Adaptor probe based on structure conversion signal and application thereof in detection of pentachlorophenol

Abstract

The application discloses an aptamer probe based on a structure conversion signal and application thereof in detection of pentachlorophenol, and belongs to the technical field of nucleic acid probes. The application provides an aptamer probe based on a structure conversion signal, which is used for specific identification of pentachlorophenol. The aptamer probe keeps a stable duplex structure with complementary antisense DNA (one FAM label and one Dabcyl label). Fluorescence is quenched due to the close distance between the two complementary chains. The presence of pentachlorophenol induces the conversion of the DNA / DNA duplex structure into a DNA / target complex structure, so that the distance between the Dabcyl label and the FAM label is increased, thereby generating a fluorescence signal, so as to realize the detection of pentachlorophenol with different concentrations. The molecular signal of the aptamer probe provided by the application is increased with the prolongation of time, the sensitivity is high, the specificity is good, and the aptamer probe can play an advantage in the application of biosensing.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid probe technology, specifically relating to an aptamer probe based on structure transition signals and its application in the detection of pentachlorophenol. Background Technology

[0002] Functional nucleic acids are mainly divided into two categories based on their mechanisms of action: the first category is called nucleic acid aptamers. Aptamers can perform recognition functions similar to protein antibodies, specifically recognizing metal ions, small molecules, proteins, cells, tissues, and even organs. Therefore, they are often used as recognition units in the construction of functional nucleic acid probes. The other category is called deoxyribonucleases (DNAzymes). Deoxyribonucleases can bind to coenzyme factors and have catalytic functions similar to proteases. Aptamers are single-stranded oligonucleotides that can selectively recognize and bind to target molecules, obtained through the in vitro exponential enrichment ligand evolution system (SELEX). Compared with antibodies, aptamers have advantages such as a wide target range, simple acquisition process, simple chemical structure, and strong stability, making them powerful tools for molecular recognition. Researchers have obtained nucleic acid aptamers that can recognize and bind to metal ions, small molecules, proteins, and pathogens through in vitro screening techniques. The multifunctionality of targets, high binding affinity and specificity, and the simplicity of in vitro selection make aptamers attractive as molecular tools for bioanalytical applications.

[0003] Pentachlorophenol (PCP) is a typical organochlorine pesticide widely used in insecticides, wood preservatives, herbicides, and fungicides. This chemically stable substance is difficult to degrade naturally after entering the environment and can remain in water bodies, soil, and other environmental media for extended periods, causing persistent water and soil pollution. Furthermore, PCP can accumulate in plants and animals through the food chain and enter the food system. Related studies have confirmed that PCP poses significant potential reproductive toxicity and carcinogenic risks to humans. Long-term exposure or ingestion can cause irreversible chronic damage to multiple organs, including the blood system, nervous system, heart, liver, and kidneys, seriously threatening human health and environmental safety.

[0004] Given the high hazard of pentachlorophenol, my country has introduced a number of stringent control standards and regulations to restrict and control it. As early as 2019, pentachlorophenol and its sodium salt were included in the list of prohibited drugs and other compounds for use in food, explicitly stating that this substance must not be detected in food; subsequently, it was also included in my country's list of key controlled new pollutants. In the field of water environment management, my country has formulated strict limits for pentachlorophenol in different water environment scenarios, including industrial wastewater discharge, surface water, groundwater, and drinking water, comprehensively constraining the environmental emissions and exposure risks of pentachlorophenol.

[0005] Currently, the mainstream methods for pentachlorophenol detection in existing technologies mainly include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and traditional electrochemical detection techniques. However, these conventional detection techniques generally have inherent drawbacks, such as high equipment costs, cumbersome pretreatment steps, long detection times, and high barriers to professional operation, making it difficult to meet the actual needs of rapid on-site screening and rapid detection of large batches of samples.

[0006] Currently, colloidal gold immunochromatography, used for the rapid detection of pentachlorophenol, relies on the principle of specific antigen-antibody binding. However, this technology is highly dependent on antibody titer and suffers from drawbacks such as complex antibody preparation processes and long preparation cycles, making it difficult to guarantee detection accuracy and stability. Furthermore, to date, there is no research or publicly available report on aptamers specifically for pentachlorophenol recognition, and a lack of novel rapid detection technologies based on aptamers is also present.

[0007] Therefore, how to provide aptamers that specifically recognize pentachlorophenol has become an important issue that urgently needs to be addressed. Summary of the Invention

[0008] Therefore, the purpose of this invention is to provide an aptamer probe based on structure transition signals and its application in the detection of pentachlorophenol, thereby solving the problem of the lack of aptamers specifically for the recognition of pentachlorophenol in the prior art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an aptamer probe based on a structure transition signal, wherein the nucleotide sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 constitute the aptamer probe based on the structure transition signal; wherein the 5' end of the nucleotide sequence shown in SEQ ID NO:8 is labeled with a fluorescent group, and the 3' end of the nucleotide sequence shown in SEQ ID NO:9 is labeled with a quencher group.

[0010] Based on the above technical solution, the fluorescent group is further defined as FAM.

[0011] Based on the above technical solution, the quenching group is further defined as Dabcyl.

[0012] Secondly, the present invention provides a detection kit comprising the above-described aptamer probe.

[0013] Based on the above technical solution, the detection kit further includes an auxiliary factor, wherein the auxiliary factor is Mg. 2+ Na+ and K + ;Mg 2+ The concentration is 20mM, Na + Concentration of 300mM, K + The concentration is 100 mM.

[0014] When mM is used as a unit of concentration, it refers to mmol / L.

[0015] Thirdly, the present invention provides the application of the above-mentioned aptamer probe in the detection of pentachlorophenol.

[0016] Based on the above technical solution, further, the conditions under which the aptamer probe specifically recognizes pentachlorophenol are: pH 7-8, temperature 25±5℃, and Mg... 2+ Na + K + As a cofactor.

[0017] Based on the above technical solution, the linear response concentration range of the pentachlorophenol is further 0.5-10 μM.

[0018] Wherein, μM specifically refers to μmol / L.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides an aptamer probe based on a structure transition signal for the specific recognition of pentachlorophenol. The aptamer probe maintains a stable duplex structure with its complementary antisense DNA (one FAM label and one Dabcyl label). Due to the close proximity of the two complementary strands, fluorescence is quenched. The presence of pentachlorophenol induces the DNA / DNA duplex structure to transform into a DNA / target complex structure, resulting in an increased distance between the Dabcyl label and the FAM label, thereby generating a fluorescence signal to achieve the detection of different concentrations of pentachlorophenol.

[0020] 2. The molecular signal of the aptamer probe provided by the present invention increases over time, exhibiting high sensitivity and specificity, which can play an advantage in biosensing applications.

[0021] 3. The aptamer probe of the present invention can report the presence of the target molecule through real-time fluorescence signal, without the need for additional separation and purification steps. Attached Figure Description

[0022] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0023] Figure 1 This is a schematic diagram of the in vitro screening process of aptamer probes based on structure transition signals in Embodiment 3 of the present invention; Figure 2 This is a schematic diagram of the aptamer probe structure transformation based on the structure transformation signal in Embodiment 3 of the present invention; Figure 3 This is a verification diagram of the in vitro screening results of Example 4 of the present invention; Figure 4 This is a sequence diagram of the PCPapt1 structure conversion aptamer of the present invention; Figure 5 This is a diagram showing the feasibility analysis results of PCPapt1 in Embodiment 4 of the present invention; Figure 6 The following is a graph showing the sensitivity analysis results of PCPapt1 to pentachlorophenol in Example 4 of this invention: A is a graph showing the fluorescence kinetics of the structure-converting fluorescent signal probe based on aptamer PCPapt1 to different concentrations of PCP; B is a linear calibration curve of fluorescence intensity versus concentration of PCP in the range of 0.5~10 μM. Figure 7 The results of the selective analysis of PCPapt1 on pentachlorophenol are shown in Figure A: A figure shows the results of the specificity verification experiment of the structure-converting fluorescent signal probe based on aptamer PCPapt1 on 100 Mm PCP and its six analogs; B is a comparison of the fluorescence intensity when the probe is applied to PCP and its six analogs respectively; the chemical structures of PCP and the six analogs are shown in the figure (NC: negative control group). Detailed Implementation

[0024] This invention provides a structure-transition signal aptamer probe that can identify pentachlorophenol through a designed in vitro screening scheme, enabling rapid and sensitive detection of pentachlorophenol.

[0025] By employing a screening strategy that fixes the library, an evolutionary selection is made of a signal aptamer whose structure-transformation mechanism functions. The invention will now be described in detail with reference to embodiments, but the implementation of the invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the invention.

[0026] In the following examples, μM refers to μmol / L when used as a concentration unit; mM refers to mmol / L when used as a concentration unit.

[0027] The following examples include DNA library construction, reverse screening, forward screening, PCR amplification, etc. The nucleic acid names and sequences involved in the examples are shown in Table 1.

[0028] Table 1: Nucleic acid names, sequences, and uses.

[0029]

[0030] Example 1 This embodiment describes the construction of a DNA library component.

[0031] A certain amount of library DNA (SEQ ID NO:1) was mixed with 1.5 molar amounts of P1 (SEQ ID NO:3), P2 (SEQ ID NO:4) and 5 molar amounts of BDNA (SEQ ID NO:2). The mixture was heated at 90°C for 5 min and cooled to room temperature for 20 min to achieve base pairing of the complementary regions of the DNA in the library during annealing. Then, a suspension of streptavidin magnetic beads was added and incubated in magnetic bead coupling buffer at room temperature for 2 h. The biotin at the 5' end of the BDNA was coupled to the streptavidin on the magnetic beads, achieving library immobilization during the screening process. After incubation, magnetic separation was performed using a magnetic rack to elute the uncoupled free DNA in the solution. The elution was performed three times each with magnetic bead buffer, ddH2O, and 1× reaction buffer to complete the construction of the DNA assembly for in vitro screening.

[0032] DNA library preparation systems include: 1) Construction of DNA library components. The reaction components of the DNA library components are shown in Table 2.

[0033] Table 2: Reaction components of DNA library components.

[0034]

[0035] 2) The library is coupled with magnetic beads, and the connection reaction components are shown in Table 3.

[0036] Table 3: Components of the Connecting Reaction.

[0037]

[0038] Example 2 This embodiment describes the preparation of target molecules.

[0039] Prepare a 10mM pentachlorophenol solution as a stock solution, and prepare all stock solutions fresh for immediate use.

[0040] Example 3 This embodiment involves in vitro screening.

[0041] In vitro screening involved steps including reverse screening, forward screening, PCR amplification, and library component construction. High-throughput sequencing was performed after 18 rounds of screening. The screening process is detailed below. Figure 1 The specific screening steps include: 1. Reverse Filtering: The DNA library assembly constructed in Example 1 was resuspended in 200 µL of ultrapure water and reacted in a reaction buffer solution. The components of the reverse screening reaction are shown in Table 4.

[0042] Table 4: Components of the reverse sieving reaction.

[0043]

[0044] Before the reaction, the constructed library components were shaken and mixed, and then 2× reaction buffer and water were added. The mixture was incubated in a DNA mixer at room temperature for 2 hours.

[0045] The 2× reaction buffer solution consists of: 100 mM HEPES, 300 mM NaCl, 100 mM KCl, 20 mM MgCl2, 0.02% Tween 20, and pH 7.5.

[0046] The reaction products are magnetically separated using a magnetic rack. Non-specific DNA sequences free in the solution are eluted, while specific DNA remains fixed on the magnetic beads.

[0047] 2. Positive screening: The uneluted DNA / magnetic bead complex obtained from the reverse screening was resuspended in 200 µL of ultrapure water and reacted in a reaction buffer containing pentachlorophenol. The components of the forward screening reaction are shown in Table 5.

[0048] Table 5: Components of the positive sieve reaction.

[0049]

[0050] Before the reaction, the recovered DNA / magnetic bead complex was shaken and mixed evenly, and then 2× reaction buffer, pentachlorophenol solution and water were added. The mixture was incubated in a DNA mixer at room temperature for 1 hour.

[0051] The 2× reaction buffer solution consists of: 100 mM HEPES, 300 mM NaCl, 100 mM KCl, 20 mM MgCl2, 0.02% Tween 20, and pH 7.5.

[0052] The reaction products were magnetically separated using a magnetic rack to recover the DNA free in the solution. After binding with sodium pentachlorophenolate, the specific DNA was eluted, and the sequence capable of structural transformation was obtained by cold ethanol precipitation.

[0053] 3. PCR amplification: Using DNA sequences capable of structural transitions recovered through forward screening as templates, the target sequences were amplified in large quantities via two-step PCR using upstream primer FP and downstream primers P2 and RP. The PCR products were purified by 10% dPAGE. Due to the spatial modification of the downstream primer RP, PCR amplification yielded sense strands (80 nt) and antisense strands (100 nt) of different lengths. The sense strands were recovered by gel excision and their content was determined. The sequence of upstream primer FP is shown in SEQ ID NO:5, and the sequences of downstream primers P2 and RP are shown in SEQ ID NO:4 and SEQ ID NO:6, respectively. The PCR amplification conditions are shown in Table 6.

[0054] Table 6: PCR amplification conditions.

[0055]

[0056] PCR reaction system: 1) The reaction conditions for PCR1 are shown in Table 7.

[0057] Table 7: PCR1 reaction conditions.

[0058]

[0059] 2) The reaction conditions for PCR2 are shown in Table 8.

[0060] Table 8: PCR2 reaction conditions.

[0061]

[0062] 4. Construction of DNA library components.

[0063] The positive-strand DNA from the PCR-obtained library was mixed with 1.5 molar amounts of P1 (SEQ ID NO:3), P2 (SEQ ID NO:4), and 5 molar amounts of BDNA (SEQ ID NO:2). The mixture was heated at 90 °C for 5 min, cooled to room temperature for 20 min, and then streptavidin magnetic beads were added. The mixture was incubated in coupling buffer at room temperature for 2 h to couple the biotin on the BDNA with the streptavidin on the magnetic beads, thus immobilizing the library during the screening process. After incubation, magnetic separation was performed using a magnetic rack to elute the uncoupled DNA in the solution. The elution was repeated three times with magnetic bead coupling buffer, H2O, and 1× reaction buffer to obtain the DNA components for the next round of screening.

[0064] High-throughput sequencing was used to select the top five sequences for experimental validation. The aptamer sequence PCPapt1 (SEQ ID NO:7) that met the target requirements was found. After constructing a molecular signaling assembly with antisense oligonucleotide chains FDNA and QDNA, the aptamer's structural conversion activity was validated. Figure 2 ).

[0065] Example 4 This embodiment characterizes the performance of the structural transformation aptamer.

[0066] 1. Validation of in vitro screening results: The top five sequences were chemically synthesized and named PCPapt1, PCPapt2, PCPapt3, PCPapt4, and PCPapt5, respectively, and their structure-transforming activity after binding with pentachlorophenol was verified. Figure 3 Real-time fluorescence signal monitoring using a microplate reader revealed that PCPapt1 showed the most significant fluorescence enhancement. After the addition of pentachlorophenol, the fluorescence intensity of PCPapt1 showed an increasing trend over time, indicating that PCPapt1 can act as a structural transition aptamer. Its sequence structure is as follows: Figure 4 As shown, it can be used as a signal molecule in biosensing applications for the detection of pentachlorophenol.

[0067] 2. Feasibility Analysis: In the feasibility analysis, a structure transition component was constructed by mixing QDNA with PCPapt1 and FDNA for real-time fluorescence analysis using a microplate reader. This allowed for the investigation of fluorescence intensity changes over time during structure transition and the determination of the equilibrium point at which the reaction reaches saturation. This process optimized the reaction time for subsequent characterization experiments. The highest fluorescence signal from PCPapt1 in response to pentachlorophenol was observed within 20 minutes, indicating that the reaction had reached equilibrium. Figure 5 ).

[0068] 3. Sensitivity analysis: While maintaining the concentration of the structure-transition aptamer and its ratio with the complementary antisense sequence in the reaction system, changing the concentration of pentachlorophenol in the reaction system allows the structure-transition probe to produce signal responses of varying intensities to different concentrations of pentachlorophenol, down to as low as 0.5 μM. Figure 6 ).

[0069] 4. Selectivity analysis: Investigating the response signal of PCPapt1 to pentachlorophenol analogs ( Figure 7PCPapt1 exhibits weak fluorescence responses to 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP), 2,4,5-trichlorophenol (2,4,5-TCP), and p-chlorophenol (MCP), but no fluorescence response to 2,6-dichlorophenol (2,6-DCP) and tetrachlorobenzoquinone (TCBQ). It produces the most significant fluorescence signal in the presence of pentachlorophenol, indicating that the structural transformation system has good specificity for pentachlorophenol.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An aptamer probe based on structure transition signals, characterized in that, Nucleotide sequences such as SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 constitute aptamer probes based on structure transition signals; the 5' end of the nucleotide sequence shown in SEQ ID NO:8 is labeled with a fluorescent group, and the 3' end of the nucleotide sequence shown in SEQ ID NO:9 is labeled with a quencher group.

2. The aptamer probe according to claim 1, characterized in that, The fluorescent group is FAM.

3. The aptamer probe according to claim 1, characterized in that, The quenching group is Dabcyl.

4. A test kit, characterized in that, Includes the aptamer probe as described in any one of claims 1-3.

5. The detection kit according to claim 4, characterized in that, The detection kit also includes a cofactor, which is Mg. 2+ Na + and K + ;Mg 2+ The concentration is 20mM, Na + Concentration of 300mM, K + The concentration is 100 mM.

6. The application of the aptamer probe as described in any one of claims 1 to 3 in the detection of pentachlorophenol.

7. The application according to claim 6, characterized in that, The conditions under which the aptamer probe specifically recognizes pentachlorophenol are: pH 7-8, temperature 25±5℃, and Mg... 2+ Na + K + As a cofactor.

8. The application according to claim 6, characterized in that, The linear response concentration range of the pentachlorophenol is 0.5-10 μM.

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