A fluorescent aptamer sensor capable of simultaneously detecting Pb2+ and Cd2+, and its preparation method and detection method

Abstract

The present invention discloses a method for simultaneously detecting Pb 2+ 、Cd 2+ The invention discloses a fluorescent aptamer sensor and its preparation and detection method, wherein the magnetic beads MBs and the substrate chain S-DNA are formed into MBs@S-DNA through biotin and streptavidin, the E-DNA and the lead ion Apt on the S-DNA are subjected to base complementary pairing to form E-DNA@Apt, and finally the S-DNA in MBs@S-DNA is combined with the E-DNA and the cadmium aptamer Apt in the E-DNA@Apt through partial base complementary pairing to finally form a Y-shaped DNA secondary structure, thus preparing a fluorescent aptamer sensor. In the fluorescent aptamer sensor of the present invention, MBs is used to reduce the background signal, and Pb 2+ substrate chain, E‑DNA and Cd 2+ The introduction of aptamers ensures the specificity of the sensor and prevents cross-reaction. FAM and Cy5 are used as signal indicators to construct the MBs@S-DNA / E-DNA / Apt fluorescent aptamer sensor to simultaneously detect Pb 2+ and Cd 2+ purpose.

Description

Technical Field

[0001] The present invention relates to the technical field of heavy metal detection, and in particular to a method for simultaneously detecting Pb 2+ 、Cd 2+ A fluorescent aptamer sensor and a preparation method and a detection method thereof. Background Art

[0002] With the increase of industrial activities, heavy metal ions will inevitably be released into the environment, making the heavy metal pollution problem increasingly serious. Among them, Pb is one of the most dangerous heavy metal environmental pollutants. When it is discharged into the environment, it will be released as Pb 2+ It exists in the form of compounds and can exist in the environment for a long time. It is widely distributed in the atmosphere, water sources, soil and other environments. It is difficult to degrade and enters the human body through biological accumulation, posing a threat to the ecosystem and human health. 2+ It can be absorbed through the skin, digestive tract or food chain and accumulate in the body, especially in the liver, kidneys and brain, causing damage to the human nervous system, digestive system, immune system and liver and kidney functions, and causing memory loss, anemia, cardiovascular dysfunction and mental retardation. Some countries have set regulations on Pb in the environment and food. 2+ There are strict requirements for Pb content in drinking water, such as the World Health Organization (WHO) and the U.S. Environmental Protection Agency (EPA) 2+ The maximum acceptable concentrations were 48 nmol / L and 72 nmol / L, respectively.

[0003] Cadmium is a non-essential element widely distributed in nature. It is highly toxic, difficult to degrade, and accumulates readily. It is the heavy metal with the largest and most serious impact on my country's arable soil pollution. It accumulates in agricultural products through crop root absorption and then enters humans and animals through the food chain, with a latent period of 10 to 30 years. Humans are exposed to cadmium through food, water, and air, with dietary intake being the primary route of cadmium exposure for non-occupational populations. Cadmium accumulates in the human body through the food chain, causing symptoms of toxicity. Cadmium poisoning primarily causes a range of damage to the kidneys, liver, respiratory system, digestive system, bones, and immune system. It also has teratogenic, mutagenic, and carcinogenic effects. In 1993, cadmium was revised by the International Agency for Research on Cancer to a Class A carcinogen, meaning it is a human carcinogen. Recent studies have reported a correlation between cadmium exposure and hypertension and diabetes.

[0004] Therefore, for Pb 2+ 、Cd 2+The detection of lead ions is of great significance to life, food, environment, and medical science. Currently, the main methods for detecting lead ions include atomic absorption spectrometry, atomic fluorescence spectrometry, and electrochemical anodic stripping voltammetry. The main methods for detecting cadmium ions include spectroscopy, electrochemical detection, and graphite furnace atomic absorption spectrometry. Although these methods have high sensitivity and accuracy, they are expensive, time-consuming, and can only measure one element at a time, resulting in low detection efficiency. They also require a high level of technical expertise from the operator and are not suitable for on-site testing. Therefore, it is very important to establish a convenient, economical, and on-site detection method for the simultaneous detection of lead and cadmium ion content in food. Summary of the Invention

[0005] The purpose of the present invention is to solve the problems existing in the prior art and provide a method for simultaneously detecting Pb 2+ 、Cd 2+ A fluorescent aptamer sensor and a preparation method and a detection method thereof.

[0006] In order to solve the above technical problems, the present invention adopts a technical solution that can simultaneously detect Pb 2+ 、Cd 2+ The invention discloses a method for preparing a fluorescent aptamer sensor, wherein the magnetic beads MBs and the substrate chain S-DNA are formed into MBs@S-DNA through biotin and streptavidin, the E-DNA and the lead ion Apt on the S-DNA are formed into E-DNA@Apt through base complementary pairing, and finally the S-DNA in the MBs@S-DNA is combined with the E-DNA and the cadmium aptamer Apt in the E-DNA@Apt through partial base complementary pairing to finally form a Y-shaped DNA secondary structure, thereby preparing a fluorescent aptamer sensor.

[0007] As a method of the present invention that can simultaneously detect Pb 2+ 、Cd 2+ Further optimization of the preparation method of the fluorescent aptamer sensor: comprising the following steps:

[0008] 1) After washing the MBs, thoroughly mix them with tris buffer and S-DNA solution, and then place them in an oven for heat preservation to obtain MBs@S-DNA for later use;

[0009] 2) Thoroughly mixing the E-DNA solution and the Apt solution, and then placing the mixture in an oven for heat preservation to obtain E-DNA@Apt, which is then set aside;

[0010] 3) MBs@S-DNA and E-DNA@Apt were fully mixed and then placed in an oven for heat preservation to prepare a fluorescent aptamer sensor.

[0011] As a method of the present invention that can simultaneously detect Pb 2+ 、Cd2+ Further optimization of the preparation method of the fluorescent aptamer sensor: the molar ratio of S-DNA, E-DNA and Apt added is 1:0.6~1.5:0.6~1.5.

[0012] As a method of the present invention that can simultaneously detect Pb 2+ 、Cd 2+ Further optimization of the preparation method of the fluorescent aptamer sensor: the conditions for the oven insulation treatment in steps 1) and 2) are: temperature 25-50° C., time ≤3 h.

[0013] As a method of the present invention that can simultaneously detect Pb 2+ 、Cd 2+ Further optimization of the preparation method of the fluorescent aptamer sensor: the sequence of the S-DNA is: 5'-TTCCACTAT / rA / GGAAGAGATTACAGTCCAAAAAA-3', wherein the 5'-end fluorescent group is Cy5 and the 3'-end is labeled with biotin.

[0014] As a method of the present invention that can simultaneously detect Pb 2+ 、Cd 2+ Further optimization of the preparation method of the fluorescent aptamer sensor: The sequence of the E-DNA is: 5'-ATAATACCACTATCTCTTCTCCGAGCCGGTCGAAATAGTGGAA-3'.

[0015] As a further optimization of the preparation method of the fluorescent aptamer sensor capable of simultaneously detecting Pb2+ and Cd2+ of the present invention: the sequence of the Apt is: 5'-GGACTGTTGTGGTATTATTTTTGGTTGTG-3', wherein the 3'-end fluorescent group is FAM.

[0016] A method for simultaneously detecting Pb 2+ 、Cd 2+ The fluorescent aptamer sensor is prepared by the above method.

[0017] A method for simultaneous detection of Pb using fluorescent aptamer sensors 2+ 、Cd 2+ Method: Add Pb to the fluorescent aptamer sensor 2+ and Cd 2+ The test solution and tris buffer solution were incubated at 25-50°C for ≤3h. After the incubation, the supernatant was removed by magnetic separation and transferred to a centrifuge tube. The supernatant was washed once with MBs cleaning solution and then transferred to the same centrifuge tube. Tris buffer was added to the volume and the tube was thoroughly mixed by vortexing. Finally, the fluorescence intensity of FAM and Cy5 was detected by fluorescence spectrophotometer.

[0018] The measurement parameters for detecting fluorescence intensity were set as follows: the maximum excitation wavelength of FAM was 480 nm, the scanning range was 500-600 nm, and the FAM fluorescence intensity was recorded; the maximum excitation wavelength of Cy5 was 625 nm, the scanning range was 645-750 nm, and the Cy5 fluorescence intensity was recorded.

[0019] The present invention has the following beneficial effects: the fluorescent aptamer sensor of the present invention can detect Pb 2+ and Cd 2+ The detection has the characteristics of high accuracy, wide linear range and low detection limit. At the same time, the test results of actual samples show that the prepared sensor has very good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the Y-shaped DNA structure of the fluorescent aptamer sensor of the present invention;

[0021] Figure 2 Schematic diagram of the detection principle of the fluorescent aptamer sensor of the present invention;

[0022] Figure 3 The sensor prepared in Example 1 was Cd-free. 2+ (dotted line), 0.01 μmoL / LCd 2+ (dashed line), in the presence of 0.10 μmoL / LCd 2+ (solid line) the fluorescence intensity of FAM;

[0023] like Figure 4 The sensor prepared in Example 1 is Pb-free 2+ (dotted line), 0.01 μmoL / LPb 2+ (dashed line), in the presence of 0.10 μmoL / LPb 2+ (solid line) is the fluorescence intensity of Cy5. DETAILED DESCRIPTION

[0024] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with examples, but the content of the present invention is not limited to the following examples.

[0025] A method for simultaneously detecting Pb 2+ 、Cd 2+The method for preparing a fluorescent aptamer sensor comprises: forming MBs@S-DNA by binding magnetic beads MBs and substrate chain S-DNA via biotin and streptavidin; forming E-DNA@Apt by binding E-DNA and lead ion Apt on S-DNA via base complementary pairing; and finally, forming a Y-shaped DNA secondary structure by binding the S-DNA in MBs@S-DNA with the E-DNA and cadmium aptamer Apt in E-DNA@Apt via partial base complementary pairing. Specifically, the fluorescent aptamer sensor comprises the following steps:

[0026] 1) After washing the MBs, thoroughly mix them with tris buffer and S-DNA solution, and then place them in an oven for heat preservation to obtain MBs@S-DNA for later use;

[0027] S-DNA is immobilized on MBs through the strong binding of biotin on S-DNA to streptavidin (SA) coated on MBs.

[0028] The sequence of S-DNA is: 5'-TTCCACTAT / rA / GGAAGAGATTACAGTCCAAAAAA-3', as shown in Sequence ID Number 1. The 5' end fluorescent group is Cy5, and the 3' end is labeled with biotin.

[0029] 2) Thoroughly mixing the E-DNA solution and the Apt solution, and then placing the mixture in an oven for heat preservation to obtain E-DNA@Apt, which is then set aside;

[0030] The sequence of E-DNA is: 5'-ATAATACCACTATCTCTTCTCCGAGCCGGTCGAAATAGTGGAA-3', as shown in Sequence ID Number 2.

[0031] The sequence of Apt is: 5'-GGACTGTTGTGGTATTATTTTTGGTTGTG-3', as shown in Sequence ID Number 3. The 3' end fluorescent group is FAM.

[0032] 3) MBs@S-DNA and E-DNA@Apt were fully mixed and then placed in an oven for heat preservation to prepare a fluorescent aptamer sensor.

[0033] The fluorescent aptamer sensor of the present invention has the following characteristics:

[0034] (1) Magnetic separation using magnetic beads can reduce background signals;

[0035] (2) DNAzyme can specifically recognize Pb 2+, and can target circulation and thus amplify the signal;

[0036] (3) The three chains of S-DNA, E-DNA and Apt hybridize to form a Y-shaped structure, which can simultaneously detect Pb 2+ and Cd 2+ Two targets.

[0037] In the fluorescent aptamer sensor of the present invention, MBs is used to reduce the background signal, Pb 2+ substrate chain, E-DNA and Cd 2+ The introduction of aptamers ensures the specificity of the sensor and prevents cross-reaction. FAM and Cy5 are used as signal indicators to construct the MBs@S-DNA / E-DNA / Apt fluorescent aptamer sensor to simultaneously detect Pb 2+ and Cd 2+ purpose.

[0038] In the absence of Pb 2+ and Cd 2+ In the presence of Pb, the fluorescent aptamer sensor will not be triggered and will remain in its original state. The fluorescence in the supernatant after magnetic separation is weak. 2+ and Cd 2+ In the presence of Pb 2+ The rA cleavage site on the S-DNA is cleaved, and Cd 2+ Binds to the cadmium aptamer, releasing the cadmium aptamer from the Y-shaped structure (e.g. Figure 2 After magnetic separation, obvious FAM and Cy5 fluorescence signals were observed in the supernatant.

[0039] <Example 1>

[0040] A method for simultaneously detecting Pb 2+ 、Cd 2+ The preparation method of the fluorescent aptamer sensor comprises the following steps:

[0041] 1) Pipette 5 μL MBs into a 200 μL centrifuge tube, wash the magnetic separator three times with 50 μL MBs washing solution, add 5 μL tris buffer, and then add 20 μL 1 μmol / L S-DNA solution, mix thoroughly with a vortex instrument, and place in an oven at 37°C for 1 hour;

[0042] The sequence of S-DNA is: 5'-TTCCACTAT / rA / GGAAGAGATTACAGTCCAAAAAA-3', where the fluorescent group at the 5' end is Cy5 and the 3' end is labeled with biotin.

[0043] 2) In another 200 μL centrifuge tube, add 20 μL of 1 μmol / L E-DNA solution and 20 μL of 1 μmol / L Apt solution, mix thoroughly using a vortexer, and place in an oven at 37°C for 1 hour.

[0044] The sequence of E-DNA is: 5'-ATAATACCACTATCTCTTCTCCGAGCCGGTCGAAATAGTGGAA-3'. The sequence of Apt is: 5'-GGACTGTTGTGGTATTATTTTTGGTTGTG-3', where the 3' end fluorescent group is FAM.

[0045] 3) Add the solution in the centrifuge tube in 2) to the centrifuge tube in 1), mix thoroughly with a vortexer, and place in an oven at 37°C for 1 hour to prepare a fluorescent aptamer sensor. Figure 1 The Y-shaped DNA structure shown.

[0046] like Figure 3 As shown, the sensor prepared in this embodiment is Cd-free. 2+ (dotted line), 0.01 μmoL / LCd 2+ (dashed line), in the presence of 0.10 μmoL / LCd 2+ (solid line) is the fluorescence intensity of FAM.

[0047] like Figure 4 As shown, the sensor prepared in this embodiment is Pb-free. 2+ (dotted line), 0.01 μmoL / LPb 2+ (dashed line), in the presence of 0.10 μmoL / LPb 2+ (solid line) is the fluorescence intensity of Cy5.

[0048] <Example 2>

[0049] A method for simultaneously detecting Pb 2+ 、Cd 2+ The preparation method of the fluorescent aptamer sensor comprises the following steps:

[0050] 1) Pipette 5 μL MBs into a 200 μL centrifuge tube, wash the magnetic separator three times with 50 μL MBs washing solution, add 5 μL tris buffer, and then add 20 μL 1 μmol / L S-DNA solution, mix thoroughly with a vortex instrument, and place in an oven at 37°C for 1 hour;

[0051] The sequence of S-DNA is: 5'-TTCCACTAT / rA / GGAAGAGATTACAGTCCAAAAAA-3', where the fluorescent group at the 5' end is Cy5 and the 3' end is labeled with biotin.

[0052] 2) In another 200 μL centrifuge tube, add 20 μL of 0.6 μmol / L E-DNA solution and 20 μL of 0.6 μmol / L Apt solution, mix thoroughly using a vortexer, and place in an oven at 25°C for 3 h.

[0053] The sequence of E-DNA is: 5'-ATAATACCACTATCTCTTCTCCGAGCCGGTCGAAATAGTGGAA-3'. The sequence of Apt is: 5'-GGACTGTTGTGGTATTATTTTTGGTTGTG-3', where the 3' end fluorescent group is FAM.

[0054] 3) The solution in the centrifuge tube in 2) was added to the centrifuge tube in 1), mixed thoroughly by vortexing, and placed in an oven at 50° C. for 0.5 h to prepare a fluorescent aptamer sensor.

[0055] <Example 3>

[0056] A method for simultaneously detecting Pb 2+ 、Cd 2+ The preparation method of the fluorescent aptamer sensor comprises the following steps:

[0057] 1) Pipette 5 μL MBs into a 200 μL centrifuge tube, wash the magnetic separator three times with 50 μL MBs washing solution, add 5 μL tris buffer, and then add 20 μL 1 μmol / L S-DNA solution, mix thoroughly with a vortex instrument, and place in an oven at 37°C for 1 hour;

[0058] The sequence of S-DNA is: 5'-TTCCACTAT / rA / GGAAGAGATTACAGTCCAAAAAA-3', where the fluorescent group at the 5' end is Cy5 and the 3' end is labeled with biotin.

[0059] 2) In another 200 μL centrifuge tube, add 20 μL of 1.5 μmol / L E-DNA solution and 20 μL of 1.5 μmol / L Apt solution, mix thoroughly using a vortexer, and place in an oven at 45°C for 2.5 h.

[0060] The sequence of E-DNA is: 5'-ATAATACCACTATCTCTTCTCCGAGCCGGTCGAAATAGTGGAA-3'. The sequence of Apt is: 5'-GGACTGTTGTGGTATTATTTTTGGTTGTG-3', where the 3' end fluorescent group is FAM.

[0061] 3) The solution in the centrifuge tube in 2) was added to the centrifuge tube in 1), mixed thoroughly by vortexing, and placed in an oven at 30° C. for 2 h to prepare a fluorescent aptamer sensor.

[0062] <Example 4>

[0063] The method for detecting actual samples using the fluorescent aptamer sensor of the present invention comprises the following specific steps:

[0064] (1) Actual sample processing: 20 μL Pb 2+ and 20 μL Cd 2+ (1mmoL / L, 10mmoL / L, 50mmoL / L) were added to three 200μL centrifuge tubes, 160μL of green tea water was added, and the mixture was thoroughly mixed by vortexing to prepare 0.100mmoL / L, 1.000mmoL / L, 5.000mmoL / L of green tea extracts containing Pb. 2+ and Cd 2+ Actual samples of green tea.

[0065] (2) Pipette 5 μL of MBs into a 200 μL centrifuge tube, wash the magnetic separator three times with 50 μL of MBs cleaning solution, add 5 μL of tris buffer, and then add 20 μL of 1 μmoL / L S-DNA solution, mix thoroughly with a vortex instrument, and place in an oven at 37°C for 1 h;

[0066] (3) Take another 200 μL centrifuge tube, add 20 μL of 1 μmoL / L E-DNA solution and 20 μL of 1 μmoL / L Apt solution, mix thoroughly with a vortexer, and place in an oven at 37°C for 1 h;

[0067] (4) Add the solution in the centrifuge tube in (3) to the centrifuge tube in (2), mix thoroughly with a vortexer, and place in an oven at 37°C for 1 hour to form a precipitate. Figure 1 Thus, a fluorescent aptamer sensor for the simultaneous detection of lead and cadmium ions based on magnetic separation technology was obtained.

[0068] (5) 2 μL of 0.100 mmoL / L, 1.000 mmoL / L, and 5.000 mmoL / L green tea samples and 6 μL of tris buffer solution were added to the fluorescent aptamer sensor, respectively. After incubation at 37°C for 30 min, the supernatant was removed by magnetic separation and placed in a 200 μL centrifuge tube. The supernatant was washed once with 10 μL of MBs cleaning solution and placed in the same centrifuge tube. The volume was then fixed to 200 μL with tris buffer solution, which was thoroughly mixed by vortexing. Finally, the fluorescence intensity of FAM and Cy5 was detected by fluorescence spectrophotometer. The measurement parameters were set as follows: the maximum excitation wavelength of FAM was 480 nm, the scanning range was 500-600 nm, and the FAM fluorescence intensity was recorded; the maximum excitation wavelength of Cy5 was 625 nm, the scanning range was 645-750 nm, and the Cy5 fluorescence intensity was recorded. The detection results are shown in the following table:

[0069]

[0070] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for simultaneously detecting Pb 2+ 、Cd 2+ The method for preparing a fluorescent aptamer sensor is characterized in that: The magnetic beads MBs and the substrate chain S-DNA are formed into MBs@S-DNA through biotin and streptavidin, and the E-DNA and the lead ion Apt on the S-DNA are formed into E-DNA@Apt through base complementary pairing. Finally, through partial base complementary pairing, the S-DNA in MBs@S-DNA is combined with the E-DNA and cadmium aptamer Apt in E-DNA@Apt to finally form a Y-shaped DNA secondary structure, thus preparing a fluorescent aptamer sensor.

2. can detect Pb simultaneously as claimed in claim 1 2+ 、Cd 2+ The method for preparing a fluorescent aptamer sensor is characterized in that: The following steps are involved: 1) After washing the MBs, thoroughly mix them with tris buffer and S-DNA solution, and then place them in an oven for heat preservation to obtain MBs@S-DNA for later use; 2) Thoroughly mixing the E-DNA solution and the Apt solution, and then placing the mixture in an oven for heat preservation to obtain E-DNA@Apt, which is then set aside; 3) MBs@S-DNA and E-DNA@Apt were fully mixed and then placed in an oven for heat preservation to prepare a fluorescent aptamer sensor.

3. can detect Pb simultaneously as claimed in claim 2 2+ 、Cd 2+ The method for preparing a fluorescent aptamer sensor is characterized in that: The molar ratio of the added S-DNA, E-DNA and Apt is 1:0.6-1.5:0.6-1.

5.

4. can detect Pb simultaneously as claimed in claim 2 2+ 、Cd 2+ The method for preparing a fluorescent aptamer sensor is characterized in that: The conditions for the oven heat preservation treatment in steps 1) and 2) are both: temperature 25-50° C., time ≤ 3 h.

5. can detect Pb simultaneously as described in claim 1 or 2 2+ 、Cd 2+ The method for preparing a fluorescent aptamer sensor is characterized in that: The sequence of the S-DNA is: 5'-TTCCACTAT / rA / GGAAGAGATTACAGTCCAAAAAA-3', wherein the fluorescent group at the 5' end is Cy5 and the 3' end is labeled with biotin.

6. can detect Pb simultaneously as described in claim 1 or 2 2+ 、Cd 2+ The method for preparing a fluorescent aptamer sensor is characterized in that: The sequence of the E-DNA is: 5'-ATAATACCACTATCTCTTCTCCGAGCCGGTCGAAATAGTGGAA-3'.

7. can detect Pb simultaneously as described in claim 1 or 2. 2+ 、Cd 2+ The method for preparing a fluorescent aptamer sensor is characterized in that: The sequence of Apt is: 5'-GGACTGTTGTGGTATTATTTTTGGTTGTG-3', wherein the fluorescent group at the 3' end is FAM.

8. A method for simultaneously detecting Pb 2+ 、Cd 2+ The fluorescent aptamer sensor is characterized in that The invention is prepared by the method according to any one of claims 1 to 7.

9. Simultaneous detection of Pb using the fluorescent aptamer sensor according to claim 8 2+ 、Cd 2+ The method is characterized in that Add Pb to the fluorescent aptamer sensor 2+ and Cd 2+ The test solution and tris buffer solution were incubated at 25-50°C for ≤3h. After the incubation, the supernatant was removed by magnetic separation and transferred to a centrifuge tube. The supernatant was washed once with MBs cleaning solution and then transferred to the same centrifuge tube. Tris buffer was added to the volume and the tube was thoroughly mixed by vortexing. Finally, the fluorescence intensity of FAM and Cy5 was detected by fluorescence spectrophotometer.

10. The detection method according to claim 9, wherein The measurement parameters for detecting fluorescence intensity were set as follows: the maximum excitation wavelength of FAM was 480 nm, the scanning range was 500-600 nm, and the FAM fluorescence intensity was recorded; the maximum excitation wavelength of Cy5 was 625 nm, the scanning range was 645-750 nm, and the Cy5 fluorescence intensity was recorded.

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