and Hg-free 2+ For the HEPES blank group, 75 nM nucleic acid aptamer FAM-Apt solution and 1500 nM Hg 2+ + Incubate with different interfering heavy metal cations for 7.5 minutes, then add 75nM complementary chain CS-BHQ-1 solution and incubate for 7.5 minutes, keeping the total reaction volume at 200μ; take samples in a black 96-well plate and use Infinite M200 Pro microplate reader to measure the fluorescence intensity, obtain F and F0 and calculate ΔF, repeat three times and calculate the average value of the fluorescence intensity difference ΔF as the vertical axis, and draw a graph with the name of each interferent as the horizontal axis, and judge the specificity of the detection system by comparing the size of each ΔF value. (2) Pb 2+ Detection sensitivity test Set up the presence of 900nM Pb 2+ + Experimental groups of various interfering heavy metal cations and Pb-free 2+ HEPES blank group, add 50nM nucleic acid aptamer FAM-Apt solution and 900nM Pb into a 1.5mL brown centrifuge tube. 2+ + Incubate with different interfering heavy metal cations for 10 minutes, then add 50nM complementary chain CS-BHQ-1 solution and incubate for 10 minutes, keeping the total reaction volume at 200μL. Samples were taken in a black 96-well plate and the fluorescence intensity was measured using the Infinite M200 Pro microplate reader to obtain F and F0 and calculate ΔF. After repeating three times, the average value of the difference in fluorescence intensity ΔF was calculated as the ordinate, and the name of each interferent was plotted as the abscissa. The specificity of the detection system was determined by comparing the size of each ΔF value. Depend on Fig. 9 It can be seen that Fig. 9 A is the relationship between different metal ions and Hg 2+ The interference effect of detection, Fig. 9 B is the difference between different metal ions and Pb 2+ The interference effect of the detection. Fig. 9 A and Fig. 9 B shows that under the optimal experimental conditions, the presence of various interfering cations does not cause significant changes in the fluorescence response of the system, indicating that the fluorescent aptamer sensor can detect Hg 2+ and Pb 2+ It has good specificity and can complete detection in complex food samples with high accuracy and reliability. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for simultaneously detecting Hg in fruits and vegetables 2+ and Pb 2+ The fluorescent aptamer sensor is characterized by: It consists of 2 components, component 1 is to detect Hg 2+ and Pb 2+ The nucleic acid aptamer probe, component 2 is the complementary chain probe.

2. A method for simultaneously detecting Hg in fruit and vegetable foods according to claim 1. 2+ and Pb 2+ The fluorescent aptamer sensor is characterized by: The nucleic acid aptamer probe comprises a specific nucleic acid aptamer Apt, the 5' end of Apt is labeled with a fluorescent group FAM, and the base sequence is 5'-GGTTGGTGTGGTTGG-3'.

3. A method for simultaneously detecting Hg in fruits and vegetables according to claim 1. 2+ and Pb 2+ The fluorescent aptamer sensor is characterized by: The complementary chain probe comprises a complementary nucleic acid chain CS, the 3' end of CS is labeled with a fluorescent quenching group BHQ-1, and the base sequence is 5'-CCAACCACACCAACC-BHQ1-3'.

4. A method for simultaneously detecting Hg in fruit and vegetable foods according to claim 1. 2+ and Pb 2+ The application method of the fluorescent aptamer sensor comprises the following steps: S1. Fluorescence detection of standard samples: The aptamer probe FAM-Apt and the complementary chain probe CS-BHQ-1 were dissolved in HEPES buffer to prepare aptamer probe solution and complementary chain probe solution, and then different concentrations of Hg 2+ or Pb 2+ The standard is incubated to obtain a mixed solution for the first incubation, and then a complementary chain probe solution is added to the mixed solution for the first incubation, mixed evenly, and incubated for the second time to obtain a sensing system, and the fluorescence intensity F of the sensing system is detected; S2. Fluorescence detection of blank control: The aptamer probe FAM-Apt and the complementary chain probe CS-BHQ-1 were dissolved in HEPES buffer to prepare aptamer probe solution and complementary chain probe solution, and then Hg-free 2+ or Pb 2+ The mixture was incubated with HEPES buffer to obtain a first incubation mixture, and then the complementary chain probe solution was added to the first incubation mixture, mixed evenly, and incubated for a second time to obtain a sensing system. The fluorescence intensity F0 of the sensing system was detected, and the fluorescence intensity difference and the Hg content were calculated. 2+ or Pb 2+ The relationship curve between the concentration of the standard substance and the concentration of the standard substance was fitted to obtain the equation; S3. Fluorescence detection of the sample to be tested: the aptamer probe FAM-Apt and the complementary chain probe CS-BHQ-1 were dissolved in HEPES buffer respectively to prepare the aptamer probe solution and the complementary chain probe solution, and then the sample to be tested was added to the aptamer probe solution for incubation to prepare a mixed solution for the first incubation, and then the complementary chain probe solution was added to the mixed solution for the first incubation, mixed evenly, and incubated for the second time to prepare a sensing system, and the fluorescence intensity of the sensing system was detected, and the Hg in the sample to be tested was calculated according to the difference in fluorescence intensity. 2+ and Pb 2+ The content.

5. A method for simultaneously detecting Hg in fruits and vegetables according to claim 4. 2+ and Pb 2+ The application method of the fluorescent aptamer sensor is characterized by: The concentrations of Apt and CS in step S1, step S2 and step S3 are all 6.25-100 nM.

6. A method for simultaneously detecting Hg in fruits and vegetables according to claim 4 2+ and Pb 2+ The application method of the fluorescent aptamer sensor is characterized by: The incubation time of step S1, step S2 and step S3 is 5-25 minutes.

7. A method for simultaneously detecting Hg in fruits and vegetables according to claim 4. 2+ and Pb 2+ The application method of the fluorescent aptamer sensor is characterized by: In the steps S1, S2 and S3, the excitation wavelength for detecting the fluorescence intensity of the sensor system is 480 nm, and the emission wavelength is 520 nm.

8. A method for simultaneously detecting Hg in fruits and vegetables according to claim 4. 2+ and Pb 2+ The application method of the fluorescent aptamer sensor is characterized by: The sample to be tested in step S3 is a sample of fruit and vegetable food after filtration, pH adjustment and constant volume.

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